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GREEN BOOK 1

Triclosan

CIR EXPERT PANEL MEETING AUGUST 30-31, 2010

Memorandum

To: From: Subject: Date

CIR Expert Panel Members and Liaisons Director, CIR


Triclosan Draft Report

30 July 2010

The literature review for Triclosan was issued in April. Several comments were received and are included in the book. They have been addressed in preparing the draft report for your review.
In addition, 3 new sources of information have been incorporated: the European Commission Scientific Committee on Consumer Products opinion on triclosan triclosan, , the European Commission Scientific Committee on Consumer Safety (same committee, new name) opinion on triclosan antimicrobial susceptibility, and a critical review of tricosan safety by Rodricks, et al. published in Critical Reviews in Toxicology. The European opinions are provided for your consideration. The Rodricks, et al. publication will be provided to you as a .pdf. One note: the references are a mix of numbered citations and author and date citations. We will make them all numbered citations in the next incarnation, but for purposes of this review, we figured you could handle a mix.

The approach CIR has taken to evaluate the safety of triclosan in cosmetics differs from the norm, in part because of the need to focus on the putative issues and i in n part because adding one more summary of relevant literature (in the face of EPA, FDA, Europe, and Australia already having done such reviews) was not considered either productive or an efficient use of time. Each of the previously available reviews discussed most of the same studies! The Rodricks et al. review and the SCCS and SCCP opinions just added to the glut of reviews.
The issues that were identified in the scientific literature review were: triclosan exposure; triclosan sourcing and dioxin impurities; photostability and dioxin photoproducts; carcinogenicity; endocrine disruption; and potential for bacterial resistance. In addition to these 6 issues, one additional issue has been raised that is inherent to the overall risk assessment: establishing a NOAEL (different values have been used ranging from 12 to 200 mg/kg/d). Are there additional issues that should have been identified for resolution?

If the above 6 issues (and any newly identified issues) are/can be resolved without additional data, then it appears that the CIR Expert Panel could issue a tentative safety assessment. If additional data are needed, they should be identified and CIR would issue a formal insufficient data announcement.

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HistoryTriclosan AttherequestofCFSAN,theCIRExpertPanelplacedtriclosanonitshighprioritylistinJuneof2009. AscientificliteraturereviewwasissuedApril9,2010.

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SearchStrategyforTriclosan None. Whenitwasdiscoveredthatmultiplereviewsoftheavailableliteraturewerealreadyavailableandthat keyquestionsregardingthesafetyoftriclosanalreadyhadbeenidentifiedbyFDAandothers,theCIR literaturereviewprimarilywasbasedonthosesecondaryreferences. Sincethatapproachwasadopted,additionalreviewshaveappearedonaregularbasisandappearto presentupdatedinformation,butacomparisonofthereferencelistscontinuestodocumentthat everyoneisreviewingthesameinformation. OnestudywascitedbytheEuropeanCommissionintheiranalysisofpublicinputontheir2009opinion ontriclosansafetythatwasnotlistedintheirbibliography,butwecontactedtheauthorsattheUSDA andobtainedacopyofthatpublication(antimicrobialresistancestudy).

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Report

Draft Report

Triclosan as used in Cosmetics August 30, 2010

The 2010 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Ronald A Hill, Ph.D. James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is F. Alan Andersen, Ph.D. This report was prepared by F. Alan Andersen, Ph.D., Director.

Cosmetic Ingredient Review 1101 17th Street, NW, Suite 412 " Washington, DC 20036-4702 " ph 202.331.0651 " fax 202.331.0088 " cirinfo@cir-safety.org

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Introduction Triclosan is a chlorinated aromatic compound with functional groups representative of both phenols and ethers. It is widely used in a variety of products, and including those regulated by the U.S. Environmental Protection Agency (EPA) and Food and Drug Administration (FDA). EPA-regulated products include those with a wide variety of antimicrobial uses (e.g., as a material preservative in a wide variety of consumer, commercial, institutional, and industrial applications), while FDA-regulated products include hand and body soaps, toothpastes, deodorants, laundry detergents, fabric softeners, facial tissues, mouthwashes, antiseptics for wound care, and medical devices. In April, 2010, the Cosmetic Ingredient Review (CIR) issued a scientific literature review of published and unpublished data relevant to assessing the safety of triclosan in cosmetics. This literature review identified a number of existing assessments of triclosan and provided information that was largely summarized from those sources. Since that time, three additional significant reviews of triclosan have been added. One is a critical review of the experimental data and development of margins of safety for consumer products published in Critical Reviews in Toxicology (Rodricks et al. 2010). Another other is European Commissions Scientific Committee on Consumer Products opinion on triclosan (European Commission 2009). And finally, the Scientific Committee on Consumer Safety (same committee, new name) issued its opinion on triclosan antimicrobial resistance (European Commission 2010). Comments were received during the open public comment period. In addition, an unpublished investigation of potential endocrine activity of triclosan was received (Environ International Corporation 2010). Although it was suggested in comments on the literature review that more original literature citations could/should be incorporated, the newly available published and unpublished reviews make it clear that all of the available data have been captured and that the only differences are in data interpretation and assumptions that are made. Creating another 63 page (Rodricks et al. 2010) or 136 page (Scientific Committee on Consumer Products of the European Commission 2009) document would be redundant. Key studies have been added, however, and a new section is now included that presents the risk assessment approaches used by Rodricks et al. (2010) and the SCCP (European Commission 2009). This draft safety assessment will broadly summarize the available data and focus on resolving those questions critical to a determination of safety. Regulation of Triclosan FDAs Center for Drug Evaluation and Research (CDER) regulates triclosan as a drug, including personal care products with antibacterial/antimicrobial claims; and FDAs Center for Devices and Radiological Health (CDRH) is responsible for regulation of devices that may contain triclosan for antibacterial/antimicrobial purposes. As defined by FDA, an antimicrobial (active) ingredient is "a compound or substance that kills microorganisms or prevents or inhibits their growth and reproduction and contributes to the claimed effects of the product in which it is included," and an antimicrobial preservative (inactive) ingredient is defined as "a compound or substance that kills microorganisms or prevents or inhibits their growth and reproduction and is included in a product formulation only at a concentration sufficient to prevent spoilage or prevent growth of inadvertently added microorganisms, but does not contribute to the claimed effects of the product to which it is added." A topical antimicrobial agent is defined, in part, as "an antiseptic-containing drug product applied topically to the skin to help prevent
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infection in minor cuts, scrapes, and burns." Table 1 identifies triclosan-containing products regulated by FDA. EPAs Office of Prevention, Pesticides and Toxic Substances regulates triclosan when used as an antimicrobial (whether as a bacteriostat, fungistat, mildewstat, deodorizer and material preservative). International health authorities in the European Union, Canada, Japan, Australia, and Norway regulate triclosan in cosmetics and other products. Tricolsan in Cosmetics In the EU and other countries, antimicrobial and antiseptic products may be considered to be cosmetics, and, as such, are controlled under cosmetic regulations which may not require pre-clearance or pre-market approval of active ingredients. In Japan, antimicrobial and antiseptic agents may be regarded as drugs subject to pre-approval. In Europe, Canada and Australia, the use of triclosan in cosmetics is limited to a maximum concentration of 0.3%; in Japan, triclosan in cosmetics is limited to a maximum concentration of 0.1%, and Norway has stated that the use of triclosan in cosmetics should be limited, but no maximum concentration is given. FDAs Center for Food Safety and Applied Nutrition (CFSAN) is responsible for the regulation of triclosan in cosmetics. CFSAN has asked CIR to undertake a review of the safety of triclosan in cosmetics. As given in the International Cosmetic Ingredient Dictionary and Handbook1, triclosan may function in cosmetic formulations as a cosmetic biocide, deodorant agent, or preservative. Cosmetic Biocides are ingredients used in cosmetic products to help cleanse the skin or prevent odor by inhibiting the growth of, or destroying microorganisms, such as bacteria, fungi or yeast. Cosmetic biocides may be cidal or static. Cidal agents kill microbiota and act as disinfectants. Static agents inhibit the growth of microorganisms but do not kill them. Ingredients used primarily for the protection of products against contamination are found in the listing of Preservatives. Ingredients used as active ingredients in OTC drug products which are intended to kill bacteria, fungi or yeast in order to treat, prevent or mitigate diseases are included in the listing of Antimicrobial Agents. Deodorants are ingredients that reduce or eliminate unpleasant odor and protect against the formation of malodor on body surfaces. Absorbents can act as deodorants if they have the ability to absorb malodorous chemicals. Also, chemical reactions can be used to destroy the malodorous substance in selected cases. Perfumes and the like can be used to mask the perception of malodor by the process of reodorization. Unpleasant odors also may be the result of microbiological activity. Thus, Cosmetic Biocides are ingredients frequently used in skin-surface deodorants. Preservatives are ingredients which prevent or retard microbial growth and thus protect cosmetic products from spoilage. Cosmetic products may support the growth of microorganisms. The use of preservatives is required to prevent product damage caused by microorganisms and to protect the product from inadvertent contamination by the consumer during use. The use of more than one preservative can sometimes increase efficacy due to synergism. Ingredients used to protect products against oxidative damage are classified as Antioxidants. Government Body Triclosan Assessments

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In preparing this report, CIR relied extensively on triclosan reviews available from various governmental sources as an alternative strategy to summarizing the large volume of original literature2-7: European Centre for Disease Prevention and Control (ECDC), European Food Safety Authority (EFSA), European Medicines Agency (EMEA), Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR). Joint Opinion on Antimicrobial Resistance (AMR) Focused on Zoonotic Infections. October 2009. http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_026.pdf European Commission Directorate-General for Health & Consumers. Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR). Assessment of the Antibiotic Resistance Effects of Biocides. January 2009. http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_021.pdf Australian Government Department of Health and Ageing (NICNAS). Priority Existing Chemical Assessment Report No. 30 Triclosan. January 2009. http://www.nicnas.gov.au/Publications/CAR/PEC/Drafts/Triclosan.asp . United States Environmental Protection Agency (EPA). Office of Prevention, Pesticides and Toxic Substances. Reregistration Eligibility Decision (RED) for Triclosan, List B, Case No. 2340. EPA 739RO-8009. September 2008. http://www.epa.gov/oppsrrd1/REDs/2340red.pdf National Toxicology Program. FDA Nomination Profile - Triclosan [CAS 3380-34-5]. Supporting Information for Toxicological Evaluation by the National Toxicology Program. July 2008. http://ntp.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/triclosan_508.pdf. United States Environmental Protection Agency (EPA). Memorandum. January 4, 2008. Triclosan: Report of the Cancer Assessment Review Committee. PC Code: 054901. http://www.epa.gov/pesticides/chemical/foia/cleared-reviews/reviews/054901/054901-2008-01-04a.pdf

Since these summaries of the available data were reviewed, a major review of triclosan safety has been published in Critical Reviews in toxicology (Rodricks et al. 2010). In addition, the European Commissions SCCP opinion on triclosan (European Commission 2009) was reviewed and the Scientific Committee on Consumer Safety (same committee, new name) issued its recent opinion on triclosan antimicrobial resistance (European Commission 2010). Information from the critical review and the two European Commission opinions has been added to provide added detail or information not included in the above governmental bodies reviews. In addition, a new section was added to present exposure assessments and margin-of-safety determinations. Report structure Because this document departs from the approaches that CIR has used in the past to initiate a safety assessment of cosmetic ingredients, a brief overview of what is included and why is appropriate. Section I addresses the relevant issues for triclosan as used in cosmetics. Section II presents technical names and synonyms, physicochemical properties, information on methods of manufacturing, chemistry methods for identification and analysis, information on impurities and photostability.

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Section III provides information on the extent of use of triclosan in cosmetics based on information provided by the industry to the FDAs Voluntary Cosmetic Registration Program (VCRP). Use concentrations based on a survey conducted by the Personal Care Products Council also are provided. Section IV provides a limited overview of triclosans absorption/toxicokinetics, distribution, metabolism, and excretion. Section V presents an overview of assessments that have been made on triclosans potential toxicological hazards, including endocrine hazards. Section VI provides information on triclosans putative mechanisms for the inhibition of bacterial growth and presents the key arguments that have been raised about triclosans potential for causing antibiotic and antibacterial resistance. New Section VII includes rationales for benchmark doses and/or no-observable-adverse-effects-levels, consumer exposures, and margins-of-safety for triclosan in consumer products. Finally, Section VIII summarizes and integrates information in the preceding sections. I. Issues to be resolved in safety substantiation of triclosan as used in cosmetics.

1. Triclosan exposure. Issue: uses of triclosan in OTC drugs may present different exposure scenarios compared to use in cosmetics. Current status: information on the number of personal care products that contain triclosan, and at what use concentration, is now available. Various analyses of the use of those products also are available. 2 Triclosan sourcing and dioxin impurities. Issue: triclosan imported from India and China reportedly may contain dioxin compounds. Current status: further information on sourcing was not available, although impurities data do indicate low levels of dioxins. Limits on the levels of dioxin compounds in triclosan as supplied for use in cosmetics could be established. 3. Photostability and dioxin photoproducts Issue: triclosan applied to the skin may photodegrade to dioxin compounds on exposure to light. Current status: no further data were provided. Empirically, this question may be addressed by the available data demonstrating the absence of phototoxicity in animal tests. 4. Carcinogenicity Issue: data from one mouse carcinogenicity study did suggest a statistically significant increase in liver carcinomas and adenomas as a function of dose, above a threshold level. Current status: the mode of action for these mouse liver tumors appears to be by a mechanism that is not relevant to human health considerations. When NTP carcinogenicity data are available, they should be considered.

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5. Endocrine disruption Issue: triclosan may bind to estrogen and/or androgen receptors and thus act as an endocrine disruptor. Current status: a mix of endpoints that may be considered as endocrine effects have been studied. These data include studies that found no effect, and other studies suggesting weak estrogenic and androgenic effects, and antiestrogenic and antiandrogenic effects, suggesting a mix of endocrine action. 6. Potential for bacterial resistance Issue: any antibiotic/antimicrobial agent potentially can be a selective agent for resistance in target organisms. Current status: while bacterial strains with resistance to triclosan can be developed in vitro, this phenomenon is not seen in surveillance studies of organisms in most use situations. While some data are available demonstrating that emergence of triclosan resistance is accompanied by resistance to common antibiotics, other studies failed to find such a link. II. Chemistry Definition and Structure The International Cosmetic Ingredient Dictionary and Handbook has established triclosan as the International Nomenclature Cosmetic Ingredient (INCI) name (to be used in cosmetic product labeling) for the substituted organic ether that conforms to the structure shown in Figure 1.1 The National Library of Medicines ChemIDPlus website at http://chem.sis.nlm.nih.gov/chemidplus/ (enter Triclosan) shows the same structure and provides other data sources, e.g. the European chemical Substances Information System, and the FDA Drug Database.8 In addition to being an INCI name, triclosan also is a INN name (International Nonproprietary Names for Pharmaceutical Substances, WHO). Although other CAS numbers have been used previously for triclosan, the current CAS number is 3380-34-5 and the EINECS number is 222-182-2. As given in the International Cosmetic Ingredient Dictionary and Handbook1, triclosan is sold under a variety of trade names and trade name mixtures that contain triclosan (supplier name given in parentheses): Trade names AEC Triclosan (A & E Connock) Amicare 100 (Cosmetic Rheologies) Dekaben TC (Dekker) Irgacare MP (Ciba Specialty Chemicals) Irgasan DP (Ciba Specialty Chemicals) Jeechem Triclosan (Jeen Int. Corp.) Mackstat TCN (McIntyre) note that this is actually triclosan in a solvent. Oletron (Sino Lion) OriStar TC (Orient Stars) Rita Triclosan (Rita) Triclosan (Kumar Organic Products) Triclosan-PC (Pretameen) Trade name mixtures

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Custoblend BAT (Custom Ingredients) Elestab 4121 (Laboratories Serobiologiques) Irgasan PG-60 (Ciba Specialty Chemicals) Lipobead Blue-T (Lipo) Lipo CD-TC (Lipo) Miracare AB-33 (Rhodia) Rovisome Acne (Rovi GMBH)

The SCCP (European Commission 2009) also lists Irgacare CF100 and Irgacide LP10 as trade names. Because triclosan is a powder, any material supplied as a liquid must be a mixture with a solvent. Also, trade name mixtures will contain chemicals in addition to triclosan, and the safety of these additional chemicals may not have been determined. Physical and Chemical Properties Triclosans physical and chemical properties are presented in Table 2. Method of Manufacture As given in the 6th edition of Ullmann's Encyclopedia of Industrial Chemistry,10 triclosan is produced by treatment of 2,4,4-trichloro-2-methoxydiphenyl ether with aluminum chloride in benzene under reflux. Conversion to chlorinated dibenzo-p-dioxins (see Impurities below and Figure 2) can occur under extreme conditions such as high alkalinity and heat. The type and purity of the starting materials in the synthesis of triclosan influenced the extent of contamination by the impurities dioxins and dibenzofurans.4 Methods of detection. Triclosan may be separated using using high performance liquid chromatography,4 and detected by infrared and/or UV (peak absorption at 281 nm)52 spectroscopy. Gas chromatography/mass spectroscopy methodology has a detection limit for triclosan of 0.5 ng/ml.53 Impurities Commercial grade triclosan is reported to be >99% pure (w/w) as the powder, and 10 to <20% (w/v) pure as a liquid solution.4 Technical grade triclosan produced by Ciba and Harmet/Vivimed is >99.0% and 99.9% pure, respectively.7 Trace level impurities identified by the US Pharmacopoeia (USP) include mono- and di-chlorophenols, as well as di-, tri-, and tetra-chlorodibenzo-p dioxins and di-, tri-, and tetra chlorodibenzofurans.11 Menoutis and Parisi tested samples of triclosan from India and China for the presence of dioxins.12 Six samples of triclosan, each of which were manufactured by a different producer in India or China (5 samples and 1 sample, respectively, from each country), were analyzed for the presence of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF). All six samples contained TCDD in excess of 1 pg/g, and 4 of the six samples contained TCDF in excess of 1 pg/g. TCDD and TCDF ranged from 17.2 pg/g to 1712.0 pg/g, and 0.43 pg/g to 207.30 pg/g, respectively, as shown in Table 3. The authors suggested that the presence of these two trace impurities may be due to the quality or purity of the starting material, the particular synthetic process, or the inability to tightly control physical synthetic parameters.

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Ciba Specialty Chemicals (now part of BASF) has reported that Irgasan DP 300 (which Ciba manufactures and distributes for topical use, specifically) and Irgasan MP, meet USPs requirements11 (see Table 4), but that generic triclosan made by other manufacturers does not necessarily comply with USP specifications.13 As noted earlier, impurities in triclosan that may be present in trace amounts include dioxins, and which are limited or not allowed by the U.S. Pharmacopoeia (USP). In addition, the government of Canada has established limits on dioxins. All these limits are presented in the Table 4. Triclosan is not on the work program of either the British Pharmacopoeia Commission or the European Pharmacopoeia Commission.14 FDA has stated that it is unaware of the purity, identity and concentration of impurities in triclosan used in cosmetics, or the sources of triclosan that is used in cosmetic formulations in the US.15 Information on impurities from all triclosan suppliers would be useful in evaluating the need to establish limits for allowable impurities in cosmetic-grade triclosan. Chemical Reactivity According to Rule et al.,16 chloroform may be produced if a soap containing triclosan comes into contact with chlorinated water. Two dish soaps, one containing triclosan (at 1.4 mg triclosan/g soap) and one without, were added to chlorinated water at a concentration of 0.25 g/L. The measured chloroform level was 15 g/L after 5 min and 49 g/L after 120 min for the triclosan-containing soap. The chloroform levels for the non-triclosan formulation were near the detection limit. Photostability In FDAs nomination of triclosan for study by the National Toxicology Program,6 the agency argued that the level of dichlorodibenzo-p-dioxins in the environment following photodecomposition of triclosan, and the levels of dichlorodibenzo-p-dioxins on skin following photodecomposition of topically applied triclosan have not been established. Lores et al. (2005)17 reported that, under artificial conditions, triclosan can photodegrade to 2,7- and 2,8dichlorodibenzo-p-dioxin (2,7/2,8-DCDD). In addition, 2,4-dichlorophenol (DCP), which is not a dioxin, has been identified as a major degradation product under artificial conditions - 93.8-96.6% of the applied triclosan degrades to DCP within 240 minutes post-treatment.18 Since the pKa for triclosan is around pH 8.1, at physiological pH, the phenolic form (shown in Figure 1) would predominate while at pH of 9, for example, the phenolate form (shown in Figure 3) would predominate. NICNAS stated that the phenolic form of triclosan is relatively photostable, whereas the phenolate form is more photodegradable. NICNAS4 included the proposal that triclosan photolysis products would include the three permutations of dichloro compounds; a dihydroxy coumpound (2,4-dichloro-2,4-dihydroxydiphenyl ether), which could further degrade to a monochloro compound, 4-chloro-2,4-dihydroxydiphenyl ether; or 4-chloro-2hydroxyphenol, which is closely related to the DCP photodegradation product noted by above. Overall, some information suggests that photodegradation, likely by UV light, may produce dioxin compounds, but other sources have postulated other photodegradation products that are not dioxin

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compounds. Since the photodegradation phenomenon appears to be pH dependent, one useful group of data would be pH values for representative cosmetic product categories that contain triclosan. III. Extent of use and use concentrations for triclosan in cosmetics. According to information supplied to FDA by industry as part of the Voluntary Cosmetic Registration Program (VCRP),19 personal hygiene products is the category with the greatest number of products containing triclosan (226 triclosan-containing products, which represents ~7% of products in this category overall). Since FDA does not verify labelers product status with regards to its VCRP database,15 it may be that some of these products are marketed with antibacterial claims, and could be considered drug products or both drug products and cosmetic products. The skin care products category ranks second, with 162 triclosan-containing products, which represents ~2% of all products in this category. FDA VCRP data for triclosan are given in Table 5. FDAs VCRP is, as the name of the program implies, voluntary. As a result, these data cannot be regarded as complete. A Personal Care Products Council (Council) survey reported that triclosan is used in cosmetics at concentrations ranging from 0.01 to 0.3%.20 It is not known that every company using triclosan is represented by these data.18 Use concentration survey responses are given in Table 5, as a function of product category. To interpret the data in Table 5, consider that, of all baby shampoos reported (total of 56), only 1 contains triclosan (~2% of all baby shampoos reported), or conversely, the vast majority do not, and the situation is similar for the baby lotions, powders, and creams category. While uses of triclosan were reported to FDA under the VCRP for each of these categories, no use concentrations were provided in the industry survey for these categories. And no uses of triclosan or use concentrations were reported for the 143 products in the other baby products category - usually this row would not be included in Table 5 because there are no data to provide, but in this case it was not deleted because information that there is a product category in which there are no products containing triclosan may be important information. Rodricks et al. (2010) reported use concentrations that are consistent with the data from the Council survey, except that a use concentration range up to 0.45% was reported for a liquid hand soap. The SCCP (European Commission 2009) reported use concentrations up to 0.3%. Table 5 presents all of the cosmetic product categories, so that a reader may see all categories in which triclosan is used and at what levels (depending on availability of those data), as well as the cosmetic product categories in which triclosan is not used. According to the Skin Deep Cosmetics Safety Database21 that lists OTC drug and cosmetic products for which triclosan is listed as an ingredient on the product label, a total of 938 products contain triclosan, one of which is in a cosmetic product categories for which no uses were reported to FDAs VCRP. For example, the label for Oscar Blandi Pronto Dry Shampoo (presumably a shampoo) lists Triclosan as an ingredient, yet no shampoos were reported to the VCRP containing triclosan. There was one baby shampoo reported, but Oscar Blandi Pronto Dry Shampoo does not appear to be a baby shampoo. Triclosan-containing rinse-off and leave-on cosmetics uses may include products that result in triclosan exposure by the dermal, inhalation, and oral routes. Dermal exposure appears to include rinse-off and leave-on cosmetics applied to adults, as well as to children.

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Cosmetic aerosols Safety of inhaled aerosols depends on the ingredient, the concentration, the duration of the exposure and where they are deposited within the respiratory system.22 The site of deposition is associated most with the particle size and density of the particle being inhaled. In general, the smaller the particle, the further into the respiratory tree the particle will deposit and the greater the impact on the respiratory system. The parameter most closely associated with this regional deposition is the aerodynamic diameter, da, defined as the diameter of a sphere of unit density possessing the same terminal settling velocity as the particle in question. In humans, particles with an aerodynamic diameter of 10 m are respirable. Particles with a da from 0.1 10 m settle in the upper respiratory tract and particles with a da < 0.1 m settle in the lower respiratory tract. 23,24 Particle diameters of 60-80 m and 80 m have been reported for anhydrous hair sprays and pump hairsprays, respectively.25 In practice, aerosols should have at least 99% of their particle diameters in the 10 110 m range and the mean particle diameter in a typical aerosol spray has been reported as ~38 m.26 Therefore, most aerosol particles are deposited in the nasopharyngeal region and are not respirable. IV. Absorption/toxicokinetics, distribution, metabolism and excretion NICNAS4 presented a review of triclosans absorption/toxicokinetics, distribution, metabolism and excretion. These data were supplemented with data reviewed by FDA, 6 specifically for data that NICNAS4 did not address, or had discounted because of shortcomings in reporting. Absorption/Toxicokinetics Most reviews have suggested that triclosan is slowly and not extensively absorbed by the dermal route, consistent with its low water solubility and log Po/w of 4.8, but is rapidly and well absorbed by the oral route. Rodricks et al. (2010) suggested that dermal absorption would likely be <10%, but that oral absorption would be complete. In human subjects, for example, daily use of triclosan-containing toothpaste for up to 65 weeks resulted in increased blood levels compared to pre-use levels, but those increased levels remained steady and returned to baseline after use. Using full thickness human skin, total absorption of triclosan was vehicle specific, with dishwashing liquid at 12%, water/oil emulsion at 11.3%, deodorant at 7.65%, and soap solution at 7.2%. These same dermal absorption figures were reported in the SCCP opinion on triclosan (European Commission 2009). Distribution Triclosan measured in rodent radioactivity studies (following oral and dermal exposures) indicate distribution at highest levels to the liver, lung, kidney, gastrointestinal tract, and gall bladder.4 Rodricks et al. (2010) suggested that differences in distribution between mice, rats, and hamsters (plasma levels are higher than liver or kidney levels in rats and hamsters, but not mice) implies that triclosan can accumulate in the mouse liver.

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Metabolism Oral and dermal routes (humans and rodents): Triclosan absorbed from the gastrointestinal tract undergoes extensive first-pass metabolism, which primarily involves glucuronide and sulfate conjugation. In both humans and rodents, at high triclosan plasma concentrations, metabolism shifts from the generation of predominantly glucuronide conjugates to sulfate-conjugates. The bioavailability of unconjugated triclosan may be limited after oral exposure because of triclosans extensive first-pass metabolism. Triclosan is also metabolized to its glucuronide and sulfate conjugates by the skin.4 FDA concluded that > 90% of absorbed triclosan is metabolized.6 Rodricks et al. (2010) noted that the glucuronide metabolite predominates in humans while the sulfate conjugate is the dominant metabolite in mice. The SCCP (European Commission 2009) also emphasized the extensive first-pass metabolism and the almost-total conversion to glucuronide and sulfate metabolites. Based on results from oral studies (e.g., toothpaste use) and dermal studies (e.g., washing with soap), there was no evidence of accumulation of triclosan in the human body. Excretion Oral and dermal routes (humans and rodents): Triclosan glucuronide is predominantly excreted in the urine, and triclosan is predominantly excreted in the feces. Triclosan that is administered orally and dermally is excreted in greater concentrations in the urine than in the feces in humans, hamsters, rabbits, and monkey. In rats, mice, and dog, the reverse is true. Up to 87% of triclosan that is administered to humans (by an unspecified route) is excreted in the urine, most of it within 72 h after dose.4 Rodricks et al. (2010) noted that elimination half-lives following repeated dermal application of triclosan (1.4 to 2.1 days) are greater than those following oral administration (10 to 20 hours). Biomonitoring Data According to Rodricks et al. (2010), several studies have reported triclosan in plasma and urine in the general population and in human breast milk in nursing mothers. Triclosan amounts in breast milk were reported to range from <20 to 300 g/kg lipid in one study and <5 to 2100 g/kg lipid in another. In a study that compared triclosan levels in women who used triclosan-containing products with those who did not, levels in breast milk were 0.022 to 0.95 g/kg lipid compared to 0.018 to 0.35 g/kg lipid, respectively. The largest bio-monitoring study was conducted as a subset of the National Health and Nutrition Examination Survey (NHANES) in which urine samples were taken from a random of the 9643 subjects yielding data on 2514 individuals (Calafat et al. 2008). For the entire sample, the geometric mean triclosan level in urine was 13 g/l. There were age differences in the findings as well as sex differences for urine concentrations, as shown in Table 6.

V. Toxicology/Safety This section presents an overview of studies performed in experimental animals models or in vitro systems (acute toxicity, dermal and eye irritation, phototoxicity, sensitization, repeat dose toxicity, reproductive toxicity, endocrine disruption, genotoxicity, and carcinogenicity), as well as in humans (skin irritation and sensitization).

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Acute toxicity Triclosan has low acute toxicity by all evaluated routes and in all evaluated species.6,7 Oral rat and mouse LD50 values were > 3700 mg/kg. Rabbit dermal LD50 values were > 9000 mg/kg and the rabbit inhalation LC50 is > 0.15 mg/L. The rat subcutaneous and intraperitoneal, and intravenous LD50 values were > 14,700 mg/kg, >1090 mg/kg, and 29 mg/kg. The dietary NOAEL for triclosan in baboons was 30 mg/kg, with a LOAEL of 100 mg/kg/day, a dose at which clinical signs were observed, such as vomiting, failure to eat, and diarrhea. Dermal and eye irritation; phototoxicity, respiratory irritation, sensitization In rabbits, triclosan was a moderate dermal irritant (Primary Irritation Index score of 3.5 @ 72 hours), as well as a moderate eye irritant.7 Triclosan in various formulations at < 0.25%, when tested dermally in a rabbit primary dermal irritation test and acute dermal lethality test did not cause dermal toxicity.29 EPA7 did not consider triclosan a sensitizer in guinea pigs, although Australia4 considered it a very weak sensitizer in the same model. Triclosan was not a phototoxicant in guinea pigs.4 NICNAS considered triclosan to be a respiratory irritant.4 Most recently, Rodricks et al. (2010) reviewed the available dermal irritation and sensitization studies. Dermal irritation was concentration dependent. For example, a single application of triclosan at 0.3% was not irritating in any animal species, but dermal concentrations of 5% were irritating to guinea pig skin and ocular concentrations of 1 to 100% produced reversible eye irritation in rabbits. Repeated dosing dermal studies (14-day) have consistently found a threshold of around 1.5% for irritation. Sensitization studies were negative in guinea pigs at 0.1% when administered subcutaneously, or topically at concentration up to 10%. Triclosan at concentrations up to 1% were not photosensitizers in guinea pigs, mice, or pigs with irradiation in either the UVA, UVB, or UVC region. The SCCP (European Commission 2009) also reviewed the available photosensitization data in guinea pigs, mice, and pigs and, while noting that all of the available studies predated good laboratory practices, suggested that there was no evidence for photosensitization. Repeat dose toxicity Triclosan repeat dose toxicity has been evaluated in the baboon and hamster (oral route), rat (oral, and inhalation routes), mouse (dermal route) and rabbit (dermal). Triclosan NOAELs based on local irritation effects tended to be < 10 mg/kg/day by all routes, except inhalation, which had a reported No-ObservedAdverse-Effect-Concentration (NOAEC) of 5 x 10-5 mg/m3. LOAELs and NOAELs based on systemic toxicity tend to be <1000 mg/kg/day, with no obvious common toxicity among studies and species. NICNAS,4 EPA,5,7 and FDA6 toxicology data summaries are presented in Table 8. NICNAS4 or FDA6,19 summary statements were not included if such statements did not provide duration of exposure or information on doses, or were fundamentally flawed (e.g., LOAEL lower than the NOAEL). Table 7 is organized hierarchically by route of exposure, duration of dosing (subacute subchronic chronic), species (monkey rat/mouse rabbit). Unless otherwise noted, NOAEL units were not standardized. Rodricks et al. (2010) summarized findings from repeated dose dermal exposures of triclosan in propylene glycol or acetone vehicles using CD-1 mice (doses from10 to 200 mg/kg/day) and Crl:CD BR rats (doses from 1.2 to 24 mg/kg/day). Responses varied as a function of vehicle, dose, species, and sex of the exposed mice. For example, in mice, liver weights were increased in males at all doses >10 mg/kg/day, independent of vehicle, but only at 200 mg/kg/day in females for the propylene glycol vehicle exposures. Pale foci were noted in the livers of male mice from the 100 and 200 mg/kg/day groups with both vehicles,

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but not in females. No significant changes in liver weights were reported in rats, nor were there any effects seen on macroscopic or microscopic examination, in either sex. Genotoxicity Trisclosan has been evaluated in a number of standard (and other) genotoxicity assays, including bacterial reverse mutation tests, in vitro mammalian cell gene mutation test, and in vitro mammalian chromosome aberration tests, a mammalian bone marrow chromosomal aberration test, and an unscheduled DNA synthesis assay in mammalian cells in culture. As FDA6 noted: the preponderance of data suggested that triclosan is not genotoxic. NICNAS4 drew a similar conclusion. EPA5 provided a detailed review of seven genotoxicity tests. It concluded that each test, except one (an in vitro cytogenetic assay with Chinese hamster lung fibroblasts), was negative. Therefore, the consensus on the weight of evidence on triclosans genotoxicity potential is that it is not genotoxic. Carcinogenicity NICNAS (2009)4 and EPA7 concluded that triclosan was not carcinogenic based on the available data. FDA, however, has concluded that the available data are not adequate to resolve the question of triclosan carcinogenicity via the dermal route of exposure seen in skin cleansing preparations.6 EPA7 specifically evaluated rat and hamster oral chronic toxicity/carcinogenicity studies and concluded that triclosan exhibited no carcinogenic potential in rats at < 3000 ppm and in hamsters at < 250 mg/kg/day. However, EPA reviewed a mouse oral chronic/carcinogenicity bioassay and found it positive for carcinogenicity based on an increased incidence of liver neoplasms in male and female mice at >30 mg/kg/day.7 Nevertheless, EPA concluded that this study did not support triclosan carcinogenicity, and that triclosan is not likely to be carcinogenic in humans. This conclusion was based on the weight of evidence that supports activation of peroxisome proliferator activated receptor alpha (PPAR) as the primary mode of action for triclosan-induced hepatocarcinogenesis in mice. Also, EPA stated that, while the data did not support either a mutagenic mode or cytotoxic mode of action, that the mode of action for liver tumors in mice is theoretically plausible in humans. Based on differences in the PPAR responses in humans compared to mice, however, EPA suggested that such a mode of action was unlikely. In referring triclosan to the NTP for study, FDA specifically commented on oral toxicity data using albino rats submitted to the agency in 1977, suggesting that the presence of test material in control animals invalidated the results and on an oral rat study conducted in 1986, suggesting that the study was inadequate based on a high rate of mortality, absence of significant body weight differences between treated and control animals, and the presence of hepatocellular lesions not consistent with the morbidity/mortality.6 In spite of the agency describing the latter study as inadequate, one FDA reviewer concluded that Triclosan was oncogenic at 3,000 ppm at 104 weeks. In 1999, FDA reviewed another carcinogenicity study (hamsters) and apparently formed no conclusion on the merits of the study because the sponsor did not respond to the Agencys request for histopathology slides of kidneys, liver, lungs, adrenals and all tumors from all animals on study for review. FDA,6 in its presentation of the rationale for NTP study of triclosan, also suggested that the only available dermal toxicity data (90 day dermal rat study) could be interpreted to suggest dose-dependent abnormalities which need subsequent study with a 2-year dermal carcinogenicity bioassay.

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Rodricks et al. (2010) reviewed chronic toxicity studies using rats, hamsters, and mice in which the incidence of tumors was evaluated. Rats were fed triclosan in the diet at 0, 300, 1000, and 3000 ppm for up to 104 weeks, with an additional group at 6000 ppm for 52 weeks. No evidence of tumors or preneoplastic lesions was found. In its discussion of these data, the SCCP (European Commission 2009) noted that the exposures were calculated to yield doses of 0, 12, 40, and 127 mg/kg/d for males and 0, 17, 56, and 190 mg/kg/d for females. The additional dose for the 52-week animals was calculated to be 247 mg/kg/d for males and 422 mg/kg/d for females. The SCCP did not disagree that no evidence of tumors or preneoplastic lesion was found, but did determine that there were significant reductions in red blood cell counts in males and females, including low-dose males at 104 weeks. Increases in mean corpuscular hemoglobin were observed in mid- and high-dose females and in males at all doses. Other hematologic parameters were also different from controls, but only in the high-dose groups. The SCCP also noted decreased absolute and relative spleen weights in mid-dose females. The SCCP considered that these hematotoxicity results and the spleen weight changes as indications of an adverse effect and established the NOAEL from this study at 12 mg/kg/d. This is the calculated lowest dose for male animals, but the SCCP did not comment on the red blood cell count changes or the increases in mean corpuscular hemoglobin that were observed in low-dose males. Rodricks et al. (2010) suggested that the statistically significant hematological changes were slight and transient (red blood cell counts were down at 13, 26, and 52 weeks, but not at 78 or 104 weeks) and that there were no other indications that the animals were anemic. These authors also noted an absence of macroscopic or microscopic evidence of an effect on the hematopoietic system and no apparent effects on homeostasis. Since this 104-week rat study will likely be a focus of discussion, CIR will try to have the original unpublished study available at the meeting. Rodricks et al. described a study in which hamsters were fed triclosan in their diet at 0, 12, 75, or 250 mg/kg/day for 90-95 weeks. Deaths in male hamsters in the high-dose group were significantly higher than in the control group. No evidence of liver damage was seen at any dose, but body weight gain was significantly reduced and nephropathy was significantly increased in both sexes at the highest dose compared to controls. In addition, hyperplasia in the fundic region of the stomach, abnormal spermatogenic cells, reduced spermatozoa, and germ-cell depletion were noted in high-dose males. The SCCP (European Commission 2009) review of these data also noted the positive findings in high-dose hamsters and set the NOAEL at 75 mg/kg/d. Rodricks et al. (2010) reviewed the study in which CD-1 mice were fed triclosan in the diet at doses of 0, 10, 30, 100, or 200 mg/kg/d for 6 months or 18 months. In the 18-month study, statistically significant increases in liver adenomas and carcinomas were seen at several dose levels compared to controls as shown in Table 8. The SCCP (European Commission 2009) did note an increased incidence in liver tumors at doses of 30 mg/kg/d, and commented that triclosan is a peroxisome proliferator in mouse liver. The SCCP adenomas

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described dose-related increases in liver weights at 30, 100, and 200 mg/kg/d in males and females, and hepatocyte hypertrophy in males at all doses. The LOAEL was established at 10 mg/kg/d. While triclosan in the diet appears to be linked to the adenomas and carcinomas in the liver of the exposed mice, the questions that arise from this finding are: (1) why did those tumors occur, and (2) is the finding relevant to human health? For example, since mice accumulate triclosan in the liver and humans dont, might this explain the causation (high accumulated levels of triclosan) and be an adequate basis for discounting the effect for human exposure? Rodricks et al. (2010) used the International Programme on Chemical Safety (IPCS) framework (Boobis et al. 2006) to assess the relevance of the mode of action (MOA) of tumor formation in the mouse study to humans. This approach posits three questions: 1. Is the weight of evidence sufficient to establish an MOA in mice for tumor formation? 2. Is the MOA relevant to human health (i.e., can it even happen in humans)? 3. Even if the MOA can happen in humans, is the MOA inconsequential on the basis of quantitative differences in either kinetic or dynamic factors between mice and humans? The first step in addressing these questions, obviously, is to postulate the MOA of triclosan in the mouse liver that produces tumors. While hepatic tumors are the most common spontaneous tumors in mice, the mouse liver is a frequent target of chemically induced tumors. MOAs for chemically induced liver tumors include genome mutation in liver cells, or non-genotoxic gene activation/deactivation, and/or receptors. As discussed earlier, the preponderance of evidence is that triclosan is not genotoxic, so the MOA is likely non-genotoxic. Activation of peroxisome proliferator-activated receptors (PPARs) is a well-characterized, non-genotoxic mechanism by which a cascade of events can lead to tumor formation. Three types of PPARs have been identified: , /, and , with the form expressed in the liver. In concept, a ligand binds to a retinoid X-receptor (RXR) in the cytoplasm, is transported to the nucleus, where the combination ligand/RXR binds to promoter sequences of peroxisome proliferation genes, activating PPAR. That alters the transcription of genes involved with peroxisome proliferation, apotosis, and lipid metabolism. Those changes increase fatty acid -oxidation which can lead to oxidative stress. In turn, increased stimulation of nonparenchymal cells and inhibition of gap junction intercellular communication can occur. Increased cell proliferation and decreased apotosis, leads to hyperplasia and hepatic tumors. So, for the mice in the triclosan carcinogenicity study, is there evidence of triclosan-related PPAR activation, cell proliferation, fatty acid -oxidation, etc? Rodricks et al. (2010) reviewed the available data and concluded that there was no direct evidence of PPAR activation, but there was evidence of triclosanrelated PPAR-dependent up-regulation of CYP3A and CYP4A, testosterone hydroxylation, and lauric acid 11-12 hydroxylation, and PPAR-dependent expression of nonperoxisomal fatty acid metabolism genes (cyanide-independent palmitoyl CoA oxidation). Peroxisome proliferation was supported by the findings of triclosan-related hypertrophy due to an increase in the number and size of peroxisomes and an increase in smooth endoplasmic reticulum. While there was no evidence of PPAR-dependent expression of cell-cycle growth and apotosis, there were triclosan dose-dependent increases in Proliferating Nuclear Cell Antigen (PNCA) labeling index, indicative of perturbation of cell proliferation and/or apotosis. Hepatocyte oxidative stress was suggested by the triclosan dose-related increases in lipofuscin in the Kupffer cell region. Kupffer cell-mediated events were suggested by triclosan-related Kupffer-cell

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activation. And finally, selective clonal expansion is suggested by the finding of triclosan-related hepatic adenomas and carcinomas. The authors considered the possibility that hepatic cytotoxicity could be the MOA. In concept, triclosan would be cytotoxic, resulting in a hyperplastic response, during which hepatic cells with DNA damage proliferate, produce preneoplastic foci, and then tumors. While cell proliferation was linked to triclosan treatment, necrosis was not. If PPAR activation is the MOA of triclosan in mice, then how would it translate to a human health risk? Expression of PPAR in the human liver is 1/10th of that in the mouse. A study of a known liver carcinogen in mice containing the gene for human PPAR compared to mice containing the normal mouse gene produced no tumors in the mice containing the human gene and the expected tumors in the mice containing the normal mouse gene. That said, activation of PPAR and expression of peroxisomal genes can occur in humans. Is there something about how humans metabolize, distribute, and excrete triclosan that suggests the mouse MOA would not be applicable? Certainly, excretion is different. In mice, triclosan is mostly excreted in the feces as unchanged parent chemical. In humans, the primary excretion route is in the urine as the glucuronide conjugate. Also, the method of excretion in mice supports the finding that triclosan can accumulate in the mouse liver. Overall, Rodricks et al. (2010) concluded that the hepatic tumors produced by a PPAR activation MOA are not relevant to predicting human health outcomes. Reproductive and Developmental Toxicity NICNAS4 reported a developmental and maternal toxicity NOAEL of 50 mg/kg/day for no specific species, but the basis for that NOAEL was unclear. Fort et al.30 examined the effect of triclosan, at larval exposure levels up to 32.39.43 g/ml (measured 21day meanSEM), on frog metamorphosis. A small marginally-significant acceleration in premetamorphic development was reported, but the effect was not thyroid-mediated. Overall there was no effect on metamorphosis. The authors suggested that the effect that was seen would be consistent with the reduced bacterial stressors that would be found in the 50 L tanks used for the study. Rodricks et al. (2010) summarized findings on developmental toxicity studies using mice, rats, hamsters, and rabbits. Only in rats and mice were significant findings reported. In a two-generation reproductive and developmental study using CRL:CD (SD)Br rats given triclosan at doses of 0, 300, 1000, and 3000 ppm, body weights were significantly decreased in F1 animals on postnatal days 14 and 21 in the high-dose group compared to controls. The viability index for high-dose F1 animals was reduced, but the difference was not significant. The SCCP (European Commission 2009) reviewed this same study and noted an absence of reproductive toxicity at the 3000 ppm dose (~200 mg/kg/day for both sexes combined), but concluded that the NOAEL for developmental effects would be 65 mg/kg/d (both sexes combined) because of pup body weight decreases at the high dose. Rodricks et al. (2010) reviewed a study in which CD-1 mice were given 0, 10, 25, 75, or 350 mg/kg/day on gestation days 6 15, fetal body weights were significantly reduced in the two highest dose groups. The

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incidence/litter of irregular skull ossification was significantly increased in high-dose litters and the litter averages for ossified forepaw and hind paw phalanges per fetus, possibly linked to developmental delay due to the reduced fetal weights. The SCCP (European Commission 2009) stated that taking maternal toxicity and fetal toxicity both into consideration, there is no evidence of triclosan developmental toxicity (teratogenicity). Endocrine Disruption In vitro studies Ahn et al.31reported results from a series of receptor-based bioassay systems for triclosan. Three receptors were stably transfected: aryl hydrocarbon receptor (AhR activates gene expression in a ligand-dependent manner); estrogen receptor (ER); and androgen receptor. In each case the reporter gene was firefly luciferase. In addition, the ryanodine receptor type 1 (RyR1) assay for compounds with potential to alter Ca2+ homeostasis was performed using primary cultures of skeletal myotubes from wild-type mice. In the AhR assay, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was used as a positive control. With TCDD activation set at 100%, triclosan at 10 M of compound was found to induce at 40.66.1%, suggesting triclosan agonist activity. With both TCDD and triclosan added at 10 M, induction reached only 70.42.1%, suggesting triclosan antagonist activity. Overall, the authors suggested triclosan would be a partial agonist against AhR. In the ER assay, triclosan exhibited no direct estrogenic activity alone, suggesting no agonist activity. When combined with estradiol, a 103 excess of triclosan reduced estradiol activity to 50% and a 105 excess of triclosan reduced estradiol activity to 20%, suggesting antagonist activity. While the authors stated that triclosan was a potent antagonist in the AR assay, no data were provided. Ryanodine binding to microsomes enriched in RhR1 was significantly increased by 1.2 M triclosan, suggesting to the authors that triclosan is a dysregulator of cell Ca2+ homeostasis. Gee et al.32 reported the estrogenic and androgenic activity of Ttriclosan using MCF7 human breast cancer cells in culture and other in vitro assays. In MCF7 breast cancer cells in vitro, estradiol binding remained >90% at 104 molar excess of triclosan. Half of estradiol binding to estrogen receptors (ER) was displaced at a 106 molar excess of triclosan. In the estrogen-triggered ERE-CAT reporter gene in MCF7 cells, a 105fold excess of triclosan over 17-estradiol effectively inhibited activation of the reporter gene and inhibited 17-estradiol-induced cell growth stimulation. In a seeming contradiction, the authors suggested that there was a small but not statistically significant increase in MCF7 cell growth in the presence of triclosan alone. In a follow-up assay in which the cultures were maintained for 21 days (the assay is normally done over 8 days), a statistically significant increase in cell growth (but still less than a was reported for triclosan at 10-6 M and at 4 x 10-6 M, but not at lower (2 x 10-7 , 6 x 10-7) or a higher concentration (8 x 10-6). The authors also performed a competitive binding assay between triclosan and testosterone to rat recombinant androgen receptor (AR) protein, with the result that triclosan, at a 103 molar excess over testosterone, reduced testosterone binding by around half and the decrease was linear when binding was plotted versus the log of the molar ratio. They also determined growth stimulation in the presence of triclosan in S115+A mouse mammary tumor cells and T24 human breast cancer cells.

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At a testosterone concentration of 10-9 M, S115+A cells are stimulated to grow and undergo a doubling in number. Triclosan at 2 x 10-5 M prevented that growth. Activation of the LTR-CAT reporter gene in stably transfected S115+A cells and in transiently transfected T24 human breast cancer cells also was studied. In S115+A cells, 10-9 M testosterone triggered activation. Triclosan at molar ratios to testosterone of 1 and 10 had no effect, but at a molar excess of 100, reduced the activation by 20%. A molar excess of triclosan of 104 reduced activation by 40%. At a testosterone concentration of 10-8 M, triggered activation in T24 cells. At a 103 molar excess of triclosan, the activation was reduced by 30% and by 75% at a molar excess of 104. James et al. (2009) postulated that triclosan is structurally related to inhibitors of estrogen sulfotransferase, such as polychlorobiphenylols. To test potential enzyme inhibition, the authors harvested placental tissue from almost term fetal sheep, homogenized the tissue and incubated the cellular material with triclosan for ~15 min. Estrone and 17-beta-estradiol were substrates and the effect of 4-hydroxy-3,3',4',5tetrachlorobiphenyl and 2'-hydroxytriclocarban on estradiol sulfonation was used for comparison. Triclosan was a very potent inhibitor of both estradiol and estrone sulfonation suggesting competitive binding of triclosan for estradiol sites on the sulfotransferase enzyme. The authors suggested that the effect of triclosan as an inhibitor of estrogen sulfotransferase activity raised concern about the possible effects of triclosan on the ability of the placenta to supply estrogen to the fetus, and in turn on fetal growth and development. Environ International Corporation (2010), in its analysis, reasoned that, were this phenomenon to be of any significance, then administration of triclosan in vivo should have an impact on successful pregnancies. In a two-generation rat reproductive and developmental toxicity study of triclosan at doses up to 3000 ppm (described earlier), there was no evidence of an impact on reproductive performance, nor were there any data to demonstrate that the ability to carry fetuses to term was compromised. In vivo studies Kumar et al.33 reported a clear no-effect level of 5 mg/kg/day (or higher) triclosan in a 60-day study of male rats treated daily. Endpoints studied included decreased body weights (no-effect level of 20 mg/kg/day); decreased testis, prostate, seminal vesicle, vas deferens and cauda epididymis weights (5 mg/kg/day); down-regulation in the testicular levels of mRNA for cytochrome P450SCC, cytochrome P450C17, 3HSD, 17-HSD, StAR and AR as compared to control (10 mg/kg/day); decreased testicular 3-HSD and 17-HSD levels in vitro (10 mg/kg/day); and decreased serum hormone levels (10 mg/kg/day). Zorilla et al.34 exposed weanling rats to 0, 3, 30, 100, 200, or 300 mg/kg/day of triclosan by oral gavage from postnatal day (PND) 23 to 53. Predicated on the idea that the separation of the foreskin of the penis from the glans penis, so-called preputial separation (PPS), is an early reliable marker of the progression of puberty in the male rat, this gross endpoint was examined beginning on PND 33. Triclosan did not affect growth or the onset of PPS. Serum testosterone and triiodothyronine (T3) were not different in a doseeffect manner. Total serum thyroxine (T4) decreased in a dose-dependent manner at 30 mg/kg and higher. Thyroid stimulating hormone was not statistically different at any dose. Liver weights were significantly increased at 100 mg/kg triclosan and above, but not in a dose-effect manner and other tissue weights were not different from controls, exemplifying the difficulty in identifying a cohesive body of work on Triclosans potential as an endocrine disruptor (or as an endocrine toxicant).

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In a follow-up study by Paul et al.,35 examined the question of how triclosan decreased serum T4 in vivo in rats. The hypothesis was that triclosan upregulates rat hepatic catabolism and alters expression of cellular transport proteins. The authors measured total serum T4, T3, and thyroid stimulating hormone (TSH). Cytochrome P450 isoforms (Cyp2b1/2 and Cyp3a1/23) were determined enzymatically and as mRNA expression levels using quantitative reverse transcriptase. Uridine diphosphate glucuronyltransferase (UGT) activity, mRNA expression of UGT isoforms and sulfotansferase (SULT) isoforms, and mRNA expression of hepatic transporters, including Oatp1a1, Patp1a4, Mrp2, and Mdr1, were also measured. All mRNA expression assays were performed using reagent kits. T4 levels decreased as expected as a function of triclosan concentration, with decreases at 100, 300, and 1000 mg/kg/day producing significant decreases, but not at 10 and 30 mg/kg/day. T3 levels were decreased at 300 and 1000 mg/kg/day, but not at the three lower exposure levels. No significant differences in TSH were found at any exposure, but the authors suggested that this may relate to T3 glucuronidation. Cyp2b1/2 (at triclosan levels of 300 mg/kg/day) and Cyp3a1/23 (at Triclosan levels of 100 and 300 mg/kg/day) gene expression were increased, No significant effect was seen at lower doses. Liver microsomal UGT activity was increased significantly only at 1000 mg/kg/day triclosan. UGT gene expression was not significantly increased for Ugt1a6 or Ugt2b5 genes, but was increased at 100 and 300 mg/kg/day for Ugt1a1 genes. SULT isoform expression was not dose-related for Sult1b1 (significantly reduced at 10 and 30 mg/kg/day, but not at 100 or 300 mg/kg/day triclosan), but Sult1c1 expression was increased significantly at 100 and 300 mg/kg/day only. No statistically significant changes were reported for mRNA expression of hepatic transporters. The authors cautioned that, while these findings support a role for hepatic catabolism of T4 in the rat as a likely mechanism of observed triclosan-induced hypothyroxinemia in the rat, the relevance to humans is not established. Allmyr et al. (2009) investigated if an everyday exposure to triclosan via triclosan-containing toothpaste for 14 days in 12 adult humans caused an increase in plasma 4-hydroxycholesterol, indicative of CYP3A4 induction, and/or alterations in thyroid hormonal status. Plasma triclosan concentrations increased from 0.0090.81 ng/g to 26296 ng/g. The authors noted that the 296 ng/g plasma triclosan level is in the range of triclosan plasma levels that could be attained with an oral dose of 4 mg triclosan. No significant changes in plasma levels of either plasma 4-hydroxycholesterol or thyroid hormones were reported during the exposure. The authors concluded that triclosan-containing toothpaste use was not likely to alter metabolism of drugs via CYP3A4 induction or cause adverse events because of thyroid disturbances in humans. Other studies suggested weak estrogenic and androgenic effects, and antiestrogenic and antiandrogenic effects (reported in fish/frogs or in vitro). Summary statements from Triclosan reviews already available from various governmental sources on the import of those data are presented in Table 9. The SCCP (European Commission 2009) did comment that data from a study using Japanese medaka fry exposed to concentrations of triclosan up to 100 g/l for 14 days showed no effect on sex ratios in the developing fish.

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Clinical studies FDA6 summarized several studies reviewed by DeSalva et al.29 and by Lyman & Furia.36 None of the studies indicated that triclosan at concentrations of < 25% causes sensitization, or that triclosan at concentrations at < 0.5% causes irritation. In contrast, NICNAS4 stated that it had reviewed several studies that had shown evidence of skin irritation, although applicable doses or references were not identified. NICNAS4 stated that there is very limited evidence for triclosan causing photosensitization in healthy volunteers or those with dermatological conditions. NICNAS4 also reported that humans orally administered triclosan at < 30 mg/day for 15 or 42 days showed no evidence of any treatment effect.

VI. Antibacterial/antimicrobial resistance FDA6 described two triclosan mechanisms of action for the inhibition of bacterial growth: 1) intercalation into bacterial cell membranes and disruption of membrane activities (without causing leakage of intracellular components, and 2) inhibition of bacterial type II fatty acid synthase enoyl-reductase (FabI gene). Triclosan is bacteriostatic at low doses and bactericidal at high doses. In a 2002 report of the Scientific Steering Committee (SSC) of the European Commission Health & Consumer Protection Directorate-General,37 the conclusion was reached that, at high (biocidal) concentrations, triclosan is very effective and unlikely to produce a major problem of anti-microbial resistance (e.g., all the microorganisms are dead). However, at sub-biocidal and bacteriostatic, concentrations, triclosan is capable of penetrating bacteria and initiating changes related to important mechanisms of antimicrobial resistance including possibly transferable mechanisms of resistance, though the scientific evidence for transferability has been disputed. Sound scientific laboratory evidence exists for the development of triclosan related mechanisms for antimicrobial resistance, but the evidence as to whether these mechanisms are shared by other antimicrobial agents or whether they are transferable to micro-organisms other than those used in the laboratory is limited and contradictory. Overall the SSC noted that no evidence of such resistance has been seen in clinical isolates, and there is no epidemiological evidence to suggest a problem in clinical practice. A study by Cole et al.43 found no relationship between the use of triclosan and other biocides and antibiotic resistance. Lambert38 analyzed minimum inhibitory concentrations (MICs) from clinical strains of S. aureus (both methicillin resistant (MRSA) and sensitive strains (MSSA)) and P. aeruginosa for changes from 1989 to 2000. While MRSA strains developed biocide resistance, MRSA antibiotic resistance has remained the same. The same was true for MSSA strains. Overall this suggested to the author that any acquisition of biocide resistance does not alter antibiotic resistance. And for P. aeruginosa, the MICs for triclosan were actually reduced in 2000 compared to 1989, although the difference was not statistically significant. The information that is available from studies of manufacturing sites39 and clinical follow up studies of dental plaque flora which have failed to show biologically significant changes in MIC values to commonly used antibiotics in patients using triclosan long term40,41 points to resistance patterns being stable over periods of three to ten years. Aiello et al.42 reviewed triclosan efficacy data along with data her laboratory developed on antimicrobial resistance in use situations. Two studies reported findings from a randomized and masked intervention trial

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of 238 households using either 0.2% triclosancontaining liquid hand soap or plain soap over one year. Neither of these studies demonstrated the emergence of antibiotic resistance associated with use of triclosan-containing liquid hand soap, compared with plain soap. Beier et al. (2008) characterized 50 vancomycin-resistant Enterococcus faecium (VRE) isolates from human wastewater effluents in Texas. These VRE isolates were also resistant to 8 fluoroquinolone antibiotics and most of the National Antimicrobial Resistance Monitoring System gram positive antibiotics. The VRE isolates were sensitive to quinupristin/dalfopristin and to linezolid antibiotics and they were sensitive to triclosan and other biocides. No cross-resistance or co-resistance between antibiotic resistance and biocide susceptibility was found. This contrasts with the work of Chen et al. who studied triclosan susceptibility to 732 pathogenic Acinetobacter baumanii clinical isolates from hospitals in China. MIC values for triclosan ranged between 0.015 and 16 mg/l. They noted that these MIC values were lower than the in-use concentrations of triclosan of 2000 to 20000 mg/l. They identified 20 (out of the 732) isolates for which the MIC was greater than 1 mg/l and declared those to have reduced susceptibility to triclosan. They then further examined those 20 isolates for antibiotic resistance and compared the results with 20 isolates with triclosan MIC values of 0.5 mg/l down to 0.03 mg/l. All 20 of the isolates with reduced susceptibility to triclosan were resistant to amikacin, tetracycline, levofloxacin, and imipenem. Among the 20 isolates with triclosan MIC values < 0.5 mg/l, 11 were resistant to amikacin and tetracycline, and 8 were resistant to levofloxacin and imipenem. These authors further examined the potential that mechanism for triclosan reduced susceptibility, including efflux pump over expression (in concept, if triclosan is removed from the bacterial cell, it is no longer available to function as a biocide), but were unable to correlate expression of efflux pump genes with triclosan reduced susceptibility. They identified mutations in the FabI (NADH dependent, enoyl-[acylcarrier-protein] reductase) gene in all 12 of the isolates with triclosan MIC values >4 mg/l and postulated that triclosan resistance was linked to mutations in that gene. Stickler and Jones (2008) studied clinical isolates of Proteus mirabilis in vitro to determine if exposure to triclosan could result in decreased sensitivity (higher MIC values) to triclosan itself and/or increased antibiotic resistance. Five strains of Proteus mirabilis were exposed in culture to triclosan at concentrations from 0.5 to 10 mg/l for 5 days. Viable colonies (mutated to reduced triclosan susceptibility) were subcultured and tested to determine triclosan MIC values and MIC values for trimethoprim, ampicillin, ciprofloxacin, nitrofurantoin, norfloxacin, cephalexin, nalidixic acid, and gentamicin. The parental strains and 2-3 mutant strains for each were tested. While mutant isolates were found with MIC values for triclosan up to 60 mg/l, none of the mutated strains showed resistance to any antibiotic that was different from the parent strain. In 2009, SCENIHR3 stated that triclosan at low concentrations acts by both inhibition of enoyl acyl reductase mechanism, inhibition of energy-dependent uptake of amino acids, and possibly discharge of membrane potential (as demonstrated in E. faecalis). The SCENIHR report concluded that current scientific evidence (including bacteriological, biochemical and genetic data) does indicate that the use of certain active substances in biocidal products in various settings may contribute to the increased occurrence of antibiotic resistant bacteria. Some mechanisms of resistance are common to both biocides and antibiotics

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(e.g. efflux pumps, permeability changes and biofilms). The selective pressure exerted by biocides may favor the expression and dissemination of these mechanisms of resistance. Most recently, the European Commissions Scientific Committee on Consumer Safety (SCCS) issued an opinion on triclosan antimicrobial resistance (European Commission 2010) with the conclusion that the available data have failed to demonstrate an increase in antibiotic resistance following triclosan use in situ. Because in vitro studies have demonstrated that resistance to triclosan in bacteria is possible (see, for example, Stickler and Jones (2008) above) and that there are mechanisms in bacterial resistance that can result in cross-resistance to biocides and antibiotics (see for example, Chen et al. (2009) above), the opinion went on note that it was not possible to draw an overall conclusion on whether the continuous use of triclosan is involved in the development of resistance. The SCCS recommended prudent use of triclosan, for example, in applications where a health benefit can be demonstrated and that additional research, for example, on mechanisms of resistance, transfer of resistance, and translational studies from in vitro to in situ situations.

VII. Exposure Assessment and Margins-of-Safety A determination of triclosan exposures and margins-of-safety presumes that a hazard has been identified. Rodricks et al. (2010) suggested that the statistically significant increases in nephropathy and stomach pathology seen in male and female hamsters and the statistically significant effects in epididymides and testes in male hamsters, all at the high-dose level of 250 mg/kg/day but not at 75 mg/kg/day could establish a no-observable-adverse-effect-level (NOAEL) for repeated dose systemic toxicity. Further, in CD-1 mice given 350 mg/kg/day on gestation days 6 15, fetal body weights were significantly reduced in the two highest dose groups. The incidence/litter of irregular skull ossification was significantly increased in highdose litters and the litter averages for ossified forepaw and hind paw phalanges per fetus. None of these effects were seen at 75 mg/kg/day, a presumptive NOAEL for developmental toxicity. In addition, Rodricks et al. (2010) modeled the high-dose levels at which significant effects were seen using the U.S. EPAs benchmark dose approach. The lowest benchmark dose (BMDL) that provided the best fit to the available male hamster nephropathy data was 46.91 (~47) mg/kg/day. All other hamster endpoints for which there were statistically significant effects yielded BMDLs higher than that. The BMDL that provided the best fit to the rat developmental toxicity data (body weight decreases in F1 animals) was 75.65 (~76) mg/kg/day. Noting that these two BMDLs were not inconsistent, the BMDL of 47 mg/kg/day was recommended. The SCCP (European Commission 2009) relied solely on a NOAEL value of 12 mg/kg/d based on hematotoxicity in a chronic exposure study using rats. The SCCP noted that the mean plasma level of 28,160 ng/ml ( 12,928) from the 12 mg/kg/d dose group could be compared to human plasma levels, were they available. Exposure Assessment Rodricks et al. (2010) noted that, for products that may be ingested, the daily triclosan intake is determined by the amount of product used per day, the percentage of triclosan in the product, the amount of triclosan absorbed by the GI tract, and the body weight of the subject. The corresponding calculation for use of dermal products varies only in replacing GI tract absorption with dermal absorption. Combined oral and

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dermal intake for adult males and females were determined. Intake for children was also determined, but utilized scaling to convert use rates for liquid body washes and body lotions from available adult usage. For toothpastes, the adult male intake was 0.005 mg/kg/day; adult female, 0.006 mg/kg/day; and children, 0.023 mg/kg/day. For mouthwashes, the adult male intake was 0.003 mg/kg/day; adult female, 0.004 mg/kg/day; and children, considered to be zero. For rinse off products such as liquid hand soap, liquid body washes, dish detergents, the adult male intake was 0.007 mg/kg/day, combined; adult female, 0.009 mg/kg/day, combined; children, 0.011 mg/kg/day, combined. For leave-on products such as body lotions, moisturizers, and deodorants, the adult male intake was 0.033 mg/kg/day, combined; female, 0.046 mg/kg/day, combined; children, 0.042 mg/kg/day. Were all the products to be used on a daily basis, the intake estimates for adult males, adult females, and children, respectively, would be 0.047, 0.064, and 0.074 mg/kg/day. For comparison purposes, data from biomonitoring levels were converted from urine concentrations to intake estimates. For the 95th percentile level (largest urine concentration levels reported), the intake estimates for adult males, adult females, and children, respectively were 0.009, 0.007, and 0.004 mg/kg/day (5 20 times less than the product usage based estimates). The SCCP (European Commission 2009) determined systemic doses for oral products assuming 100% availability of whatever triclosan was present, toothpaste use levels of 2,750 mg/d, mouthwash use levels of 30,000 mg/d, and triclosan content of 0.3% for toothpaste and 0.3% or 0.2% for mouthwashes. The resulting systemic dose for toothpaste was 0.0234 mg/kg/d and for mouthwash was either 0.10 or 0.15 mg/kg/d for mouthwashes. For leave-on cosmetics, the SCCP determined systemic doses using dermal absorption based on in vitro data, triclosan content (ranged from 0.15 to 0.3%), surface area exposed (e.g., deodorant stick = 200 cm2 and body lotion = 15670 cm2), and one application per day. The resulting systemic dose for deodorant stick was 0.0015 mg/kg/d, for body lotion at 0.15% and 0.3% triclosan was 0.0823 and 0.1646 mg/kg/d, respectively. Face powder systemic doses ranged from 0.004 to 0.006 mg/kg/d and blemish concealer doses ranged from 0.0003 to 0.0006 mg/kg/d. For rinse-off cosmetics, the SCCP determined systemic doses using dermal absorption based on in vitro data, a 10x dilution of triclosan in the product in use situations, and an 860 cm2 exposure area for hand soap or 17500 cm2 for shower gel/body soap. The resulting systemic dose for hand soap was 0.0066 mg/kg/d and, for shower gel/body soap, was 0.0268 mg/kg/d. Table 10 presents a list of the systemic doses determined by the SCCP as a function of product type. Margin of Safety Rodricks et al. (2010) determined a margin of safety (MOS) by dividing the BMDL by the daily intake estimate. For such a determination to be the most conservative, the daily intake should be the largest value supportable by the available data and the BMDL should be the lowest value supportable by the available data. For example, the MOS for body lotion usage for adult males is l808, for adult females is 1237, and for children is 1119. These were the lowest MOSs reported. Were the biomonitoring levels (95 percentile)

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used instead, the MOS for body lotion usage for adult males would be 5222, for adult females would be 6714, and for children would be 11750. Consideration was also given to the use of multiple products on a daily basis. Were each of the oral care products, rinse-off products, and leave-on products applied on a daily basis, and were each of them preserved with triclosan (unlikely, given the data in Table 5, where only 2% of baby shampoos contain triclosan), then the MOS for adult males would be 1000, for adult females would be 732, and for children would be 634. Rodricks et al. (2010) concluded that exposure to triclosan in consumer products is not expected to result in adverse health effects in children or adults who use these products as intended. The SCCP (European Commission 2009) used the rat NOAEL (which SCCP determined to be 12 mg/kg/d) divided by the systemic dose delivered by products containing triclosan as given in Table 9 to determine an MOS. For toothpaste, the MOS was 513 and for combined use of toothpaste, deodorant sticks, and hand soap, the MOS was 381. For all products usage, which includes body lotion, the MOS values ranged from 49 to 32. The SCCP concluded that use of triclosan up to a maximum concentration of 0.3% in toothpastes, hand soaps, shower gels/body soaps, and deodorant sticks is safe, any additional use in face powders and blemish concealers at concentrations up to 0.3% also is considered safe, but use in other leave-on products (e.g., body lotions) and in mouthwashes is not considered safe for consumer use. Table 11 provides a side-by-side comparison of MOS determinations by Rodricks et al. (2010) and the SCCP (European Commission 2009).

VIII. Summary Triclosan is a chlorinated aromatic compound with functional groups representative of both phenols and ethers. Its IUPAC name is 5-Chloro-2-(2,4-dichlorophenoxy)phenol. Triclosan may function in cosmetic formulations as a cosmetic biocide, deodorant agent, or preservative. At ambient temperatures, triclosan is a crystalline powder, so any material supplied as triclosan in a liquid form, must, by definition, be a mixture with a solvent. Triclosan is supplied to cosmetic formulators under several trade names and in several trade name mixtures. Information on the use concentration of triclosan in cosmetics as a function of cosmetic product type is available from the VCRP maintained by the FDA, but this information is likely underreported. Use concentration data as a function of product type is limited (not all reported uses have use concentrations), but use concentrations in cosmetics appear to be in the 0.01 - 0.3% range. Triclosan also is used in some product categories that raise the possibility of user exposure to aerosols. Most aerosol particles from cosmetic products, however, are sufficiently large such that they are deposited in the nasopharyngeal region and are not respirable. Analysis of triclosan imported from India and China uncovered the presence of dioxin and furan impurities. USP and the government of Canada have established limits for such impurities. Independent of the presence of dioxin impurities in triclosan as supplied to cosmetics formulators, there is a question regarding the possibility that triclosan in cosmetic formulations applied to the skin may photodegrade to dioxin compounds on exposure to light. Triclosan can photodegrade to 2,7- and 2,8dichlorodibenzo-p-dioxin, and 2,4-dichlorophenol, but the effect is pH dependent. The relevance of these photodegradation products to the safety of triclosan in cosmetics is not established.

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Triclosan is slowly and not extensively absorbed by the dermal route, but is rapidly and well absorbed by the oral route. Triclosan measured in rodent radioactivity studies (following oral and dermal exposures) indicate distribution to the liver, lung, kidney, gastrointestinal tract, and gall bladder. Triclosan absorbed from the gastrointestinal tract undergoes extensive first-pass metabolism, which primarily involves glucuronide and sulfate conjugation. Triclosan is also metabolized to its glucuronide and sulfate conjugates by the skin. Triclosan glucuronide is predominantly excreted in the urine, and triclosan is predominantly excreted in the feces. Triclosan administered orally and dermally is excreted in greater concentrations in the urine than in the feces in humans. Triclosan has low acute toxicity by all evaluated routes in all evaluated species. Repeat dose toxicity has been evaluated in the baboon (oral route), rat (oral, and inhalation routes), mouse (dermal route), rabbit (dermal), and hamster (oral). Triclosan NOAELs based on local irritation effects tend to be < 10 mg/kg/day by all routes, except inhalation, which has a reported NOAEL of 5 x 10-5 mg/m3. LOAELs and NOAELs based on systemic toxicity tend to be <1000 mg/kg/day, with no obvious common toxicity among studies and species. Statistically significant increases in nephropathy and stomach pathology were reported in male and female hamsters and statistically significant effects were reported in epididymides and testes in male hamsters, all at the high-dose level of 250 mg/kg/day but not at 75 mg/kg/day. Triclosan does not appear to have significant reproductive/fertility/developmental toxicity. Triclosan has been linked to hypothyroxinemia in rats and has been suggested as having potential to disrupt the thyroid axis in amphibians. In rats, hypothyroxinemia via a hepatic catabolism mechanism has been suggested, but the implications for human exposure is unclear. One recent study in frogs reported a marginal acceleration of pre-metamorphic development by a non-thyroid mechanism in amphibians, with no overall alteration in metamorphosis. In CD-1 mice given 350 mg/kg/day on gestation days 6 15, fetal body weights were significantly reduced in the two highest dose groups. The incidence/litter of irregular skull ossification was significantly increased in high-dose litters and the litter averages for ossified forepaw and hind paw phalanges per fetus. None of these effects were seen at 75 mg/kg/day, a presumptive NOAEL for developmental toxicity. In various assays for endocrine disruption effects, triclosan gave weak responses, although one study did report competitive binding to the estrogen receptor sufficient to support growth of an estrogen-dependent cell line and another study reported binding to the thyroid hormone receptor. Trisclosan has been evaluated in a number of standard (and other) genotoxicity assays, including bacterial reverse mutation tests, in vitro mammalian cell gene mutation test, and in vitro mammalian chromosome aberration tests, a mammalian bone marrow chromosomal aberration test, and an unscheduled DNA synthesis assay in mammalian cells in culture --- except in one (an in vitro cytogenetic assay with Chinese hamster lung fibroblasts), the findings were negative. Based on the weight of evidence, triclosan is not genotoxic. Rat, mouse, and hamster carcinogenicity studies are available and have been reviewed extensively with mixed interpretations. Rat and hamster oral chronic toxicity/carcinogenicity studies found no carcinogenic potential for triclosan in rats at < 3000 ppm and in hamsters at < 250 mg/kg/day. However, a mouse oral chronic/carcinogenicity bioassay was positive for carcinogenicity based on an increased incidence of liver neoplasms in male and female mice at >30 mg/kg/day. Presuming that activation of peroxisome

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proliferator activated receptor alpha is the primary mode of action for triclosan-induced hepatocarcinogenesis in mice, these findings did not support either a mutagenic mode or cytotoxic mode of action. FDA has nominated triclosan for dermal carcinogenicity study under the NTP. In rabbits, triclosan is a moderate dermal irritant as well as a moderate eye irritant, but when tested at concentrations < 0.5% in various formulations did not cause dermal toxicity. Triclosan is, at most, a weak sensitizer, but when tested at concentrations of < 25% in formulation, no sensitization was reported. Triclosan is not a phototoxicant in guinea pigs. Triclosan is bacteriostatic at low concentrations and bactericidal at high concentrations. At high (biocidal) concentrations, Triclosan is very effective and unlikely to produce a major problem of anti-microbial resistance (e.g., all the microorganisms are dead). However, at sub-biocidal and bacteriostatic, concentrations, triclosan is capable of penetrating bacteria and initiating changes related to important mechanisms of antimicrobial resistance including possibly transferable mechanisms of resistance. In actual usage, however, no evidence of such resistance has been seen so far in clinical isolates, and there is no epidemiological evidence to suggest a problem in clinical practice. Although, the stability and persistence of triclosan biocidal resistance has not been widely studied, the limited information available points to resistance being stable over a three to ten year period. One study found no relationship between the use of triclosan and other biocides and antibiotic resistance in homes where biocidal products were or were not being used. Different approaches have been described for determining the systemic dose that would result from use of triclosan-containing products, although the maximum use concentration of triclosan in those products is given by 0.3% by all. Resulting systemic doses from use of triclosan-containing products have been compared to the dose determined in animal studies to be a NOAEL, but the NOAEL value chosen in one case was 12 mg/kg/d based on hematotoxicity in rats and in another case was 47 mg/kg/d based on nephropathy in hamsters. Resulting MOS values have ranged from a low of 32 for use of all products by one analysis to a high of 47000 for hand soap use by another analysis.

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1 2 3 4

OH O

Cl

5 6 7 8 9 10 11 12

Cl

Cl

Figure 1. Triclosan (5-Chloro-2-(2,4-dichlorophenoxy)phenol (IUPAC name))

Cl O O

Cl

Cl

OH

13

Cl

Cl

Cl

14 15 16 17 18 19 20 21 22 Triclosan High pH + heat Trichlorodibenzo-p-dioxin

Figure 2. Conversion of triclosan to trichlorodibenzo-p-dioxin (for illustrative purposes, the structure of triclosan is pictured in Figure 2 with the phenol moiety rotated around the O bond so that the proximity of the hydroxyl group and conversion to a dioxin compound can be more readily seen).

Cl O

23 24 25

Cl

O-

Cl

Figure 3. Phenolate form of Triclosan at pH > 8.1.

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Table 1. Regulatory Decisions on Triclosan.


Authority Product Name or Category Concentration Limit/Restriction Comment

U.S. (FDA)44-47 Drugs Total Toothpaste (ColgatePalmolive Co.) Acne therapeutic 0.30% OTC dentifrice to treat gingivitis44

Proposed: leave-on: 0.2-0.5%rinse-off: 0.3-1.0% Antibacterial soaps generally contain < 0.3% triclosan.45 NR

Under review as an OTC 45

Soap and deodorant

Antibacterial claim

Devices

TempBond Clear with Triclosan (Sybron Dental Specialties, Inc.) MONOCRYL* Plus (Poliglecaprone 25) Antibacterial Suture (Ethicon Inc.)

Regulated as a temporary dental cement. 46

< 2360 g/m

Regulated as an absorbable surgical suture47

Europe48 Canada49

Cosmetic products Cosmetic products (mouthwashes excluded) All oral products

< 0.3% < 0.3% in other cosmetic products

< 0.03% in mouthwashes

not to be used by children < 12 years of age and labeled do not swallow

Japan 50

Cosmetic products as a preservative Cosmetic products

polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) impurities <0.1 ng/g 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran; and <10 g/g total other PCDD/PCDF impurities, with no individual impurity greater than 5 g/g <0.10 g/100 g product (<0.1%)

Australia4

< 0.3%

Provisional recommendation. Eye, skin, respiratory system irritant. Recommendation for compliance with USP limits for dioxins and dibenzofurans (synthesis impurities). Concern about bacterial resistance to triclosan and to clinically important antimicrobial agents.

Norway51

Cosmetic products

should be restricted.

NR = not reported

2 3

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Table 2: Triclosan Physicochemical Properties


Property Molecular weight Physical state Specific Gravity Density Acid Dissociation Constant (pKa) pH 289.541 white crystalline powder 1.55 X 103 kg/m3 1.55 g/cm3 at 22 C 8.14 at 21 C Not available Neat triclosan is stable to UV radiation Triclosan solutions are not stable to chlorine Stable under normal storage conditions (ambient temperature) when tested after 4 and after 9 years Melting point 56.5 C 54 57.3 C Boiling point 280 290 C (decomposes) 0.012 g/l at 20 C Water solubility 20 mg/l at 20 C 4.8 at 25 C Octanol-water partition coefficient (Log Po/w) 4.76 5.2 x 10-6 mm Hg at 25 C
11 13 13

Value and Conditions


11

Reference

4,13

11

11

11

Stability

European Commission 2009


11

13

13

11

11

Vapor Pressure

2.2 x 10 mm Hg at 20 C 4 x 10-6 mm Hg at 20 C
13

-6

2 3

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1 2

Table 3. Measured 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-Tetrachlorodibenzofuran (TCDF) impurities in Triclosan from India and China.52
Sample # 1 2 3 4 5 6 Country India China India India India India TCDD (pg/g)a 17.2 95.4 111.8 41.5 1712.0 18.9 TCDF (pg/g) a 0.70 7.13 3.43 8.51 0.43 207.3

3 4
a

those values in excess of USP specifications11are highlighted.

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Table 4. USP and Health Canadas Limits on Triclosan Impurities


Impurity 2,4 Dichlorophenol 3-Chlorophenol 4-Chlorophenol USP 32 (2009)11 < 10 g/g (< 10 ppm or 0.001%) < 10 g/g (< 10 ppm or 0.001%)) < 10 g/g (< 10 ppm or 0.001%) Canada49 NA

2,3,7,8 Tetrachlorodibenzo-p-dioxin 2,3,7,8 Tetrachlorodibenzo-furan 2,8-Dichlordiobenzofuran 2,8-Dichlorodibenzo-p-dioxin 1,3,7 Trichlorodibenzo-p-dioxin 2,4,8 Trichlorodibenz-furan Other

<1 pg/g (1 ppt)a <1 pg/g (1 ppt)a < 0.25 g/g (< 0.25 ppm or 0.000025%) < 0.5 g/g (< 0.5 ppm or 0.00005%) < 0.25 g/g (< 0.25 ppm or 0.000025%) < 0.5 g/g (< 0.25 ppm or 0.000025%) Contains not less than 97.0% triclosan calculated on the anhydrous basis. Not more than 0.5% of total impurities. Not more than 0.1% of any individual impurity.

< 0.1 ng/g < 0.1 ng/g < 10 g/g total other PCDD/PCDF impurities, with no individual impurity greater than 5 g/g

manufacturers must possess: raw material specifications for triclosan; identification of analytical method used to determine PCDD and PCDF levels; and finished product specifications

NA = Not applicable or not specified PCDD/PCDF = polychlorinated dibenzo-p-dioxin / polychlorinated dibenzofuran


a

Calculated as follows: (1 pg/l) x (10 l) x (1/30 g) x (30 l) = 1 pg/g = 1 ppt52

2 3

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Table 5. Frequency of use and use concentrations of Triclosan in cosmetics.


Product Category Total number of products in each product category53 Number of products containing Triclosan in each product category53 Concentration of Use (%) 20

Baby Productsa Shampoos Lotions, oils, powders, and creams Other Baby products subtotal Bath products Oils, tablets, and salts Bubble baths Capsules Other Bath products subtotal Eye Makeup Eyebrow pencils Eyeliners Eye shadow Eye lotions Eye makeup remover Mascara Other Eye makeup subtotal Fragrance products Colognes and toilet waters Perfumes Powders Sachets Other Fragrance products subtotal 1377 666 221 12 566 2842 27 (2%) None reported 5 (~2%) None reported 10 (~2%) 42 (~2%) 0.1% None reported None reported None reported None reported 0.1% 144 754 1215 254 128 499 365 3359 None reported None reported 18 (~1.5%) 2 (~1%) None reported 2 (<1%) 6 (~2%) 28 (~1%) None reported None reported 0.05% None reported None reported None reported None reported 0.05% 314 169 4 234 721 1 (<1%) None reported None reported None reported 1 (<1%) None reported None reported None reported None reported None reported 56 137 143 336 1 (~2%) 3 (~2%) None reported 4 (~1%) None reported None reported None reported None reported

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1
Product Category Total number of products in each product category53 Number of products containing Triclosan in each product category53 Concentration of Use (%) 20

Non-coloring hair care products Conditioners Sprays/aerosol fixatives Straighteners Permanent waves Rinses Shampoos Tonics, dressings, etc. Wave sets Other Non-coloring hair care products subtotal Hair coloring productc Dyes and colors Tints Rinses Shampoos Color sprays Lighteners with color Bleaches Other Hair coloring products subtotal 2393 21 40 40 7 21 149 168 2839 None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None reported None Reported 1226 312 178 69 33 1361 1205 51 807 5242 1 (<1%) None reported None reported None reported None reported None reportedb 1 (<1%) None reported 1 (<1%) 3 (<1%) 0.05% None reported None reported None reported None reported 0.04 - 0.2% 0.1% None reported None reported 0.04 0.2%

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1
Product Category Total number of products in each product category53 Number of products containing Triclosan in each product category53 Concentration of Use (%) 20

Makeup Blushers (all types) Face powders Foundations Leg and body paints Lipsticks d Makeup bases Rouges Makeup fixatives Other Makeup subtotal Nail care products Basecoats and undercoats Cuticle softeners Creams and lotions Extenders Nail polishes and enamels Nail polish and enamel removers Other Nail care products subtotal Oral hygiene products Dentifrices Mouthwashes and breath fresheners e Other Oral hygiene products subtotal 59 74 86 219 None reported None reported e None reported None reported None reported 0.04% None reported 0.04% 79 27 14 2 333 24 138 617 None reported None reported None reported None reported None reported None reported 1 (~1%) 1 (<1%) None reported None reported None reported None reported None reported None reported None reported None Reported 434 661 589 29 1883 117 102 45 485 4345 1 (<1%) None reported 5 (~1%) None reported None reportedd 1 (1%) None reported None reported 3(<1%) 10 (<1%) 0.2% 0.2% 0.1% None reported None reported None reported None reported None reported 0.3% 0.1 0.3%

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1
Product Category Total number of products in each product category53 Number of products containing Triclosan in each product category53 Concentration of Use (%) 20

Personal hygiene products Bath soaps and detergents Underarm deodorants Douches Feminine deodorants Other Personal hygiene products subtotal Shaving Products Aftershave lotions Beard softeners Mens talcum Preshave lotions Shaving cream Shaving soap Other Shaving products subtotal Skin care products Skin cleansing creams, lotions, liquids, and pads Depilatories Face and neck creams, lotions, etc. Body and hand creams, lotions, etc. Foot powders and sprays Moisturizers Night creams, lotions, powder and sprays Paste masks/mud packs Skin fresheners Other 1446 42 1583 1744 47 2508 353 441 259 1308 31 (2%) Not reported 30 (2%) 27 (1.5%) 6 (~13%) 28 (1%) 14 (4%) 12 (~3%) 3 (~1%) 11 (~1%) 0.01-0.3% None reported 0.1% 0.1% None reported None reported None reported None reported None reported None reported 367 3 3 22 122 10 134 661 2 (<1%) None reported None reported None reported 3 (2.5%) None reported 6 (4.5%) 11 (~2%) None reported None reported None reported None reported None reported None reported None reported None Reported 1665 580 14 19 792 3070 45 (~3%) 162 (28%) None reported None reported 19 (~2%) 226 (~7%) None reported 0.2 - 0.3% None reported None reported 0.3% 0.2 - 0.3%

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Skin care products subtotal Product Category

9731 Total number of products in each product category53

162 (~2%) Number of products containing Triclosan in each product category53

0.01-0.3% Concentration of Use (%) 20

Suntan products Suntan gels, creams, liquids and sprays Indoor tanning preparations Other Suntan products subtotal Total usage/concentrations-of-use range across all product categories
a

107 240 62 409

2 (~2%) 1 (<1%) None reported 3 (<1%)

None reported None reported None reported None reported

34391

491 (~1.5%)

0.01-0.3%

For baby shampoos reported, only ~2% contain Triclosan, or conversely, the vast majority do not, and the situation is similar for the baby lotions, powders, and creams category. While uses of Triclosan were reported to FDA under the VCRP for each of these categories, no use concentrations were provided in the industry survey. And no uses of Triclosan or use concentrations were reported for the 143 products in the other baby products category. b Triclosan is reported to be listed on the label of Oscar Blandi Pronto Dry Shampoo ( http://www.cosmeticsdatabase.com ) c None of the 2839 hair coloring products were reported to contain Triclosan in the VCRP and no use concentrations were reported by industry. d Triclosan is reported to be listed on the label of Revlon Colorstay Overtime Lip Color Glossy Topcoat ( http://www.cosmeticsdatabase.com ) e While no reported uses were submitted to the VCRP, a use concentration was reported in the Council survey, so it must be presumed there is at least one use.

35

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Table 6. Triclosan in urine from NHANES subjects. Calafat et al. (2008).


Urine concentrations (g/L) 50th percentile 95th percentile 9.2 459.0 5.9 148.0 10.2 649.0 10.3 491.0 6.5 386.0 11.7 566.0 7.4 363.0

Group All 6 - 11 years 12 - 19 years 20 - 59 years 60 years All male All female

Sample size 2514 314 713 950 537 1228 1286

Geometric mean 13.0 8.2 14.5 14.7 10.3 16.2 10.6

36

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Table 7. Triclosan route-specific and species-specific repeat-dose NOAELs.4-7


Route, Duration, Species Dermal Subacute Rat Rat 3.0 mg/kg/day 7.5 mg/kg/day, males 3.5 mg/kg/day, females Mouse 0.6 mg/animal (100 g/cm2) Rabbit Subchronic Rat Rat 40 mg/kg 80 mg/kg NOAEL based on systemic toxicity, characterized as occult blood in urine (EPA) or lack thereof (NICNAS). Each Agency may have drawn a different conclusion from the data, which NICNAS stated were unreliable evidence of systemic toxicity. NOAEL from above study, but based on local irritation reversible after a 20 day recovery period. None 15% None. LOAEL = 1.5 mg/kg/day (dermal irritation and increased absolute and relative liver weights) LOAEL = 6.0 mg/kg/day (local effects at application sites) Basis for NOAEL selection: irritation NOAELa Comments

Rat

10 mg/kg/day

Rat Inhalation All Durations Rat

2.5%, 5%

None assigned

LOAEL = 3.21 mg/kg/day, males, 9.91 mg/kg/day females. Based on increased total leucocyte count and serum alkaline phosphatase.

Subacute Rat NOAEC (irritation): 5 x 10-5 mg/m3 b Oral Subacute Baboon Subchronic Dog 12.5 mg/kg Effects observed at all doses; NOAEL based on reversal of serum alkaline phosphatase elevations after 28-day recovery period. LOAEL: 3000 ppm (168.0 mg/kg/day) based on liver histopath. LOAEL: 25 mg/kg/day. Based on hematology parameters, relative liver weights and total cholesterol. NICNAS excluded 30 mg/kg None LOAEL (systemic): 1300 mg/m3, clinical signs of toxicity and death after 2 days of dosing

Rat Mouse

1000 ppm (52.4 mg/kg/day) None assigned

37

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Route, Duration, Species

NOAELa

Comments this study from its risk assessment due to mechanistic differences between humans and mice.

Chronic Baboon Rat 30 mg/kg 40 mg/kg/day, males 56 mg/kg/day, females Rat 1000 ppm (52.4 mg/kg/day) LOAEL of 3000 ppm (168.0 mg/kg/day) based on significant body weight decreases (both sexes) and non-neoplastic liver changes in males. Based on neoplasms (both sexes) at 30 mg/kg/day [interpreted as mouse-specific and PPAR-,mediated] Based on LOAEL of 250 mg/kg/day (both sexes) based on decreased body weight gains, mortality, nephropathy, and histopathology (stomach and testes). Based on diarrhea at LOAEL of 100 mg/kg/day NOAEL based on histopath. changes in male liver and a trend for reduced body weight in females (LOAEL not reported).

Mouse

10 mg/kg/day (systemic)

Hamster

75 mg/kg/day

1 2 3 Table 8. Hepatic carcinomas and adenomas in mice as a function of triclosan dose.


Number of tumor bearing micea Adenoma Males 5 (5) 7 (7) 13 (16)b 22 (24) c 26 (26) c Females 0 1 3 (3)b 6 (6) c 11 (11) c

Carcinoma Dose (mg/kg/day) 0 10 30 100 200 Males 2 3 (3) 6 (3) 11 (9)c 24 (22) c Females 0 0 1 (1) 1 (1) 14 (14) c

Combined Females 0 1 3 (3)b 6 (6) c 20 (20) c

4 5 6 7 8

Males 6 (6) 10 (10) 17 (17)c 32 (32) c 42 (42) c

results in parentheses are based on a pathology peer review conducted after the study using the liver slides prepared during the study. statistically significant at p0.05. statistically significant at p0.01.

b c

38

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1 2

Table 9. Observations made in various governmental reviews regarding triclosan endocrine disruption effects in fish, frog, and in vitro preparations.
Fish Weakly androgenic, weakly estrogenic, toxic (altered fin length, sex ratio, etc.) 5 Preliminary data indicate that triclosan (or metabolite) is not potently estrogenic to freshwater fish but it may be weakly estrogen, anti-estrogen or androgenic. 4 Frog Induces estrogen antagonism following intraperiotoneal injection of high doses; reduced testosterone levels at lower doses. Binds to thyroid hormone receptor.5 Competively binds to estrogen receptor and supports growth of this estrogen-dependent MCF-7 cell line cell line. Binds to rat androgen receptor 5

In vitro

39

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1 2

Table 10. Systemic triclosan doses determined as a function of product type containing triclosan (European Commission 2009).
Product Triclosan content (%) 0.3 0.3 0.3 0.3 0.2 0.3 Face powder 0.2 0.3 Body lotion 0.15 0.3 Blemish concealer 0.15 0.3 Toothpaste, hand soap, shower gel/body soap, deodorant combined Mouthwash, body lotion, face powder, blemish concealer combined 0.3 Systemic triclosan dose (mg/kg/d) 0.0234 0.0066 0.0268 0.0015 0.1000 0.1500 0.0040 0.0060 0.0823 0.1646 0.0003 0.0006 0.0583

Toothpaste Hand soap Shower gel/body soap Deodorant Mouthwash

0.15 - 0.3 0.3

0.1866 0.3212 0.2449 0.3795

All products

0.15 - 0.3 0.3

3 4

40

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Table 11. Comparison of margin of safety (MOS) determinations.


Product MOS from Rodricks et al. (2010) adult male adult female child MOS from SCCP (European Commission 2009) 513 80 - 120 1118 448 73 - 146 8000 32 49

toothpaste mouthwash hand soap body washes body lotion deodorant combined exposures

9400 15667 47000 11750 1808 15667 1000

7834 11190 47000 9400 1237 15667 732

2043 ND 9216 7833 1119 ND 634

2 3 4
ND = not determined

41

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

REFERENCES
1. Gottschalck TE and Bailey JE. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal Care Products Council, 2010. 2. Eurpoean Centre for Disease Prevention and Control (ECDC), European Food Safety Authoirty EFSA European Medicines Agency EMEA Scientific Committee on Emerging and Newly Identified Health Risks SCENIHR. Joint Opinion on Antimicrobial Resistance (AMR) Focused on Zoonotic Infections. 2009. http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_026.pdf. Date Accessed 10-15-2009. 3. European Commission Directorate-General for Health & Consumers.Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR). Assessment of the Antibiotic Resistance Effects of Biocides. 2009. http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_021.pdf. Date Accessed 10-15-2009. 4. Australian Government, Department of Health & Ageing (NICNAS). Priority Existing Chemical Assessment Report No. 30. "Triclosan". 1-1-2009. http://www.nicnas.gov.au/Publications/CAR/PEC/PEC30/PEC_30_Full_Report_PDF.pdf. Date Accessed 10-15-2009. 5. United States Environmental Protection Agency (EPA). Office of Prevention, Pesticides and Toxic Substances. Reregistration eligibility decision (RED) for Triclosan, List B, Case 2340. 9-1-2008. http://www.epa.gov/oppsrrd1/REDs/2340red.pdf. Date Accessed 10-15-2009. Report No. EPA 739-RO-8009. 6. National Toxicology Program. United States Food and Drug Administration (FDA). Department of Health and Human Services. Nomination Profile. Triclosan. [CAS 3380-34-5]. Supporting Information for Toxicological Evaluation Toxicology Program. 2008. http://ntp.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/triclosan_508.pdf. Date Accessed 10-15-2009. 7. United States Environmental Protection Agency (EPA). Memorandum. January 4, 2008. Triclosan: Report of the Cancer Assessment Review Committee. PC Code: 054901. http://www.epa.gov/pesticides/chemical/foia/cleared-reviews/reviews/054901/054901-2008-0104a.pdf Date Accessed 10-15-2009. 8. United States National Library of Medicine.ChemIDplus Lite. http://chem.sis.nlm.nih.gov/chemidplus/. Accessed 10-22-2009. 9. HSDB, PubMed. National Library of Medicine. Triclosan [33857-26-0]. 8-6-2002. http://toxnet.nlm.nih.gov/cgi-bin/sis/search/r?dbs+hsdb:@term+@rn+@rel+33857-26-0. Date Accessed 11-12-2009. 10. Phenol derivatives. 2002. Ullmann's Encyclopedia of Industrial Chemistry. John Wiley & Sons Inc. 11. United States Pharmacopeia. USP 32. The National Formulary. NF 27. Official Monographs. "Triclosan.". 2009. 12. Menoutis, J and Parisi, Al. Testing for dioxin and furan contamination in triclosan. Cosmetics and Toiletries Magazine. 2002;117:(10):75-78. 13. Ciba.United States Pharmacopeia (USP): Standards of Quality and Purity. http://www.ciba.com/ind-pctriclosan-global-steward-ups.htm. Accessed 10-14-2009. 14. Turner, Richard British Pharmacopeia Secretariat. Written communication. 11-2-2009.

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15. Havery, Don. Personal communication. 11-2-2009. 16. Rule, KL, Ebbett, VR, and Vikesland, PJ. Formation of chloroform and chlorinated organics by freechlorine-medicated oxidation of triclosan. Environ Sci Technol. 2005. 39:(9): pp.3176-3185. 17. Lorres, M and et al. Short Communication: Confirmation of the formation of dichorodibenzo-p-dioxin in the photodegradation of triclosan by photo-SPME. Anal Bioanal Chem. 2005;381:1294-1298. 18. Eisenmann, Carol. Written communication. 10-26-2009. PCPC. 19. National Toxicology Program. FDA Nomination Profile - Triclosan [CAS 3380-34-5]. Supporting Information for Toxicological Evaluation by the National Toxicology Program. 2008. http://ntp.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/triclosan_508.pdf. Date Accessed 10-15-2009. 20. Bailey, John. Written Communication. 10-26-2009. PCPC. 21. Triclosan. http://www.cosmeticsdatabase.com/browse.php?containing=706623. 22. Industrial Hygiene. 1993. Aerosol Measurement: Principles Techniques and Applications. Willeke K and Baron PA. New York: John Wiley and Sons, Inc. 23. Oberdorster G, Oberdorster E, and Oberdorster J. Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles. Environmental Health Perspectives. 2005;113:(7):823-839. 24. James AC, Stahlhofen W, Rudolf G, and et al. Annexe D. Deposition of inhaled particles. Annals of the ICRP. 1994;24:(1-3):231-232. 25. Bower D. Unpublished information on hair spary particles provided at the September 9, 1999 CIR Expert Panel Meeting. 26. Johnson MA. The influence of particle size. Spray Technology and Marketing. 2004;(November):24-27. 27. Bremmer, HJ, Prud'homme de Lodder, LCH, and van Engelen, JGM.RIVM Report 320104001/2006. Cosmetics Fact Sheet to assess the risks for the consumer. Updated version for ConsExpo 4. http://www.rivm.nl/bibliotheek/rapporten/320104001.pdf. 28. Sheridan RL and et al. J Burn Care Rehabil. 1995. 16:(Nov-Dec): pp.605-606. 29. DeSalva, SJ, Kong, M, and Lin, Y-J. Triclosan: a safety profile. American Journal of Dentistry. 1989;2:186196. 30. Fort DJ, Rogers RL, Gorsuch JW, Navarro LT, Peter R, and Plautz JR. Triclosan and Anuran Metamorphosis: No Effect on Thyroid-Mediated Metamorphosis in Zenopus laevis. Toxicological Sciences. 2010;114:392-400. 31. Ahn KC, Zhao F, Chen J, Cherednichenko G, Sanmarti E, Denison MS, Lasley B, Pessah IN, Kultz D, Chang DPY, Gee S, and Hammock BD. In vitro biologic activities of the Antimicrobials triclocarban, its analogs, and triclosan in bioassay screen: receptor-based bioassay screens. Environ Health Perspect. 2008;116:1203-1210. 32. Gee RH, Charles A, Taylor N, and Darbre PD. Oestrogenic and androgenic activity of triclosan in breast cancer cells. J A[[ Toxicol. 2008;28:78-91. 33. Kumar V, Chakrabortya A, Kural MR, and Roya P. Alteration of testicular steroidogenesis and histopathology of reproductive system in male rats treated with triclosan. Reprod Toxicol. 2009;27:(2009):177-185.

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34. Zorrilla LM, Gibson EK, Jeffay SC, Crofton KM, Setzer WR, Cooper RL, and Stoker TE. The effects of triclosan on puberty and thyroid hormones in male wistar rats. Toxicological Sciences. 2009;107:(1):56-64. 35. Paul KB, Hedge JM, DeVito MJ, and Crofton KM. Short-term exposure to triclosan decreases thyrozine in vitro via upregulation of hepatic catabolism in Young Long-Evans rats. Toxicological Sciences. 2010;113:367-379. 36. Lyman, FL and Furia, T. Toxicology of 2,4,4'-trichloro-s'hydroxy-diphenyl ether. Industrial Medicine. 1969;38:45-52. 37. Scientific Steering Committee of the European Commission Health & Consumer Protection DirectoratGengeral, 27-28 June 2002. 2002. http://ec.europa.eu/food/fs/sc/ssc/out269_en.pdf 38. Lambert RJW. Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000. Journal of Applied Microbiology. 2004;97:699-711. 39. Lear et al. Journal of Pharmacy and Pharmacology. 2001;52:126S. 40. Walker C, Borden LC, Zambon J, Bonta CY, DeVizio W, and Volpe AR. The effects of a 0.3% triclosancontaining dentifrice onthe microbia composition of supragingival plaque. J Clin Periodontol. 1994;21:334-341. 41. Dunford RG. Efficacy of a triclosan/NAF dentifriceint he control of plaque and gingivitis and concurrent oral microflora monitoring. Am J Dent. 1998;11:259-270. 42. Aiello, AE, Larson, EL, and Levy, SB. Consumer antibacterial soaps: effective or just risky? Clin Infect Dis. 2007. 45: pp.S137-S147. 43. Cole EC, Addison RM, Rubino JR, and et al. Investigation of antibiotic and antibacterial agent crossresistance in target bacteria from homes of antibacterial product users and nonusers. Journal of Applied Microbiology. 2003;95:664-676. 44. Draft Guidance for Industry on Gingivitis: Development and Evaluation of Drugs for Treatment or Prevention; Availability. Federal Register. 6-28-2005;70:37102-37103.

45. ICIS Chemical News.Ciba Defends antibacterial triclosan in soap. http://www.icis.com/Articles/2008/06/23/9132724/ciba-defends-antibacterial-triclosan-in-soap.html. Accessed 11-18-2009. 46. Sybron Dental Specialties. Section III 510(k) Summary of Safety and Effectiveness. 2-3-2006. http://www.accessdata.fda.gov/cdrh_docs/pdf5/K053565.pdf. Report No. K053565. 47. Ethicon Inc. Summary of Safety and Effectiveness. 6-29-2005. http://www.accessdata.fda.gov/cdrh_docs/pdf5/K050845.pdf. Report No. K050845. 48. European Commission. Volume 1. Cosmetics legislation. Cosmetic products. 1999 Edition. Directive 76/768/EEC, Annex VI, part 1. 1999. 49. Health Canada. List of Prohibited and Restricted Cosmetic Ingredients. Canada's Cosmetic Ingredient Hotlist. 9-1-2009. http://www.hc-sc.gc.ca/cps-spc/person/cosmet/info-ind-prof/_hot-listcritique/hotlist-liste-eng.php. Date Accessed 10-12-2009. 50. Japan Ministry of Health, Labour and Welfare. Evaluation and Licensing Division. Pharmaceutical and Food Safety Bureau and Ministry of Health and Welfare. "Standards for Cosmetics". Notification No.331

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of 2000, 2006. 2010. http://www.mhlw.go.jp/english/topics/cosmetics/index.html. Date Accessed 11-18-2009. 51. Norwegian Scientific Committee for Food Safety. Risk Assessment on the Use of Triclosan in Cosmetics. 131-2005. Report No. #04/406-16. 52. Gilbert, Brian. Written communication. 11-10-2009. Manager, Technical Services, USP Department of Standards Development. 53. US Food and Drug Administration (FDA).Uses of triclosan as a function of product category form the Voluntary Cosmetic Registration Program database. Allmyr, M., G. Panagiotidis, E.D. Sparve, U. Diczfalusy, and G. Sandborgh-Englund. Human Exposure to Triclosan via Toothpaste does not change CYP3A4 Activity or Plasma Concentrations of Thyroid Hormones. Basic & Clinical Pharmacology & Toxicology. 2009;105(issue5):339-344. Beier, R.S., S.E. Duke, R.L. Ziprin, R.B. Harvey, M.E. Hume, T.L. Poole, H.M. Scott, L.D. Highfield, W.Q. Alali, K. Andrews, R.C. Anderson, D.J. Nisbet. Antibiotic and disinfectant susceptibility profiles of vancomycin-resistant Entercoccus faecium (VRE) isolated from community wastewater in Texas. Bull. Environ. Contamin. and Toxicol. 2008; 80(3):188-194. Boobis, A.R., S.M. Cohen, V. Dellarco, D. McGregor, M.E. Meek, C. Vickers, D. Wilicocks, and W. Farland. IPCS framework for analyzing the relevance of a cancer mode of action for humans. Crit Rev Toxicol. 2006;36:781-792. Calafat, A.M., X. Ye, L-Y. Wong, J.A. Reidy and L.L. Needham. Urinary Concentrations of Triclosan in the U.S. Population: 2003-2004. Environ Health Perspect. 2008;116(3):303-307. Chen, Y., B. Pi, H. Zhou, Y. Yu, and L. Li. Triclosan resistance in clinical isolates of Acinetobacter baumannii. J. Med. Microbiol. 2009; 58:1086-1091. Environ International Corporation. Written communication - investigation of potential endocrine activity of triclosan. 05-17-2010. European Commission. 2009. Directorate General for Health and Consumer Protection. Scientific Committee on Consumer Products (SCCP). Opinion on Triclosan, adopted 21 January 2009. http://ec.europa.eu/health/archive/ph_risk/committees/04_sccp/docs/sccp_o_166.pdf Date accessed 06-22-2010. European Commission. 2010. Directorate General for Health and Consumers. Scientific Committee on Consumer Safety (SCCS) Opinion on triclosan antimicrobial resistance, adopted 22 June 2010. http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_023.pdf. Date accessed 07-20-2010. James, M.O., W. Li, D. Summerlot, L. Rowland-Faux, C.E. Wood. Triclosan is a potent inhibitor of estradiol and estrone sulfonation in sheep placenta. Environment International. 2009 epub. http://www.ncbi.nlm.nih.gov/pubmed/19299018. Date accessed 06-25-2010. Rodricks, J.V., J.A. Swenberg, J.F. Borzelleca, R.R. Maronpot and A.M. Shipp. Triclosan: A critical review of the experimental data and development of margins of safety for consumer products. Crit Rev Toxicol. 2010;1-63, Early Online. Stickler, D.J. and G.L. Jones. Reduced Susceptibility of Proteus mirabilis to Triclosan. Antimicrobial Agents and Chemother. 2008; 52(3):991-994.

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Data

Green Book 1

Tab: Data

Panel Book Page

Data/Document Title or Description

51

Environ Endocrine Description Commentary

94

Rodricks et al

157

SCCP opinion on safety

283

SCCP opinion response to public comments

299

SCCS opinion on antimicrobial resistance

355

Council comments on SLR

359

Environ comments on SLR

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Pre p ve

EJo

ci bd/

Or

Investigation of Potential Endocrine Activity of Triclosan


1. BACKGROUND
Recent studies have investigated the effect of triclosan on thyroid hormone levels (Crofton et al. 2007; Paul et al. 2009; Zorrilla et al. 2009) or other serum hormone levels, e.g., testosterone (Kumar et al. 2008, 2009; Zorrilla et a]. 2009) or estradiol (James et al. 2009), in studies conducted to assess the potential that triclosan could function as an endocrine disruptor in mammalian species. Triclosan has been tested in an extensive battery of tests to include studies with human subjects on safety and tolerability and studies in experimental animals conducted over a wide range of doses and durations in several species to assess the potential for general systemic toxicity, carcinogenicity, mutagenicity, neurotoxicity, and reproductive andlor developmental toxicity (as reviewed by Rodricks et al. 2009). None of these tests reported effects that were specifically attributed to endocrine disruption activity; however, the tests were not designed to evaluate subtle modulations in endocrine function. Yet, these data along with recently published studies that did investigate the potential for endocrine action by triclosan could be used, in an integrated manner, to assess the following: if triclosan were acting by modulation of endocrine function and targeting endocrine-responsive organs, i.e., the thyroid or reproductive system, then what would be the expected signs of toxicity that should have been manifested in the studies already conducted? An important consideration in this investigation was the applicability of the data reviewed to assess impacts on human endocrine system function. To address this, a hierarchical approach was taken and value assigned in descending order to the following types of studies: 1) studies in human volunteers; 2) studies conducted in vivo in mammalian species; 3) studies conducted in vitro with primary cells or tissues harvested from mammalian species; and. 4) in vitro studies in immortalized cells, in particular immortalized cancer cell lines whether human or animal cell lines. Use of this latter category, immortalized cell lines, requires careful scrutiny. In order to use in vitro data to be predictive of human health outcomes, extrapolation from an in vitro system to an in

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Investigation of Potential Endocrine Activity of TrIclosan September 24, 2009


*

vivo model and in the case of non-primate cells, extrapolation across species, e.g., animal

to human, must be considered. More importantly is consideration of the genomic behavior of immortalized cells, in particular immortalized cancer cell lines, especially when transcriptional and receptor activation may be involved. Gentry et al. (2009) reported that the genomic activation profile in response to xenobiotics may be considerably different in immortalized and cancer cell lines than in primary cells even at comparable doses of the compound. The approach taken in this investigation was to: Review recent in vivo and in vitro data for each endpoint that could be impacted, e.g., the thyroid and reproductive system; Define what would be expected to be seen in terms of signs and symptoms if triclosan were acting to disrupt endocrine function; Compare the expected outcome to the data that have been reported in the human safety and experimental studies to identif those reported effects that could be the consequence of endocrine disruption; and, Discuss the potential for biologically significant effects in humans following long-term, low-level exposure.

The investigation of the recent literature for triclosan identified two major systems that could be impacted if triclosan was acting as an endocrine disruptor, i.e., the thyroid and the reproductive system. This investigation focused on those two potential targets for triclosan-induced endocrine activity.

2.0 2.1

POTENTIAL EFFECTS ON THE THYROID Recent Thyroid Hormone Studies

Three studies have been conducted in rats that evaluated the effects of triclosan on total serum thyroid hormone concentrations (Crofton et al. 2007; Paul et al. 2009; Zorrilla et al. 2009) and a study in human volunteers has been conducted (Alimyr et al. 2009). Studies in rats in which changes in thyroid hormone levels and alteration in thyroid histopathology are evaluated serve as an initial screen of the potential for a xenobiotic to alter thyroid function in humans (DeVito et al. 1999). However, changes in thyroid hormone levels in rats do not necessarily mean that thyroid function in rats will be altered

2
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Investigation of Potential Endocrine Activity of Triclosan September 24, 2009


-

or that physiological systems in rats will be adversely affected. Nor do these changes in serun-i thyroid hormone levels in rats translate quantitatively to similar changes in humans because of the differences between rats and humans in both the capacity to keep thyroid hormones within normal physiological ranges but also because of the decreased sensitivity of humans to these changes. As discussed in the following sections, changes in thyroid hormone levels in rats, in particular in short-term studies, in which initial adaptive mechanisms may be operative, are reviewed in the context of all of the experimental data for triclosan.

2.1.1

Review ofstudies in rats The study by Crofton et a!. (2007) was conducted to evaluate the effects of

triclosan on circulating levels of thyroxine 4 (T ) . The protocol was designed based on data for other chemicals in which a 4-day exposure to rats had previously been used to assess the potential for an effect on thyroid hormone levels (Zhou et al. 2001; Craft et al. 2002; Crofton 2004). In the Paul et al. (2009) study, the effect of triclosan on T 4 levels in dams and in offspring was investigated. The Zorrilla et al. (2009) study was designed to investigate the effects of triclosan exposure during pubertal development on thyroid hormone levels and sexual development in male rats. In the Crofton et a!. (2007) study, young female Long-Evans rats (8 to 16 rats per group) age 27 to 29 days were randomly assigned to treatment groups and administered 0, 10, 30, 100, 300, or 1000 mg triclosanlkg/day by oral gavage in corn oil for 4 consecutive days. No clinical signs of toxicity were seen in any animals during treatment. Twenty-four hours after the last dose, rats were sacrificed, trunk blood was collected and body and liver weights were determined. No treatment-related effects on body weight gains were found. Absolute and relative liver weights were increased in the 1000 mgilcg/day dose group but not in any of the other treatment groups. No other organs were weighed nor were histopathological examinations conducted. After 4 days of treatment, dose-dependent decreases in total serum T 4 (bound and free) concentrations were seen in rats treated with triclosan at concentrations of 100, 300. or 1000 mg/kglday with decreases of 28%, 34%, and 53%, respectively. The study authors did not offer an opinion as to the consequences of decreases in circulating total T 4

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levels nor whether a reduction of 53% in circulating total T 4 levels would affect physiological function in these female rats. Serum concentrations of triiodothyronine (T ) 3 . the more bioactive thyroid hormone, or thyroid stimulating hormone (TSH) were not measured. In the Paul et a]. (2009) study pregnant Long Evans rats (age was not specified) received triclosan by gavage in corn oil at doses of 0, 30, 100, or 300 mg/kg/day beginning on gestational day (GD) 6 and continuing daily until post-natal day (PND) 21. At PND 22, there was a significant decrease (3 0%) in total T 4 serum concentration in dams given 300 mg/kg/day triclosan but other doses were not significantly different from control values (number of animals per dose group was not given). A similar percentage reduction in total T 4 serum concentrations of approximately 30% was seen in offspring of dams in the 300 mg/kg/day dose group when measured at PND 4; however, no effects on total T 4 serum concentrations were seen in these offspring when T 4 levels were measured at PND 14 or 212 and no effects on serum T 4 levels were seen in offspring in the two lower dose groups at any time point measured postnatally. The similarity in pattern of total T 4 concentrations in offspring at PND 4 with maternal T 4 levels at PND 22 suggested, according to the authors, that the dam supplied thyroid hormones to the fetus. While maternal T 4 serum concentrations were not measured on PND 4, given the rapid metabolism and excretion of triclosan from the body (Rodricks et al. 2009), steady state levels, and hence effects on T 4 levels, may have been reached in that short time-frame, e.g., PND 4. The lack of changes in T 4 in offspring on PNDs 14 and 21 (note only the dams were continued on treatment) were interpreted by the authors to indicate that lactational transfer did not play a significant role in maintaining thyroid homeostasis and that changes seen at PND 4 were transient. In a pubertal study, Zorrilla et al. (2009) treated groups of male Wistar rats (8 to 15 per group) with 0, 3, 30, 100, 200, or 300 mg/kg/day triclosan in corn oil by oral gavage from PND 23 to 53(31 treatments). According to the authors, the protocol used was that defined in the Endocrine Disruptor Screening Program (EDSP) (USEPA 2009) to evaluate male pubertal effects from exposure to xenobiotics. No visible signs of
Only a poster presented at the Society of Toxicology annual meeting in 2009 was available for review. Note that the Figure from which these data were obtained was not consistent with the text of the abstract of this poster that states that T4 levels were not affected in pups at any PND evaluated.
2

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toxicity were noted in any of the treated animals over the course of treatment. No treatment-related changes were seen in body weight gain (growth) or in terminal body weights at necropsy. Kidney and adrenal weights were unaffected, while liver weights were significantly increased in the 100, 200, and 300 mg/kg/day, a finding consistent with all of the subchronic and chronic toxicity data for triclosan (Molitor et al. 1992; Eldridge 1993; Auletta 1995; Molitor and Pershon 1993; Pershon and Molitor 1993; Yau

and Green 1986).


In the Zorrilla et al. (2009) study, significant dose-dependent decreases in the mean serum total T 4 concentrations were seen at 30, 100, 200, and 300 mg/kg/day with no effect at 3 mg/kg/day. Serum concentrations of total T 4 were reduced to approximately 58% and 44% of control in the 30 and 100 mg/kg/day groups, respectively, and to approximately 25% and 22% of control values in the 200 and 300 mg/kg/day groups, respectively. The lack of a clear monotonic dose-response may be an artifact of the protocol. Two control groups were used and designated as Block 1 and Block 2 by the authors. Block 1 contained the 0, 3, 30, and 300 (n10) mg/kg/day dose groups and Block 2 contained the 0 (n1 5), 100 and 200 (both n8) mg/kg/day dose groups. The change in T 4 levels between control groups was significantly different and, therefore, the absolute change in T 4 levels for treated groups was compared to their respective controls. It is unclear from the Figures in the paper (Figures 6A and 6B) that the percent of control for each dose group was determined from the mean of both control groups combined or to the mean of the respective control group to the treated groups. Also in the Zorrilla et al. (2009) study, no consistent, treatment-related changes in total serum T 3 concentrations were seen (there was a significant increase in the 3 mg/kg/day group and a significant decrease at 200 mg/kg/day but T 3 plasma concentrations were not affected in 30, 100, or 300 mg/kg/day dose groups). Also, no treatment-related changes in thyroid stimulating hormone (TSH) were seen in any dose group tested. At the histological examination, no significant changes were seen in the follicular epithelial height in any dose group. Decreases in colloid area were seen in thyroid gland sections but only in the 300 mg/kg/day dose group, which, according to Zorrilla et al. (2009), indicated a compensatory release of active thyroid hormones from the colloid.

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Changes in thyroid colloid area in combination with an increased height of follicular cells along with hypertrophy and hyperplasia of the follicular cells could indicate persistent TSH stimulation (Khan et al. 1999). However, no changes in follicular height or signs of hypertrophy or hyperplasia were seen at any dose level. Further, 4 T levels were decreased at doses of 30 mg/kg/day and higher but no changes in colloid morphology were noted in rats receiving triclosan at doses less than 300 mg/kg/day. Also, consistent, dose-related changes in levels ofT 3 were not seen and TSH levels were not affected by treatment with triclosan. Morphological changes in colloid area in the absence of follicular cell changes are of uncertain relevance. 2.1.2 Review of a human study In contrast to the rat studies conducted by Crofton et al. (2007), Paul et al. (2009) and Zonilla et al. (2009), triclosan, administered in toothpaste to healthy volunteers did not result in an increase in thyroid hormone levels (Allmyr et al. 2009). Twelve volunteers (5 men and 7 women), who discontinued use of any triclosan-containing products two weeks prior to the initiation of the study, were instructed to brush twice daily using an over-the-counter toothpaste (Colgate Total) for 14 days. The concentration of triclosan in the toothpaste was 0.3% (w/w) and the amount applied was standardized (2 cm), which was estimated by the authors to result in a dose of swallowed triclosan of 0.01 mg/kg/day. This dose was based on the estimates of intake of triclosan from daily brushing (Bagley and Lin 2000). The subjects were instructed to brush for 3 minutes per event and no toothpaste was to be swallowed intentionally. The protocol was designed to provide an estimate of the upper range of exposures to triclosan from the use of toothpaste that could be expected in a normal human population. In the Allmyr et al. (2009) study, plasma triclosan levels were measured before the experiment and after 14 days of brushing with plasma concentrations of triclosan (both unconjugated and conjugated forms) ranging from 0.009 to 0.81 ng/g before exposure to 26 to 296 ng/g after 14 days of brushing. After exposure, the median triclosan concentration in plasma was 54 ng/g, which was approximately 3 times higher than the triclosan concentrations measured in serum samples in populations in Sweden and Australia (Allmyr et al. 2006. 2008). Based on this comparison, Allmyr et al. (2009)

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concluded that the instructions for brushing used in their study, e.g.. the duration and the amount, overstated the amount of triclosan exposure from standard brushing procedures. According to kinetic studies, approximately 70% of the triclosan in plasma is in the form of the sulphate or glucuronide conjugates (Sandborgh-England 2006), which are assumed to be biologically inactive (Alimyr et al. 2009). After the exposure period, Ailmyr et al. (2009) measured plasma concentrations of 43-hydroxycholestero1, free plasma concentration of T , and TSH. No significant 4 ,T 3 changes in plasma concentrations of any of the thyroid hormones were seen compared to baseline levels measured prior to initiation of treatment nor were any outside of normal reference values. No significant changes were seen in plasma concentrations for 4f3hydroxycholesterol, which is a cholesterol metabolite and an endogenous marker for CYP3A4 activity (Kanebratt et al. 2008; Wide et a!. 2008; Diczfalusy et a!. 2009). No marked deviation from the results found for the entire group was noted in the individual in this study with the highest triclosan plasma concentration of 296 ng/g, which is in the range of plasma levels obtained after an oral dose of 4 mg triclosan (Sandborgh-Englund 2006).

2.1.3

Comparison among recent thyroid studies

Given that the above studies in rats were conducted by the same group, a number of comparisons can be made that may be useful in assessing the relevance of these findings to human health outcomes. In a comparison of the Crofton et al. (2007) and Paul et al. (2009) studies, both examined female Long Evans rats given triclosan at similar concentrations but ages at first exposure were different (young weanling rats at 23 to 25 days of age v. sexually mature rats) and for different durations (4 days compared to 36 days). Despite the longer duration of dosing and older females tested in the Paul et al. (2009) study compared to that reported by Crofton et a!. (2007), significant decreases in maternal T 4 levels were only seen in the 300 mg/kg/day dose group and were comparable with a 34% v. 30% decrease in the Crofton et al. (2007) and Paul et al. (2009) studies, respectively. These data would indicate that with an increased duration of treatment, 4 T levels were not further depressed. However, there was a significant difference in response in the 100 mg/kg/day dose groups [28% v. 8% in the Crofton et al.(2007) and

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Paul et al. (2009) studies, respectively] that may indicate that exposure at a younger age would shift the dose-response curve with significant responses occurring at lower doses; however, no statistical analyses were conducted to discern if the response in the 100 mg/kg/day dose group was significantly different from that in the 300 mg/kg/day dose group in the Crofton et al. (2007) study. The reduction in T 4 levels was comparable in both studies for the 30 mg/kg/day group (non-significant decreases of 7% v. 8%). These differences may reflect natural variability due to the small numbers of animals tested [16 per group in the Croflon et al. (2007) study; the number of animals per dose group was not given in the Paul et al. (2009) poster] but does introduce uncertainty as to an estimate of a threshold for this effect. When the results of the Paul et al. (2009) and Crofton et al. (2007) studies are compared with those reported by Zorrilla et al. (2009), there are differences in responses. In the Zorrilla et al. (2009) study, significant changes in total T 4 serum concentrations were seen in weanling male Wistar rats in the 30, 100, 200 and 300 mg/kg/day dose group given triclosan for 31 days (approximately 50% reduction in 30 and 100 mg/kg/day groups and 80% reduction in the 200 and 300 mg/kg/day groups). In the Paul et al. (2009) study a significant decrease in total T 4 serum concentrations was noted in adult females only in the 300 mg/kg/day dose group (a decrease of approximately 30% from control values) given triclosan in the same manner for 36 days. The key differences in the Zorrilla et al. (2009) study and that conducted by Paul et al. (2009) were the gender difference, the rat strain tested, and the age at initiation of dosing, which may not be a determining factor as noted in the comparison between the Crofton et al. (2007) and the Paul et al. (2009) studies mentioned in the preceding paragraph. Differences in total T 4 serum concentrations following triclosan treatment were also seen between the results of Crofton et al. (2007) and Zorrilla et al. (2009) studies in

which the animals were the same approximate age at the initiation of the study but the
duration, gender, and species were different. The duration of exposure, at least in females, was not a factor, as noted above. Gender differences in rats are known to exist, e.g., male rats may develop more thyroid tumors than female rats due to greater changes in TSH or to complex interactions with testosterone (USEPA 1998). However, TSH and testosterone levels were unaffected at any dose tested in the Zorrilla et al. (2009) study.

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The levels of TSH are higher in male rats than in female rats, i.e., the demand on the thyroid gland of the male rat is higher than in female rats, and chemicals that interfere with thyroid hormone homeostasis would likely have more of an impact in male rats than in females (USEPA 1998). Another factor is the difference in the rat strains tested; differences in sensitivity have been noted for other endpoints, such as the production of Leydig cell tumors in Fisher 344 rats or mammary carcinomas in Sprague Dawley rats compared to other strains. However, all of these differences may also reflect inherent variability in this assay due to the use of total (bound and unbound) T 4 concentration rather than free T 4 as the metric. The results in the rat studies can be compared to those reported in a study of human volunteers. As noted above, serum concentrations of total T 4 were reduced in female rats at doses between 100 and 300 mg/kg/day (Crofton et al. 2007), while T 4 levels were significantly reduced in male rats at doses of 30 mg/kg/day and higher (Zorrilla et al. 2009). No changes in serum T 4 concentrations were seen in volunteers in the Ailmyr et al. (2009) study. There are a number of reasons for the lack of agreement between the changes in 14 levels in the rat studies and the results reported by Alimyr et al. (2009). The most obvious reason is the difference in the dose of triclosan. In the rat studies, the dose at which a change in T 4 levels was significantly different from controls ranged from 30 mg/kg/day to 300 mg/kg/day. The estimated dose to the subjects in the Alimyr et al. (2009) study was estimated to be 0.01 mg/kg/day, a dose that could result from typical use of triclosan-containing toothpaste. Perhaps the more important reason, as discussed in Section 2.3, is the differences in homeostatic controls in the rat and human. It is important to note that serum concentrations of T , the biologically active 3 thyroid hormone in both species, and TSH were not altered in either the Zonilla et al. (2009) study in rats or the Alimyr et al. (2009) study in human volunteers. While it is impractical to establish a threshold in an experimental study, the data in rats do demonstrate that there are administered doses of triclosan below which no treatment-related changes in 14 levels were measured. Further, in the rat studies, total (bound and unbound) 14 concentrations were measured, while only free 14 levels were measured in humans. Use of free 14 as the relevant metric has been recommended by DeVito et al. (1999), who reported the summary of the working group on screening

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methods for thyroid hormone disruption. Use of total 14 determinations was not recommended by this working group as a screen for thyroid function but rather free 14

and TSR levels because changes in bound 14 could occur without changes of the
biologically available 14 levels (DeVito et al. 1999).

2.2

Comparisons to Clinical and Experimental Data

The safety of triclosan has been evaluated in studies with human volunteers designed to assess tolerability and/or kinetics and has been extensively studied in experimental animals primarily by the oral and dermal routes of exposure with acute, subacute, subchronic, and chronic administration as reviewed by Rodricks et al. (2009). None of these studies was specifically designed to evaluate thyroid hormone levels or thyroid function. Under the assumption that decreases in serum 14 concentrations could lead to or be a marker for hypothyroidism, as suggested by Crofton et al. (2007) and Zorrilla et al. (2009), a number of adverse human health outcomes could be hypothesized. Thyroid hormones influence normal metabolic functions in most tissues in the body. The consequences of decreased thyroid hormone levels can be manifested in a number of ways, e.g., overt clinical signs, such as fatigue or subtle changes in biochemical parameters, and the nature and extent of the effects could depend on the affected organ and the timing or age at which hypothyroidism occurs. Therefore, clinical signs and symptoms that could be the consequences of hypothyroidism were evaluated in these clinical and experimental studies. The potential effects were grouped into four major categories: Overt signs of toxicity and/or changes in metabolic or biochemical parameters; Continued pressure on thyroid homeostasis resulting in the production of thyroid hyperplasia and neoplasia; and,
Effects resulting

from altered thyroid homeostasis during critical windows of development: o Neurobehavioral deficits resulting from in utero exposure, and o Alterations in reproductive competence resulting from exposure during the sensitive developmental times to include neonatal and juvenile periods.

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2.2.1

Overt signs of toxicity and/or changes in metabolic or biochemical parameters A number of studies have been conducted to specifically evaluate human safety

and tolerability of triclosan in oral care products (DeSalva et al. 1989; Lucker et al. 1990; Safford 1991; Barnes 1991 a; Barnes 1991 b). DeSalva and coworkers published a review of triclosan safety that summarized the results of clinical studies of oral exposure to triclosan-containing toothpaste or mouthwash products (DeSalva et al. 1989). Although individual studies were not identified, this publication reported results from 1,246 participants in clinical studies in which toothpaste or mouthwash with triclosan at concentrations ranging from 0.0 1% to 0.6% had been used for 1 day to 12 weeks. No difference between control and exposed populations with respect to hematology and clinical chemistry findings were reported, including tests for liver and kidney function. Safford (1991) evaluated hematological and clinical chemistry changes in 112 subjects who brushed with toothpaste containing 0.2% triclosan for 65 weeks. Subjects (n=392) in the Barnes et al. (199la; 1991b) study brushed normally with 0.2% triclosan containing toothpaste for 52 weeks followed by a 13-week period without triclosan containing toothpaste. In a study designed to evaluate the kinetics of triclosan, 20 subjects were given increasing amounts of triclosan for 52 days; doses increased from 1 mg/day to 30 mg/day over a 2-week period for a total of 10 days, followed by 15 mg/day for 30 days (Lucker et al. 1990). These studies evaluated changes in clinical biochemical and hematological endpoints in humans and, in the case of Lucker et al. (1990), urinary parameters. In all of these studies, no clinically relevant, sustained, treatment-related changes in hematological or clinicallbiochemical parameters were observed in more than 2500 subjects following the daily use of toothpaste or other oral products containing 0.01% to 0.6% triclosan for periods ranging from I day up to 4 years. Similarly, in the numerous experimental studies conducted there were no overt clinical signs of toxicity following acute, subchronic or chronic administration of triclosan at very high doses (Rodricks et al. 2009). Further, there were no treatmentrelated, consistent effects on any hematological parameters or clinical chemistry (non liver) parameters, When present, alterations in enzymes associated with liver function were found to include decreases in cholesterol, consistent with the proposed mode of

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action of triclosan as a PPARCL agonist in the liver, the main target organ identified in all of the numerous experimental studies (Rodricks et al. 2009). 2.2.2

Production of thyroid hyperplusia and neoplasia

Chemicals known to interfere with thyroid hormone homeostasis in rats are structurally diverse and interfere with thyroid hormone homeostasis by different mechanisms (USEPA 1998). The general hypothesis is that an alteration in the balance of thyroid hormones. i.e., decreased circulating levels due to either decreased production of 4 or increased metabolism and excretion ofT T 3 or T , triggers, through feedback loops, 4 the release of thyrotropin releasing hormone (TRH) from the hypothalamus, with the resulting cascade leading to an increase in TSH release from the pituitary. The sustained stimulus by TSH on the thyrocytes is to produce more thyroid hormones. The end result is that in rodents, primarily rats, following the administration of these chemicals, thyroid hormone levels (T 3 and T ) can be decreased, resulting in a compensatory increase in 4 TSH in order to maintain T 3 and T 4 concentrations at physiological levels, With continued chemical interference, the elevated levels of TSH would be sustained resulting in a persistent signal to the follicular cell to produce T , thereby, promoting the stimulus 4 to hyperplasia and neoplasia in rats (LJSEPA 1998). Free levels of T 3 or 14 are subject to rapid metabolism and degradation. Unlike humans, rats lack specific thyroid hormone-binding globulins and, therefore, lack the reserve capacity to release thyroid bound hormones in response to decreases in free serum thyroid hormone levels. The rat thyroid gland is at near maximum stimulation in order to maintain physiological levels of thyroid hormone. The addition of a chemical that results in decreased serum levels of T 3 and T 4 with a compensatory increase in TSH would further stress a system that was near capacity. If the TSH stimulation was sustained, hyperplasia and possibly progression to neoplasia could result in rats (USEPA 1998). This progression by non-genotoxic modes of action to thyroid tumors has not been seen in humans (USEPA 1998). In the rat study conducted by Zorrilla et al. (2009) no sustained treatment-related alterations in T 3 or TSH levels were reported. Under the assumption that 31 days may not have been sufficient to see a sustained change that could result in compensatory

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increases in TSH with resulting biological consequences, other experimental data for triclosan were reviewed. Thyroid histopathology has been evaluated in several of the subchronic and chronic rodent toxicity studies. No adverse effects on the thyroid have been noted in any of these studies suggesting that the reductions of serum T 4 in the shortterm studies by Crofton et al. (2007) and Zorrilla et al. (2009) would not have lead to altered thyroid histopathology following chronic exposure. In those studies in which thyroid weights and/or histopathological evaluations were conducted, there was no evidence of an effect on the thyroid in subchronic studies in mice at doses up to 900 mg/kg/day (Trutter 1993), rats at doses up to 300 mg/kg/day (Goldsmith and Craig 1983), dogs at doses up to 25 mg/kg/day (Leuschner et al. 1970) or Rhesus monkeys treated dermally with a 0.1% triclosan solution daily (Dalgard et al. 1979; Parkes. 1979). No effects on thyroid weight or histopathology were seen in chronic/oncogenicity studies following administration of triclosan in the diet in mice at doses up to 200 mg/kg/day (Auletta 1995). rats at doses up to 150 mg/kg/day (Yau and Green 1986), hamsters at doses up to 250 mg/kg/day (Chambers 1999) or baboons at doses up to 300 mg/kg/day (Drake 1975). Triclosan did not produce thyroid tumors in

any species tested when administered at doses that were above those that were reported to
produce changes in T 4 levels in the short-term studies reported by Crofton et al. (2007), Zorrilla et al. (2009), and Paul et al. (2009). 2.2.3 Altered thyroid homeostasis during critical windows of development: 2.2.3.1 Neurobehavioral deficits resulting from in utero exposure It has been proposed that hypothyroidism in general, and hypothyroxinemia in particular, in women during the first trimester of pregnancy could affect normal development of the fetal brain and lead to neurobehavioral deficits in children born to these mothers (Morreale de Escobar et al. 2000). Hypothyroidism has been defined by
Morreale de Escobar et al. (2000) reviewed literature drawn primarily from studies in geographical areas with a high incidence of cretinism due to maternal iodine deficiency or that reported an association between th women with maternal hypothyroidism (defined as having TSH levels higher than the 98 percentile of the normal range) or hypothyroxinemia (undefined as to extent) and neuropsychological deficits in their offspring. In contrast, Soldin et al. (2003) found no evidence to suggest that in utero thyroid status as reflected by neonatal T 4 levels had an impact on the neurological disorders diagnosed in childhood. In the Soldin et al. (2003) study, to determine if there was an association between childhood neurobehavioral

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the American Thyroid Association as decreased free T 4 concentrations accompanied by increased serum TSH concentrations sufficient to disrupt thyroid homeostasis (DeVito et al. 1999). Potential neuropsychological effects in offspring from maternal exposure to chemicals may only occur at doses that produce sufficient hypothyroidism or hypothyroxinemia in the mother, such that maternal transfer ofT 4 to the fetus is sufficiently impaired (DeVito et al. 1999). For example, decreases in T 4 levels (55%) in the early postnatal period were not sufficient to alter synaptic transmission in the dentate gyrus of the hippocampus of rat pups (Gilbert et al. 2002). Consequently, in the absence of maternal hypothyroidism or hypothyroxinemia, subsequent neurobehavioral deficits as a result of impaired thyroid function are unlikely to occur. According to DeVito et al. (1999) changes in thyroid hormone levels and histopathological changes would occur at lower doses than those required to detect behavioral changes. Therefore, changes in free 4 and TSH levels are recommended as a screen for neurobehavioral potential rather than T developmental assays (DeVito et al. 1999). When triclosan was administered to pregnant females, decreases in T 4 levels were only seen in the 300 mg triclosan/kg/day dose group and not in those females receiving 30 or 100 mg/kglday (Paul et al. 2009). In this study, a transient decrease in T 4 levels was seen only in pups born to dams in the 300 mg/kg/day dose group and only when measured at PND 4 but not on PND 14 or 21, and no changes in maternal or fetal T 4 levels were seen at maternal doses up to 100 mg/kg/day. Further, no alterations in T 3 ,T 4 or TSH levels were seen in human volunteers given triclosan-containing toothpaste that resulted in doses relevant to those that may result for the use of triclosan-containing
products (AIlmyr et al. 2009). At doses of triclosan, such as those encountered as a result of use of triclosan-containing consumer products, alterations in thyroid hormones are

unlikely to occur and consequently, neurobehavioral effects are unlikely to occur at doses relevant to potential human exposures.

deficits, such as attention deficit hyperactivity disorder (ADHD). learning disorders, or autism, that could be attributed to fetal thyroid hormone deficits, neonatal T 4 levels were obtained from a neonatal hypothyroidism screening program. All cases were diagnosed at medical school diagnostic clinics. No significant differences were found between neonatal T 4 levels of cases and controls for any neurobehavioral condition.

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2.2.3.2 Alterations in reproductive competence resultingfrom exposure during the sensitive developmental times to include neonatal andjuvenile periods Another potential concern is that hypothyroidism induced in the early neonatal period can result in effects on reproductive development and function in male rats manifested as alterations in sexual maturation, testicular and reproductive tract development and changes in gonadotrophin and steroid concentrations and by having an impact on both Sertoli cells and Leydig cells (Zorrilla et al. 2009). This topic is discussed further in Section 3. 2.3 Relevance to Human Health Outcomes The potential for altered T 4 levels seen in the rat studies to occur in humans and have an adverse effect on health is dependent on the mode of action for these decreases in 4 levels and the relative susceptibility to changes in homeostasis between species that T could result. There are a number of steps in the normal production of thyroid hormones and the maintenance of thyroid homeo stasis that could be the targets of xenobiotics in rats. Changes in either the production of T , the metabolism ofT 4 , or the binding 3 4 to T andlor the elimination ofT 4 from the circulation could impact homeo stasis and, consequently, engage the normal physiological feedback loop to compensate for such changes; the latter two general steps are mediated by liver enzymes. In biochemical and mechanistic studies, triclosan has been shown to induce selected liver enzyme systems (Molitor et al. 1992; Eldridge 1993; Molitor and Persohn 1993; Persohn and Molitor 1993; Persohn 1994; Thomas 1994). Triclosan produced an increase in CYP3AI or CYP3A2 4 in mice (Molitor et al. 1992) and rats (Molitor and Persohn 1993) but not in hamsters (Thomas 1994). The CYP3A family of monooxygenases catalyzes many reactions including the synthesis of cholesterol, steroids, and other lipids (Smith et al. 1998). CYP3A mediates the metabolic activation of uridine diphosphoglucuronosyl transferase (UDPGT) (Allmyr et al. 2009). This could result in an increase in glucuronidation ofT 4 after conjugation with hepatic microsomal UDPGT, which could result in an increased clearance ofT 4 in
The CYP3A family serves a common function across species to include metabolism of steroid and lipids. There are slight variations across species (Carr et al. 2006). The predominant forms are CYP3AI or CYP3A in rats; CYP3AI2 and CYP3A26 in dogs, CYP3A6 in rabbits, CYP3A64 in rhesus monkeys, and CYP3A4 and CYP3A5 in humans (Carr et al. 2006)

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the bile (Saghir et al. 2008), one potential way in which serum T 4 levels could be reduced. Triclosans effects on selected enzymes were assessed in rats (Zorrilla et al. 2009; Paul et al. 2008, 2009). In rats in the Zorrilla et al. (2009) study, an increase in UDPGT activity was seen in the highest dose group (300 mg/kg/day) compared to controls. Although the authors stated that the increase was not statistically significant, UDPGT levels were at least 2 to 3 times that in the control group or the two lower dose groups tested (3 and 30 mg/kg/day). Decreases in T 4 levels were noted at a dose of 30 mg/kg/day and higher; however, UDPGT activity was not elevated in the 30 mg/kg/day group; the 100 or 200 mg/kg/day dose groups were not evaluated for UDPGT induction. The authors noted that this type of inconsistent relationship between UDPGT induction and T 4 levels has been reported previously by Craft et al. (2002), de Sandro Ct al. (1992), and Hood and Klaassen (2000) and was likely due to variability inherent in this assay. Transcription of CYP3A is mediated by the human nuclear receptor hPXR (Ailmyr et al. 2009) and by PPARL agonists (Klaunig et al. 2003; Molitor et al. 1992; Molitor and Pershon 1993). Triclosan was found to be a medium affinity ligand to hPXR when tested in vitro in the human hepatoma cell line (HuH7) transfected with human PXR (Jacobs et al. 2005). However, no significant changes were seen in plasma concentrations of 43-hydroxycholesterol, a cholesterol metabolite and an endogenous marker for CYP3A4 activity in humans following use of triclosan-containing toothpaste compared to pre-exposure levels (Ailmyr et al. 2009). The lack of an increase in a defmitive marker of CYP3A4 activity in subjects in the Ailmyr et al. (2009) study would indicate that at the triclosan exposures at environmentally relevant amounts, activation of CYP3A4 and resulting biological consequences, such as the increased excretion of T 4 in the bile is not occurring. The significance of these findings is relevant to the difference in species response to triclosan. Induction of hepatic enzymes that may be involved in thyroid hormone metabolism and catabolism may also be mediated by triclosans demonstrated activity as a PPARa agonist, a mode of action that is not likely relevant to humans (Rodricks et at. 2009). Regardless of the specific mode of action, there is considerable evidence that humans are more resistant to changes in circulating thyroid hormone levels than rats

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because of the significant buffering capacity of thyroxine-binding proteins (TBG) in humans (USEPA 1998). There are also species differences in the plasma half-lives of thyroid hormones. T 3 and T 4 are bound to plasma proteins, such as albumin, in most mammalian species. Protein-bound thyroid hormone is unavailable for metabolism and serves as a buffer for changes in peripheral T 3 and T 4 levels. However, in addition to albumin, some species, including humans and primates, have high affinity T 3 and T 4 binding globulins and a large percentage of T 3 and T 4 is bound to these proteins (USEPA 1998). In contrast, rats and mice lack these binding globulins, and a smaller fraction of 3 and T T 4 is bound to protein in rats and mice. Unbound or free T 3 and T 4 is available for metabolism, resulting in faster hormone turnover in rodents than in humans, with plasma half-lives of 12 to 24 hours and 5 to 9 days reported for T 4 in rats and humans, respectively (USEPA 1998). Further, TSH levels are from 6 to 60 times higher in rats than in humans (USEPA 1998). Therefore, due to the rapid hormonal turnover, the rat thyroid gland must work harder and is continuously stimulated in order to maintain T 3

and T 4 within physiological levels. Consequently, the rat thyroid gland would be more
susceptible to chemical perturbation of thyroid hormone homeostasis than the human thyroid gland (USEPA 1998). A number of compounds have been shown to affect thyroid hormone levels, including the PPARa-agonist, clofibrate, without an impact on normal thyroid function in humans (Visser et al. 1993).

3.0 3.1

POTENTIAL EFFECTS ON REPRODUCTION AND DEVELOPMENT Recent Studies in Mammalian Systems Recent studies have been conducted to evaluate the potential for triclosan to

impact the reproductive/developmental system. These have included studies conducted following in vivo (Kumar et al. 2009; Zorrilla et al. 2009) or in vitro (Kumar et al. 2008; James et al. 2009; Gee et al. 2008; Chen et al. 2007; Ahn et al. 2008) administration of triclosan. As noted above for the changes in serum thyroid hormone concentrations, changes in levels of circulating hormones associated with male or female reproductive systems in rats serve as a screen for the potential of a xenobiotic to have an adverse impact on reproductive/developmental competency in humans. Such changes in these

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screening assays should be viewed in parallel with in vivo tests for reproductive/developmental effects, when conducted as they have for triclosan, to determine if changes in hormone levels measured in these screening studies were sufficient to affect function.

3.1.1

Review ofstudies conducted in vivo Two studies have been conducted in the same strain of male rats (Wistar) in

which many of the same parameters were evaluated with conflicting results (Kumar et al. 2009; Zorrilla et a!. 2009). These studies are discussed in this section. Kumar et al. (2009) investigated potential antiandrogenic effects in male Wistar rats administered triclosan in phosphate buffered saline via gavage once a day for 60 days at concentrations of 0, 5, 10, or 20 mg/kg/day (n=8 per group). Treatment was initiated when the animals were 10 weeks old, with sacrifice 5 occurring 24 hours following the final treatment. As described below a number of endpoints were evaluated; however, for the majority of these analyses only the control and high dose group were tested and for a number of those, testes from the control or the high dose group were homogenized and the pooled samples tested. Kumar et al. (2009) reported a significant decrease in the weight of testes and all sex accessory tissues evaluated following administration of 10 or 20 mg/kg/day but not in the 5 mg/kg/day, compared to controls. The authors reported significant decreases in the enzymatic activity of both 3 j3-hydroxysteroid dehydrogenase (3 13-HSD) and I 7j3hydroxysteroid dehydrogenase (1 7f3-HSD) in the two highest dose groups with decreases of 27% and 39% in 313-HSD in the 10 and 20 mg/kg/day dose groups, respectively, and decreases of 31% and 46% in 1 7J3-HSD, respectively. Significant decreases in the following endpoints were reported in the 20 mg/kg/day dose group (the only treatment group evaluated): serum levels of leutinizing hormone (LH) (38.5%), follicle stimulating hormone (FSH) (17%), pregnenolone (31%), testosterone (41%), and cholesterol (35%). Decreases were also seen in: daily sperm production (DSP) (34%); mRNA for all genes

The methods section reports that rats were sacrificed by cervical dislocation under ether anesthesia. However, the hormone serum concentrations were said to be determined from blood collected from decapitated animals by cardiac puncture. Typically, truck blood is collected from decapitated animals and cardiac puncture if performed on animals euthanized.

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evaluated (P45Oscc. 313-HSD, l7f3-HSD. and P450c , androgen receptor (AR) and 17 steroidogenic acute regulatory (StAR); levels of protein for AR and StAR protein (all of which were from pooled samples). Histopathology examinations were conducted for the control and 20 mg/kg/day dose group in the Kumar et al. (2009) study. Reduced sperm density was noted in the lumina of the epididymal tubule as well as malformations of the vas deferens (lumen showed presence of sterocilia detached from the epithelium and presence of eosinophillic bodies) and degenerated and empty folliculli in prostate tissues. No noticeable histopathological changes were reported in the seminal vesicles of the 20 mg/kg/day treated rats. Several questions arose in the review of the Kumar et al. (2009) study that, when considered with all of the other experimental evidence, cast considerable uncertainty as to the interpretation of these results. The authors noted that the reason for reporting only 20 mg/kg/day group for many parameters was because of similar responses in 10 mg/kg group; however, as noted above, the method section clearly stated that for all but two of the analyses only the high dose group was evaluated. The methods section describes five different analyses in which the testes were either fixed in paraffin sections (analysis of StAR protein) or were homogenized and pooled (niRNA to evaluate gene expression using RT-PCR, levels of AR and StAR protein using Western blot analysis). It is not entirely clear from the protocol description if each whole testes was homogenized with a sample drawn from each for an n=8, as is may be the case for the analysis of DSP, or as is more likely the case for other assays, the testes of all 8 animals were homogenized for an ntl and then replicates of that pooled sample were evaluated. In the latter case (n1 with 3 or 4 replicates), the statistical methods applied, i.e., Anova and students t-test, may be inappropriate because what is being assessed is the variability in the test method not the variability among test subjects. Another concern is because of the various techniques required for these analyses, i.e., each homogenization procedure was different requiring different reagents and protocols, obviously with two testes per animal, two different procedures could have been performed. However, with five separate analyses, as noted above, it is questionable that

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one testes from each of the eight males in the control or high dose groups could have been evaluated in the same assay. Consequently, considerable uncertainty remains. Another question deals with pre-and post-treatment body weights in these animals. The final body weights of the male Wistar rats in the control and treated groups appeared to be much lower than the average body weight of this strain of rat. The average body weight of a male Wistar rat at 10 weeks of age is approximately 373 grams (Derelanko and Hollinger 2002), which is greater than the average body weight of the control animals (168 6.3 g) reported by Kumar et al. (2009). At 18 to 19 weeks of age, male Wistar rats should weigh approximately 500 grams (Derelanko and Hollinger 2002); however, the body weights of the control animals in the Kumar et al. (2009) study averaged approximately 187 grams. While it is unclear how these differences may have impacted the results, it does raise questions regarding the age of the animals tested and the methods and results provided by Kumar et al. (2009). Very different results were reported by Zorrilla et al. (2009) who investigated many of the same endpoints. Zorrilla et al. (2009) conducted a study in male Wistar rats to determine the potential effects of triclosan on pubertal development and on indices of reproductive competence. Animals were administered triclosan daily via oral gavage in corn oil at doses of 0, 3, 30, or 300 mg/kg/day (Block 1, n10) or 0, 100 or 200 mg/kg/day (Block 2, n=1 5 for controls and n8 for treated groups) . Dosing began on 6 PND 23 and continued to PND 53, when necropsies were performed. Triclosan did not affect normal growth; body weights were approximately 300 g at necropsy. Administration of triclosan did not affect the age of preputial separation at any dose tested. Nor did triclosan affect the growth of reproductive tract organs; weights of the testes, ventral prostate, seminal vesicle, epididymides. and levator ani bulbocavernosus muscle in the treated groups were comparable to controls. No consistent, dose-related change in testosterone was measured. While a significant decrease in serum testosterone concentration was observed in the 200 mg/kg/day group compared to the combined controls, testosterone levels were not decreased in the 300 mg/kg/day group. Nor were there significant dose-related decreases
When the values in the control groups for an endpoint under consideration were not statistically significantly different, the controls were combined and results in the all treated groups were compared to the mean of the control values.
6

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in serum androstenedione, a testosterone precursor, or in serum or pituitary LH or prolactin (PRL) in treated groups compared to controls, indicating that the hormonal triggers to the production of testosterone by Leydig cells were not affected. Histological examination did not reveal any significant treatment-related lesions or alteration in either the testes or the epididymides. The authors reported that a few animals in the high dose group had multinucleated giant cells within the seminiferous tubule epithelium; however, these changes were characterized as minimal and did not correlate with testosterone levels or testes weight in the individual animals. Both Kumar et a!. (2009) and Zorrilla et al. (2009) evaluated changes in LH and testosterone concentrations, both indicators of a potential impact on steroidogenesis. Kumar et al. (2009) reported significant decreases in LH, testosterone, and testes weight following administration of 10 and 20 mg/kg; however, these results are in contrast to those reported by Zorrilla et al. (2009). No significant changes in testes weight or LH (serum or pituitary) were reported by Zorrilla et al. (2009) at any dose tested (up to 300 mg/kg/day). While a significant decrease in serum testosterone concentrations was observed by Zorrilla et al. (2009) in the 200 mg/kg/day dose group, no significant change was observed in the high dose group (300 mg/kg/day) and the changes observed in all dose-groups were not dose-related. In addition, Zorrilla et al. (2009) did not see any histopathological changes in the reproductive tissues examined. More importantly, Zorrilla et al. (2009) reported no change in the age of onset of preputial separation, an indicator of the onset of puberty, which would have been expected if the changes in serum hormones, i.e., testosterone were impacted. In comparison, the range of doses administered was different, with a much broader range of doses administered by Zorrilla et al. (2009) (3 to 300 mg/kg/day) compared to Kumar et al. (2009) (5 to 20 mg/kg/day). The life stage or window of exposure was also different. Zorrilla et al. (2009) administered triclosan to weanling rats during the period of pubertal development (PND 23 to 53), while Kumar et al. (2009) administered triclosan to adult rats starting at 10 weeks of age. The duration of exposure was different with a 31-day exposure in the Zorrilla et al. (2009) study and a 60-day exposure in the Kumar et al. (2009) study. However, these differences are unlikely to explain the considerable differences in response between the two studies.

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The comparison of these results calls into question those results reported by Kumar et al. (2009), especially since the animals in the Zorrilla et al. (2009) study were administered triclosan during the period of development of sexual maturity, typically considered a sensitive window of exposure. The increased duration of exposure would not be expected to result in such a difference in the dose-response in these studies. Because of the rapid metabolism and elimination of triclosan with little potential for bioaccumulation, the longer duration of exposure should not have been a factor, as noted above for evaluation of thyroid hormone levels (see Crofton et al. 2007 and Paul et al. 2009) Kumar et al. (2009) concluded that the effects observed in their study indicated that triclosan may pose a hazard to human health by acting through various mechanisms that result in endocrine disruption. As this relates to the reproductive/developmental system, no evidence of an impact on this organ system in animals has been demonstrated after chronic exposure in rats (Yau and Green 1986), in a two-generation reproductive study in rats (Morseth 1988), or in developmental studies in rats (Schroeder and Daly 1 992a; Denning 1992) or in mice (Christian and Hoberman 1992), hamsters (Piekacz 1978) or rabbits (Schroeder and Daly 1992b).
3.1.2

Review of in vitro studies

In vitro studies have been conducted with triclosan in primary Leydig cells (Kumar et al. 2008), in cultured sheep placental tissues (James et al. 2009), and immortalized non-target cells (Chen et al. 2007) or cancer cell lines (Ahn et al. 2009; Gee et al. 2008). In vitro tests are useful for screening purposes and for forming hypotheses on the mode(s) of action of a chemical that can then be tested in more robust in vivo systems. Interpretation of these results and extrapolation of these results to be predictive of in vivo responses should be done with care and without extending this interpretation beyond what the data are capable of providing. In particular, the results in immortalized cancer cell lines are of uncertain relevance because of the potentially altered genomic responsiveness to xenobiotics (Gentry et al. 2009). These in vitro data are presented for completeness but must be considered in concert with the short-term in vivo studies assessing the same endpoints, as discussed in Section 3.1.1, and, more importantly, the

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results of long-term experimental studies, as discussed in Section 3.2. Further, only those aspects of studies that evaluated the potential of triclosan to affect endocrine but not nonendocrine signaling systems are discussed in this section.

3.1.2.1 Primary Cell Lines Kumar et al. (2008) conducted an in vitro study in Leydig cells from male Wistar rats in order to assess potential antiandrogenic effects of triclosan. The isolated cells were incubated with 0. 0.001, 0.01, 0.1, 1, or 10 tM triclosan for 2 hours in the presence or absence of LH (100 ng!ml). Changes in testosterone biosynthesis, cell viability, adenylyl cyclase activity, cAMP production, P450c , 3f3-HSD, 1713-HSD and StAR gene 17 expression, and activity of three enzymes (P45Oc , 3-HSD, and 17f3-HSD) involved in 17 testosterone biosynthesis were measured. No significant reduction in cell viability, cellular proliferation (an indicator of cytotoxicity) or alterations in cellular morphology were noted at any concentration tested other than the highest tested (10 tiM). A concentration-related significant decrease in LH-induced production of testosterone by Leydig cells was measured at concentrations tested from 0.01 to 10 jiM. In the absence of LH, no impact on testosterone by triclosan administration (1 jiM), compared to untreated controls, was reported. The authors also reported a significant decreased expression and activity of P450i , 3f3-HSD, 17J3-HSD 7 and expression of StAR protein at concentrations of 0.01 jiM and greater, in the presence of LH stimulation. These data suggested a decreased delivery of intermediates in the biosynthesis of testosterone contributing to the decrease in testosterone synthesis noted. In a separate experiment, cells from both triclosan-treated and vehicle controls were treated with forskolin (an adenylyl cyclase activator) or SQ22536 (an adenylyl cyclase inhibitor), alone or in combination with triclosan (0.00 1 to 10 jiM) for 2 hours. Kumar et al. (2008) reported that triclosan produced a dose-dependent decrease in the activity of adenylyl cyclase and cAMP at concentrations from 0.01 jiM to I jiM (no further decrease in activity was noted at the highest triclosan concentration). Administration of forskolin increased testosterone production, which was returned to control values (LH-treated only) with 1 jiM of triclosan; however, triclosan did not further decrease testosterone levels produced by SQ22536. The authors suggested that the mode

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of action for triclosans antiandrogenic activity was a direct effect on adenylyl cyclase. Given that adenylyl cyclase and cAMP are secondary messengers in many, if not all, cells, a direct effect on adenylyl cyclase levels or ability to activate cAMP is unlikely. Understanding homeostatic control by way of a specific feedback loop 7 is critical to interpreting the results provided by Kumar et al. (2008). The in vitro system employed by Kumar et al. (2008) lacked the homeostatic mechanisms that would be active in vivo to regulate LH levels in response to decreasing testosterone concentrations. While doserelated decreases in testosterone were reported by Kumar et al. (2008) with increasing concentrations of triclosan starting at micromolar concentrations (0.01 jiM), similar results were not observed in vivo following administration of concentrations in the mg/kg range. No effects on testosterone levels were reported by Kumar et al. (2009) following administration of 5 mg/kg triclosan via oral gavage for 60 days or by Zorrilla et al. (2009) following administration of doses as high as 300 mg/kg for 32 days in rats undergoing pubertal development. This indicates that the concentrations of triclosan employed in the in vitro assay would not be sufficient to overwhelm compensatory mechanisms in vivo.

3.1.2.2 Tissue Culture

In a study conducted by James et al. (2009), an in vitro sheep placental model was used to evaluate the potential for triclosan to inhibit the sulfonation of estradiol and estrone by placental sulfotransferases in the SULT family of enzymes, and to examine the possibility that triclosan could be detoxified by glucuronidation or sulfonation in the placenta. The placental tissue used in the assay was harvested from fetal sheep at 126 to
Steroidogenesis, specifically testosterone production in the Leydig cell, is primarily dependent on LH secreted from the anterior pituitary (Clark and Cochrum 2007). LH stimulates the production of Leydig cell testosterone via activation of cAMP-dependent protein kinase A (PKA) resulting in two distinct responses: 1) immediate cholesterol delivery to the mitochondria and 2) activation of genes encoding the steroid hydroxylase enzymes of the biosynthesis pathway (Figure 1). Once produced, testosterone then feeds back to the hypothalamus and pituitary to suppress transiently increased LH and thus testosterone production (Zirkin and Chen 2000). If testosterone levels are reduced, LH, as well as Gonadotrophin releasing hormone (GnRH), is produced to stimulate testosterone production (Zirkin and Chen 2000). This feedback mechanism controls homeostasis allowing the body to compensate for variability in Leydig cell testosterone concentrations. The feedback loop is assumed to function under nomial physiological conditions; however, when levels of cholesterol are significantly reduced beyond normal physiological levels, then even in the presence of adequate LH, key enzymes in the testosterone biosynthetic pathway will be down-regulated and testosterone biosynthesis will be reduced.

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130 days of gestation (3 sheep), with term being approximately 147 days. Placental tissue was homogenized and cytosolic (sulfonation) and microsomal (glucuronidation) fractions isolated and incubated with triclosan at final concentrations of 0.1 to 10 tM for approximately 15 minutes. Triclosan was a substrate for sulfonation with an apparent Km of 1.14 and apparent Vmax of 160 pmol/minlmg protein. There was no detectable glucuronidation in sheep placental microsomes. Sheep placental sulfotransferases formed only estradiol-3-sulfate and not 1 7J3estradiol-sulfate suggesting that the sheep placenta contained SUET I, a homolog of

human SULT1EI. Addition of triclosan at concentrations ranging from 0 to 2.5 nM


altered the kinetic constants for estrogen sulfotransferase resulting in a dose-dependent increase in apparent Km for estradiol (1 nM) and a decrease in the apparent Vm. Triclosan also inhibited the sulfonation of estrone (2 nM) in a dose-dependent manner. Inhibition was considered to be primarily competitive, with triclosan competing and blocking the binding of estradiol and estrone to the active binding site on the enzyme. The authors suggested that changes in extent of sulfonation of estrogen or estrone, which are the predominant forms delivered to the fetus where the active steroid is released, could result in an impact on successfully maintaining pregnancy. This outcome is highly unlikely at environmentally relevant exposure to triclosan for a number of reasons: 1) the role of placental estrogen sulfotransferases in the maintenance of pregnancy in humans is not known with certainty (James et al. 2009); 2) competitive inhibition does not mean a total cessation of the production of estrogen or estrone sulfate conjugates and it is not known what level of inhibition would be outside of the homeostatic range or the ability to deliver enough of the conjugate to maintain a normal pregnancy, if such were necessary; 3) this in vitro analysis tested a specific estrogen to triclosan ratio and it is unclear what that ratio would be with normal physiologically levels of estrogen compared to concentrations of triclosan found in human populations (the higher the estrogen to triclosan ratio, the less the expected competitive inhibition); 4) at least in the humans, redundant sulfotransferases are operative, e.g., SULT1A1 and SULT2A1 (Stanley et al. 2001); and 5) because detoxification by glucuronidation and sulfonation, primarily in the liver, are the major metabolic pathways in all species, in particular in the human liver (Wang et al. 2004), the

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systemic bioavailability of free triclosan to interfere with placental estrogen metabolism at environmentally relevant triclosan exposures such that an impact on the fetus is unlikely. Most importantly, if triclosan were actively interfering with estrogen transport into the fetus, then administration of triclosan in vivo should result in an impact on successful pregnancies; however, as discussed in the following section. there is no evidence of an impact on reproductive performance or in the ability of rats to carry fetuses to term (Morseth 1988). While the observation that triclosan can be a competitive substrate for sulfotransferase enzymes is not surprising given that sulfate and glucuronide conjugates are the major detoxifying metabolites in all species (Wang et al. 2004), a significant impact on reproductive/developmental function at triclosan concentrations detected in human populations is unlikely.

3.1.2.3 Immortalized or Cancer Cell Lines Chen et al. (2007) have developed a protocol that is designed to maximize any potential effect of an antagonist, even if in an in vivo system the compound would be a weak antagonist at the anticipated concentrations. In this in vitro study with human embryonic kidney (HEK) 293 cells, a stably transfected cell line that lacks critical steroid metabolizing enzymes, the androgenic/antiandrogenic activity of triclosan was evaluated in a cell-based human AR-mediated bioassay. The cells were treated with 1 or 10 .tM of triclosan alone, testosterone alone (0.125 nM), or a combination of testosterone and triclosan at these designated concentrations. No cytotoxicity was reported when triclosan was tested alone at 10 iM or in combination with 0.125 nM testosterone. No statistically significant differences in cell proliferation were noted. Triclosan at a concentration of 10 jiM inhibited transcriptional activity of testosterone by more than 92%, and by 38.8% at a concentration of 1.0 pM. However, details of this assay were not provided. Androgenic activity was not exhibited at concentrations up to 10 pM in the absence of testosterone. A number of aspects of the Chen et al. (2007) study limit its usefulness to be predictive of triclosan effects in humans at environmentally relevant exposures. The cells employed in this bioassay lacked critical steroid metabolizing enzymes and were transfected with PCDNA6-hAR and an MMTV-Luc.neo plasmid containing a luciferase

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reporting gene. The resulting cells are reported to be highly responsive to endogenous steroids and synthetic compounds. The authors stated that the concentration of testosterone used in the assay was relatively low (0.125 nM) compared to the levels of testosterone that the authors reported as mean circulating concentration in humans (3.5 to 35 nM) (Chen et al. 2007). The authors noted that because only approximately 2 to 3% of circulating testosterone is free and considered bioactive this low testosterone concentration was assumed to be biologically relevant; however, in the whole animal free and bound testosterone are in dynamic equilibrium such that depletion of free testosterone can be compensated by the release of bound testosterone to maintain homeostasis. The authors noted that at this testosterone concentration, it is likely that antagonist activity would be enhanced, which is an attribute useful for screening assays but of uncertain quantitative relevance to normal in vivo behavior. The authors stated that it was clear that the relative binding efficiency of triclosan for ARs was orders of magnitude below that of the natural ligand (testosterone). Therefore, the responses of this isolated cell system are likely to be different than that of cells in normal physiological situation. In a study by Ahn et al. (2008), an in vitro assay was conducted with recombinant cells that contained stably transfected estrogen (ER) receptors to evaluate the potential biological activity of triclosan. In the cell-based ER-mediated assay (conducted in recombinant human ovarian cancer cells containing a stably integrated ER-a-responsive firefly luciferase reporter plasmid), cells were incubated with one of three concentrations of triclosan (10 nM, 1 or 100 .tM) with or without I tiM estradiol for 24 hours. Triclosan exhibited a concentration-dependent decrease of E2-dependent reporter gene expression in the presence of estradiol, with 50% inhibition observed at a concentration of luM (20% at 10 nM and 80% at 100 .iM). When tested alone, triclosan exhibited no estrogenic activity at any concentration in the absence of estradiol. Gee et al. (2008) conducted an in vitro assay with MCF7 McGrath human breast cancer cells to evaluate the ability of triclosan to bind to estrogen (ERa, ERJ3) receptors or in the androgen-responsive LTR-CAT reporter gene in Si 15 mouse mammary tumor cells in competitive binding assays. Assays were conducted to evaluate the ability of triclosan to bind to estrogen-responsive or androgen-responsive reporter genes and for

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triclosan s ability to increase the growth of estrogen- or androgen-responsive immortalized breast cancer cells. In these assays, either estradiol or testosterone was added to the cells and the amount of triclosan, expressed as molar equivalents that could effectively compete for the specific binding sites was assessed. Triclosan was added to these cultured cells at levels that exceeded endogenous estradiol or testosterone levels, on a molar basis, ranging up to 10,000.000 times the concentration of the normal substrate. Triclosan was able to inhibit estradiol binding to the ER in the cytosol of MCF7 cells. MCF7 cells were incubated with 0.4 nM estradiol and the extent of inhibition of binding was determined using increasing concentrations of triclosan. Binding of estradiol to the ER receptor could be inhibited by triclosan; however, triclosan concentrations required to do so were expressed as a molar excess of 100,000- to 10,000,000- fold the concentration of triclosan to estradiol that resulted in an approximate 25% and 72% inhibition, respectively. When evaluating recombinant human ERa and ERI3, rather than cytosolic ER, 63% inhibition was achieved at 100,000 molar excess of triclosan. Comparing the ability of triclosan to inhibit recombinant ERa and ERf3, at 10,000-fold and 20,000-fold molar excesses, greater inhibition was observed with ERa (22 and 34%, respectively) than ER3 (0 and 12%, respectively). Triclosan was also able to inhibit the stimulation of the estrogen-responsive ERE-CAT reporter gene in these cells, but only at 100,000 fold molar excess concentrations of triclosan, compared to estradiol. Separate assays to evaluate triclosan s ability to bind to the AR receptor and regulate androgen-responsive reporter genes were conducted with recombinant ligand binding protein of rat AR and stably transfected androgen-sensitive reporter genes (LTR CAT) in Si 15 +A mouse mammary tumor cells, respectively (Gee et al. 2008). In the competitive binding assay with the recombinant rat AR protein, triclosan inhibited testosterone binding by an average of 49% at 1000-fold molar excess and 77% at 10,000fold molar excess. In mouse mammary tumor cells, triclosan was able to inhibit the induction of the androgen-responsive LTR-CAT reporter gene, but only at concentrations that were 100-fold molar excess of testosterone. While these in vitro studies suggest potential activity of triclosan at certain receptor sites, it must be considered that the cells used in some of these experiments were conducted in inunortalized or cancer cell lines that are not expected to respond in a

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similar manner to normal or primary cells (Gentry et al. 2009). In addition, cell lines, such as those derived from the kidney, are not the target organ of concern and may not behave in a manner similar to cells from the appropriate organ. The concentrations of triclosan and endogenous steroids used in some of these assays (i.e., Chen et al. 2007) were selected by the authors to achieve a maximum response and are not similar to those expected in vivo. Therefore, these in vifro results must be interpreted with caution when extending them to the whole animal. It should be noted that no effects on reproduction or development were reported in the battery of tests for triclosan conducted in vivo in rats at doses of triclosan ranging from 15 to 150 mg/kg in a two-generation reproductive study (Morseth 1988) or at doses of 15 to 300 mg/kg/day in one-generation developmental studies (Schroeder and Daly 1992a, Denning 1992). In addition, no developmental effects have been noted in mice administered doses up to 25 mg/kg/day (Christian and Hoberman 1992), or in rabbits administered doses up to 50 mg/kg/day (Schroeder and Daly 1 992b) or in hamsters administered doses up to 80 mg/kg/day (Piekacz 1978).
3.2 Comparison of New Studies to Other Experimental Data

Recent in vivo studies (Kumar et al. 2009; Zorrilla et al. 2009) conducted in male Wistar rats at different life stages (adult versus prepubertal) focused on the potential for triclosan to impact endocrine function related to reproductive competence. The results of these studies differed in their conclusions. Further, a comparison of these results with the results from a battery of studies conducted in experimental animals to determine the potential safety of triclosan is provided to put these recent studies in perspective. Kumar et al. (2009) suggested that the combined results from their studies in Wistar rats indicated that exposure to triclosan could result in an impact on several key steps involved in the biosynthesis of testosterone, which in turn could have an impact on reproductive function or development of offspring. However, the results reported by Kumar et al. (2009) were not duplicated in developing male rats of the same strain, as reported by Zorrilla et al. (2009). Even if the adult Wistar rat were more sensitive to the changes reported by Kumar et al. (2009), Zorrilla et al. (2009) administered doses approximately 15-fold greater (300 mg/kg/day) than those administered by Kumar et al.

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(2009) and still reported no significant change in critical parameters, including testosterone and LH concentrations, following triclosan administration. According to Kumar et al. (2009), signs of potential antiandrogenic effects, e.g., decreases in testosterone, in male rats occurred at low doses; however, the overall toxicity database for triclosan comprised of reproductive/developmental studies and chronic studies do not support effects on reproduction or development in this low-dose region. In a two-generation reproductive study conducted by Morseth (1988) in CRL:CD(SD)Br rats, no significant treatment-related effects were observed in animals administered triclosan in the diet at doses ranging from 15 to 150 mg/kg/day from 10 weeks of age prior to mating with exposure continued throughout mating and then gestation and lactation for females. No impact on reproductive performance in either males or females was reported nor was there any evidence of toxicity during the growth phase of either generation. The Kumar et al. (2009) results are inconsistent with Zorrilla et al. (2009), as well as the results from a classic two-generation reproductive study, both of which administered much higher doses to the animals than those administered by Kumar et al. (2009). It is important to note that the majority of the parameters evaluated by Kumar et al. (2009) are expected to have some biological variability because of normal compensatory mechanisms that maintain many physiological and biochemical parameters within normal ranges and without impairment of function. Comparison of the Kumar et al. (2009) results with those from the reproductive, developmental, subchronic and chronic triclosan studies conducted in rats, as well as other species, suggests that if triclosan was having an impact on the parameters measured by Kumar et al. (2009) in this low dose region, these changes were not extensive enough to result in any impairment in reproductive function or development following chronic exposure or exposure during a critical windows of exposure.

3.3

Relevance to Human Health

Recent in vivo (Kumar et al. 2009; Zorrilla et a!. 2009) and in vitro (James et al. 2009; Chen et al. 2007; Gee et al. 2008; Ahn et al. 2008) studies have been conducted to screen for the potential for triclosan to have an impact the endocrine system. As noted,

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the Kumar et al (2009) study the authors suggested that triclosan could be an endocrine disruptor and affect reproductive systems; however, these results were not confirmed in the study conducted by Zorrilla et al. (2009). While in vitro studies have been conducted to evaluate the potential for triclosan to inhibit hormones, enzymes or receptors critical to steroidogenesis or reproduction (Kumar et al. 2008; Chen et al. 2007; Ahn et al. 2008; Gee et al. 2008), these results must be interpreted with caution. In many cases, concentrations of endogenous compounds in the in vifro system were not comparable to physiological conditions. In addition, some of the studies were conducted in immortalized or cancer cells, which do not always have the same active genes or react in a similar manner to normal cells (Gentry et al. 2009). For many of these assays, the results were meant to be used as a screen, requiring additional testing in the whole animal to confirm results, if such were not already conducted unlike triclosan. As noted above, the lack of reproductive/developmental effects reported in several triclosan studies in experimental animals do not support the potential for triclosan to result in adverse reproductive effects in rats or humans. More importantly, humans have high levels of naturally occurring estrogen and testosterone. As with thyroid hormones, humans have a critical buffering capacity to bind these hormones and act as a reserve to release free biologically active estrogen or testosterone, as well as critical feedback loops involving the pituitary-hypothalamic axis to respond to decreased or increased levels of estrogen or testosterone. Both the capacity in humans to bind and release these hormones, with a much higher capacity in humans than in rodents, and the feedback loops act to maintain homeostasis. As with the thyroid, changes in hormone status that could occur with exposure to a xenobiotic can be compensated for in humans by these active systems that maintain homeostasis, especially at environmentally relevant exposures to triclosan.

4.0

SUMMARY AND CONCLUSIONS


Crofton Ct al. (2007) and Zorrilla et al. (2009) reported dose-dependent decreases

in serum T 4 levels in rats administered triclosan in the diet for 4 days at doses of 100, 300. or 1000 mg/kg/day (Crofton et al. 2007) or after 31 days of treatment at doses of 30

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to 300 mg/kg/day (Zorrilla et al. 2009) when administered to young weanling rats. No consistent changes in T 3 levels were seen and TSH levels were unchanged after 31 days of treatment (Zorrilla et al. 2009). Zorrilla et al. (2009) reported that there were no treatment related changes in the follicular epithelial height in any dose group following exposure to triclosan but decreases in colloid area (colloid depletion) were seen in thyroid gland sections. but only in the high dose group (300 mg/kg/day). DeVito et al. (1999) stated that changes in histology, in particular changes in follicular structures were less impacted by confounding than serum thyroid hormone levels and perhaps a better indicator of alterations in potential thyroid function. Morphological changes in colloid area in the absence of follicular cell changes are of uncertain relevance. In contrast to the animal studies, Ailmyr et al. (2009) did not find significant changes in thyroid hormones in the plasma of volunteers after using triclosan-containing toothpaste. As noted above, no clinical signs of toxicity or changes in various clinical or biochemical parameters were seen in human volunteers following the use of triclosan containing products. The weight-of-the evidence of all of the triclosan data is that in the rat model, triclosan can affect circulating levels of total T 4 at high doses. While T 4 changes have been seen in rats, signs of either hypothyroidism or other effects on the thyroid, e.g., changes in thyroid weight, or signs of hypertrophy or hyperplasia, have not been seen in several species administered triclosan for subchronic and chronic durations at doses comparable to those administered in the short-term studies reported by Crofton et al. (2007) and Zorrilla et al. (2009). A number of compounds including the PPARct-agonist, clofibrate, a cholesterol-lowering drug, affect thyroid function in rodents without a similar impact in humans. Moreover, humans have both sensitive feedback mechanisms and extensive binding capacity, both of which maintain thyroid hormone levels with a homeostatic range. Because of the robust nature of the human thyroid hormone system, that is, resistance to changes in thyroid hormone levels, humans are expected to be less sensitive to these changes than rodents. This conclusion is reinforced by the results of Alimyr et al. (2009) where no changes in thyroid hormone levels were seen in humans receiving doses of triclosan that are in the relevant range encountered from the use of consumer products.

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As discussed above. Kumar et al. (2009) and Zorrilla et al. (2009) investigated the effects of triclosan on the levels of testosterone, LH and associated critical enzymes in the production of testosterone in rats. Due to methodological questions in the Kumar et al. (2009) study, the results of Zorrilla et al. (2009) are considered more reliable to discern the potential effects of triclosan on these biochemical indices of the potential for reproductive impacts. The results of Zorrilla et al. (2009) are in agreement with the experimental data for reproductive effects reported by Morseth (1988). As discussed in the Zorrilla et al. (2009) study, triclosan did not affect critical parameters, e.g., testes weight, testosterone and LH production or normal development of puberty at concentrations up to the highest dose tested, 300 mg/kg/day, which is higher than the highest dose tested in the 2-generation reproduction study conducted by Morseth (1988). While the in vitro studies provide insights into the potential ways in which triclosan could affect various enzymes involved in testosterone biosynthesis, these studies are only useful as screens to generate hypotheses for such effects not as definitive evidence of endocrine disruption leading to reproductive toxicity. In light of the classic negative reproductive/developmental toxicity tests, the changes noted in these in vitro tests are of interest and may explain changes at high triclosan concentrations when normal physiological processes, e.g., feed-back loops, are not operative but are not relevant to or predictive of human health outcomes at environmentally relevant exposures. It is important that these results be put in perspective, not only as they compare to the results from a battery of classic reproductive/developmental studies that have been conducted in experimental animals, but also as these exposures compare to those anticipated for users of consumer products containing triclosan. A large biomonitoring study, which includes triclosan, has been conducted by the CDC as part of the NHANES survey, which has been conducted annually since 1976. The NHANES study was designed to measure the health and nutritional status in the general US population (Calafat Ct al. 2008). Recently, the survey has included data from urine and plasma samples to assess exposure to environmental chemicals. In the 2003 -2004 NHANES survey (CDC 2005), which included examinations of 9.643 subjects, urine specimens were collected from a random one-third of the subjects
(n=2,5 17 subjects) and analyzed for triclosan (Calafat et al. 2008). A single spot urine

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sample was collected from each subject during one of three daily examination sessions, either morning, afternoon or evening. The samples were analyzed for concentrations of free plus conjugated triclosan (total triclosan) in the urine and adjusted for volume or total creatinine. These data provided actual measurements of triclosan from a large

number of subjects of various ages that potentially used multiple triclosan-containing


products. Because of the pharmacokinetic properties of triclosan in the human (i.e., approximately 90% urinary excretion of parent and metabolites and no bioaccumulation), the urinary biomonitoring data provided by the NHANES survey (CDC 2005; Calafat et al. 2008) can be used to provide estimates of the absorbed daily intake of triclosan resulting from exposure to triclosan, irrespective of source. A similar approach has been used to estimate pesticide exposure (Mage Ct al. 2004). If it is assumed that the individuals have a relatively constant use of triclosan products resulting in a constant daily intake, the samples taken as part of the NHANES survey would represent a steady state concentration of triclosan in the urine. These urinary concentrations can be adjusted by creatinine and body weight of the subject to estimate a daily intake in units of mg/kg/day. The resulting daily triclosan intake estimates based on the 50th percentile, assumed to represent the average user, urinary concentrations of triclosan reported in the NHANES 2003-2004 (CDC 2005) survey were approximately 0.0002, 0.0002 and 0.0001 mg/kg/day for men, women, and children, respectively (Rodricks et al. 2009). Based on these estimates, the highest estimated daily intake of triclosan from consumer products would be approximately 0.2 jig/kg/day, which is more than tenthousand-fold lower that the No Observed Adverse Effect Levels (NOAELs) in experimental animals based on changes in hormone levels, which was 3 mg/kg/day for the lack of changes in thyroid hormone levels or 300 mg/kg/day for the lack of effects on hormones in the reproductive system, both of which were reported by Zorrilla et al. (2009). While some changes in hormone levels associated with thyroid function or with male reproductive function in rats have been reported, it is unlikely that triclosan will have similar effects in humans because: I) humans have considerable redundancy and robust mechanisms to maintain homeostatic functioning; and 2) expected exposures to and resulting average daily intakes from the use of consumer products containing

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triclosan are orders of magnitude below the levels at which changes in hormone levels

and other indices of endocrine action have been reported in animals. Therefore, the
recent studies in rats and in vitro systems that evaluated the potential effect of triclosan on the endocrine system, specifically endocrine function as it relates to thyroid function

and reproductive function, are unlikely to be relevant to humans and so do not pose a
safety concern for triclosan use in humans.

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5.0

REFERENCES

Ahn, K., Zhao, B., Chen, J., Cherednichenko, G., Sanmarti. E., Denison, M.. Lasley, B., Pessah, I., Kultz, D., Chang, D., Gee, S., and Hammock, B. (2008). In vitro biologic activities of the antimicrobials triclocarban. its analogs, and triclosan in bioassay screens: receptor-based bioassay screens. Environmental Health Perspectives. 116(9)1203-1210. Alimyr, M., Adolfsson-Erici, M., McLachlan, M., and Sandborgh-Englund, G. (2006). Triclosan in plasma and milk from Swedish nursing mothers and their exposure via personal care products. The Science ofthe Total Environment. 372(1) 87-93. Alimyr, M., Harden, F., Toms, L., Mueller, J., McLachlan, M.. Adolfsson-Erici. M., and Sandborgh-Englund, G. (2008). The influence of age and gender on triclosan concentrations in Australian human blood serum. The Science ofthe Total Environment. 393(1) 162-167. Ailmyr, M., Panagiotidis, G., Sparve, E., Diczfalusy, U., and Sandborgh-Englund, G. (2009). Human Exposure to Triclosan via Toothpaste does not change CYP3A4 Activity or Plasma Concentrations of Thyroid Hormones. Basic and Clinical Pharmacology and Toxicology. [Epub ahead of print]. Auletta, C. (1995). An 18-month oral oncogenicity study of tricolsan in the mouse via dietary administration. Pharmaco LSR. Study No. 93-2260. Bagley, D., and Lin, Y. (2000). Clinical evidence for the lack of triclosan accumulation from daily use in dentifrices. American Jourmal ofDentistiy. 13(3) 148-152. Barnes, E. (1991 a). Mentadent P Toothpaste: Background Study in Man Haematological and Biochemical Data. Environmental Safety Laboratory Unilever Research. Study number TT88 1017. Barnes, E. (1991b). Mentadent P Toothpaste: Triclosan Toothpaste Study in Man Haematological and Biochemical Data. Environmental Safety Laboratory Unilever Research. Study number Tf880669. Calafat, A., Ye, X., Wong, L., Reidy, J., and Needham, L. (2008). Urinary concentrations of triclosan in the U.S. population: 2003-2004. Environmental Health Perspectives. 116(3) 303-3 07. Carr, B., Norcross, r., Fang, Y., Lu, P., Rodrigues. D., Shou, M., Rushmore, T., Booth Geathe, C. (2006). Characterization of Rhesus monkey CYP3A64 enzyme: Species comparisons and CYP3A specificity and kinetics using Baculovirus expressed recombinant enzymes. Drug Metabolism and Deposition. 34:1703 to 1712,

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CDC. (2005). National Health and Nutrition Examination Survey NHANES 20032004. Atlanta, GA: Centers for Disease Control and Prevention. Available at: http ://www.cdc.gov/nchs/aboutlmajor/nhanes/nhanes2003-2004/nhanesO3_04.htm

Chambers, P. (1999). Fat 80023/S Potential Tumorigenic and Chronic Toxicity Effects in Prolonged Dietary Administration to Hamsters Huntingdon Life Sciences Ldt.

Chen, J., Ahn, K., Gee, N., Gee, S.. Hammock, B., and Lasley, B. (2007). Antiandrogenic properties of parabens and other phenolic containing small molecules in personal care products. Toxicology and Applied Pharmacology. 221(3) 278-284.
Christian. M., and Hoberman, A. (1992). Developmental Toxicity (Embryo-Fetal Toxicity and Teratogenic Potential) Study of C-P Sample No. 38326 Administered Orally Via the Diet to Cr1: CD1(ICR) BR Presumed Pregnant Mice. Argus Research Laboratories. Protocol number 403-010. Sponsors Study number 92-001. Clark, B., and Cochrum, R. (2007). The steroidogenic acute regulatory protein as a target of endocrine disruption in male reproduction. Drug Metabolism Reviews. 39(2-3) 353-370. Craft, E., DeVito, M., and Crofton, K. (2002). Comparative responsiveness of hypothyroxinemia and hepatic enzyme induction in Long-Evans rats versus C57BL/6J mice exposed to TCDD-like and Phenobarbital-like polychlorinated biphenyl congeners. Toxicological Sciences. 68(2) 372-380. Crofton, K. (2004). Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Analysis. 24(6) 1665-1671. Crofton, K., Paul, K., DeVito, M., and Hedge, J. (2007). Short-term in vivo exposure to the water contaminant triclosan: Evidence for disruption of thyroxine. Environmental Toxicology and Pharmacology. 24 194-197. Dalgard, D. (1979). 90-Day Bathing of Newborn Rhesus Monkeys with Triclosan Soap Solution Ha.zelton Laboratories. April 26. de Sandro, V., Catinot, R., Kriszt, W., Cordier, A., and Richert. L. (1992). Male rat hepatic UDP-glucuronosyltransferase activity toward thyroxine. Activation and induction properties--relation with thyroxine plasma disappearance rate. Biochemical Plwrmacology. 43(7) 1563-1569. Denning, H., Sliva, S., and Wilson, G. (1992). Triclosan: Effects on Pregnancy and Post Natal Development in Rats. Environmental Safety Laboratory. Unilever Research. Document Reference 92-105.
Derelanko, M., and Hollinger, M, Eds. (2002). Handbook of Toxicology. Boca Raton,

Florida, CRC Press.

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DeSalva, S., Kong, B., and Lin, Y. (1989). Triclosan: a safety profile. American Journal ofDentistry. 2 (Spec No) 185-196. DeVito, M., Biegel, L., Brouwer, A., Brown, S., Brucker-Davis, F., Cheek, A., Christensen, R., Colborn, T., Cooke, R, Crissman, J., Crofton, K., Doerge, D., Gray, E., Hauser, P., Hurley, P., Kohn, M., Lazar, J., McMaster, S., McClain, M., McConnell, E., Meier, C., Miller, R., Tietge, J., and Tyl, R. (1999). Screening methods for thyroid hormone disruptors. Environmental Health Perspectives. 107(5)407-415. Diczfalusy, U., Olofsson, K., Carisson, A., Gong, M., Golenbock, D., Rooyackers, 0., Flaring, U., and Bjorkbacka, H. (2009). Marked up-regulation of cholesterol 25hydroxylase expression by lipopolysaccharide. Journal ofLipid Research. [Epub ahead of print]. Drake, J. (1975). 1 Year Oral Toxicity Study in Baboons with Compound Fat 80 023/A. Ciba-Geigy Limited. July 26. Eldridge, S. (1993). Cell proliferation in rodent liver. Final Report. Pathology Associates, Inc. January 13. Gee, R., Charles, A., Taylor, N., and Darbre, P. (2008). Oestrogenic and androgenic activity of triclosan in breast cancer cells. The Journal ofApplied Toxicology. 28(1) 78-91. Gentry, P., McDonald, T., Sullivan, D., Shipp, A., Yager, J., and Clewell, H, 3rd. (2009). Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicity. Environmental and Molecular Mutagenesis. [Epub ahead of print]. Gilbert, M., Taylor, E., and Crofton, K. (2002). Developmental exposure to polybrominated diphenyl ethers does not alter synaptic transmission or LTP in hippocampus. The Toxicologist. 66(1-S) No. 644. Goldsmith, L., and Craig, D. (1983). 90-Day Oral Toxicity Study in Rats with Fat 80023/H. Final Report. Litton Bionetics. LBI Project Number 22188. October. Hood, A., and Klaassen, C. (2000). Differential effects of microsomal enzyme inducers on in vitro thyroxine (T4) and triiodothyronine (T3) glucuronidation. Toxicological Sciences. 55(1) 78-84. Jacobs, M., Nolan, G., and Hood, 5. (2005). Lignans, bacteriocides and organochiorine compounds activate the human pregnane X receptor (PXR). Toxicology and Applied Pharamcology. 209(2) 123-133.

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James, M., Li, W., Summerlot, D., Rowland-Faux, L., and Wood, C. (2009). Triclosan is a potent inhibitor of estradiol and estrone sulfonation in sheep placenta. Environment International. [Epub ahead of print]. Kanebratt, K., Diczfalusy, U., Backstrom. T., Sparve, E., Bredberg, E.. Bottiger, Y., Andersson, T., and Bertilsson, L. (2008). Cytoebrome P450 induction by rifampicin in healthy subjects: determination using the Karolinska cocktail and the endogenous CYP3A4 marker 4beta-hydroxycholesterol. Clinical Pharmacology and Therapeutics. 84(5) 589-594. Khan, M.. Davis, C., Foley, G., Friedman. M., and Hansen, L. (1999). Changes in thyroid gland morphology after acute acrylamide exposure. Toxicological Sciences. 47(2) 15 1-157. Klaunig, J., Babich, M., Baetcke, K., Cook, J., Corton, J., David, R., DeLuca, J., Lai, D., McKee, R., Peters, 3., Roberts, R., and Fenner-Crisp, P. (2003). PPARalpha agonist-induced rodent tumors: modes of action and human relevance. Critical Reviews in Toxicology. 33(6) 655-780. Kumar, V., Balomajumder, C., and Roy, P. (2008). Disruption of LH-induced testosterone biosynthesis in testicular Leydig cells by triclosan: probable mechanism of action. Toxicology. 250(2-3) 124-131. Kumar, V., Chakraborty, A., Kural, M., and Roy, P. (2009). Alteration of testicular steroidogenesis and histopathology of reproductive system in male rats treated with triclosan. Reproductive Toxicology. 27(2) 177-185. Leuschner, F., Leuschner, A., Schwerdtfeger, W., and Dontenwill, W. (1970). 90 Days Oral Toxicity Study in Beagle Dogs with CH 3565. Labortorium fur Pharmakologie und Toxikologie. July 10. Lucker, P., Wetzelsberger, N., Wieckhorst, G., and Sturm, Y. (1990). Safety (tolerance) of pharmacokinetics of triclosan (TCS) an expertise. Ciba-Geigy AG, Basel, Switzerland.

Mage, D., Allen, R., Gondy, G., Smith, W., Barr, D.. and Needham, L. (2004). Estimating pesticide dose from urinary pesticide concentration data by creatinine correction in the Third National Health and Nutrition Examination Survey (NJ-lANES-Ill). Journal ofExposure Analysis and Environmental Epidemiology. 14(6) 457-465. Molitor, E., and Persohn, E. (1993). The effects of fat 80023/Q (IRGASAN DP 300) on selected biochemical and morphological liver parameters following subchronic dietary administration to male rats. Ciba-Geigy Limited. Laboratory Report No. CB 92/28.

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Molitor, E., Persohn, E., and Thomas, H. (1992). The effect of fat 80023/Q (Irgasan DP 300) on selected biochemical and morphological liver parameters following subchronic dietary administration to male and female mice. Ciba-Geigy Limited. Laboratory Report No. CB 91/18. Morreale de Escobar, G., Obregon, M.. and Escobar del Rey, F. (2000). Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? The Journal of Clinical Endocrinology and Metabolism. 85(11) 3975-3 987. Morseth, S. (1988). Two-Generation Reproduction Study in Rats Fat 80023 A. CibaGeigy Corporation. Hazieton Laboratories America, Inc. HLA Study No. 23 86100. March 18. Parkes, D. (1979). Irgasan DP 300 ninety day bathing study in newborn rhesus monkeys. Final Analytical Report. Hazelton Laboratories. June 4. Paul, K., Hedge. J., Crofton. K., and DeVito, M. (2008). Triclosan alters thyroid hormone homeostasis via up-regulation of hepatic catabolism. Toxicologist. 102(1) No. 93. Paul, K., Hedge, J., DeVito, M., and Crofton, K. (2009). Triclosan disrupts thyroxine mechanisms and life-stage susceptibility. Toxicologist. 108(1) No. 163. Persohn, E., and Molitor, E. (1993). The effect of Fat 80023/Q (Irgasan DP 300) on replicative DNA Synthesis in Hepatocytes Following Dietary Administration to Male Rats Chemicals Division, Ciba-Geigy Limited. Laboratory Report CB 92/28-2. September 17. Persohn, E. (1994). Fat 80023/, Assessment of replicative DNA synthesis in the course of a 13-week oral toxicity study in the hamster. RCC project 356490. Piekacz, H. (1978). Effects of certain preservative agents on the course of pregnancy and fetal development in experimental animals with preliminary toxicological characters. Roczn Pzh. 29(5) 469-481. Rodricks, J., Swenberg, J., Borzelleca, J., Maronpot, R., and Shipp, A. (2009). Triclosan: A critical review of the experimental data and development of margins of safety for consumer products. Critical Reviews in Toxicology. (Submitted). Safford. B. (1991). A critical assessment of the 65-week in-use human trial with toothpaste containing 0.2% triclosan. Environmental Safety Laboratory Unilever Research. Document Reference: D9l/007.

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Saghir, S., Charles, G., Bartels, M., Kan, L., Dryzga, M., Brzak, K., and Clark, A. (2008). Mechanism of trifluralin-induced thyroid tumors in rats. Toxicology Letters. 180(1) 38-45. Sandborgh-Englund. G., Adolfsson-Erici, M., Odham, G., and Ekstrand, J. (2006). Pharniacokinetics of triclosan following oral ingestion in humans. Journal of Toxicology and Environmental Health. Part A. 69(20) 1861-1873. Schroeder, R., and Daly, I. (1992a). A Segment II Teratology Study in Rats with Irgacare MP (C-P Sample No. 38328). Bio/Dynamics. Project No. 91-3665. ColgatePalmolive Study No. 9 1-005. April 16. Schroeder, R., and Daly, 1. (1992b). A Segment 11 Teratology Study in Rabbits with Irgacare MP (C-P Sample No. 38328). Bio/Dynamics. Project No. 9 1-3666. Colgate-Palmolive Study No. 91-006. April 16. Smith, G., Stubbins M., Harries, L., and Wolf, C. (1999). Molecular genetics of the human cytochrome P450 monooxygenase super family. Xenobiotica. 28(12):1 129-65. Soldin, 0., Lai, S.. Lamm, S., and Mosee, S. (2003). Lack of a relation between human neonatal thyroxine and pediatric neurobehavioral disorders. Thyroid 13(2) 193198. Stanley, E., Hume, R., Visser, T., and Coughtrie, M. (2001). Differential expression of sulfotransferase enzymes involved in thyroid hormone metabolism during human placental development. The Journal ofClinical Endocrinology and Metabolism. 86(12) 5 944-5955. Thomas, E. (1994). The effect of fat 80024/R and the model inducers phenobarbitione, 3-methyicholanthrene, pregnenolone- 1 6x-carbonitri1e and napenopin on selected biochemical and morphological liver parameters in the Syrian hamster. CibaGeigy Limited, CH-4002 Laboratory Report No. CB 93/40. Trutter, J. (1993). 13-week subchronic oral toxicity study of tricolsan in CD-i mice. Hazieton Washington, Inc. Report No. HWA 483-287. USEPA. (1998) Assessment of Thyroid Follicular Cell Tumors. US EPA, Risk Assessment Forum, Washington, DC. EPAJ63O/R-97/002, March. USEPA. (2009). http://epa.gov/scipoly/oscpendo/index.htm. Visser, T., Kaptein, E., van Toor, H., van Raaij, J., van den Berg, K., Joe, C., and Engelen, J. (1993). Glucuronidation of thyroid hormone in rat liver: effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions. Endocrinology. 133(5): 2177-2186.

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Wang, IQ., Falany CN., James MO. (2004). Triclosan as a substrate and inhibitor of 3phosphoadenosine 5 -phosphosulfate-sulfotransgerase and UDP-glucuronosyl transferase in human liver fractions. Drug Metabolism Dispo. 32:1162-1169. Wide, K., Larsson, H., Bertilsson, L., and Diczfalusy, U. (2008). Time course of the increase in 4-beta-hydroxycholesterol concentration during carbamazepine treatment of paediatric patients with epilepsy. British Journal of Clinical Pharmacology, 65(5) 708-7 15. Yau, E., and Green, J. (1986). Fat 80?023 2-Year Oral Administration to Rats (MIN 833005). Ciba-Geigy Limited. April 28. Zhou, T., Ross, D.. DeVito, M., and Crofton, K. (2001). Effects of short-term in vivo exposure to polybrominated diphenyl ethers on thyroid hormones and hepatic enzyme activities in weanling rats. Toxicological Sciences. 6 1(1) 76-82. Zirkin, B., and Chen, H. (2000). Regulation of Leydig cell steroidogenic function during aging. Biology ofReproduction. 63(4) 977-981. Zorrilla, L., Gibson, E., Jeffay, S., Crofton, K., Setzer, W., Cooper, R., and Stoker, T. (2009). The effects of triclosan on puberty and thyroid hormones in male Wistar rats. Toxicological Sciences. 107(1) 56-64.

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cholesterol 3 H CH CH

CH

STAR,TSPO,A1

CYPIIA1 DHEA 0 androstenediol CH I r-hydroxypregnenolone 3 H CHNz:zzO 0

pregnenolone 3 H CHjzzO

_cjI:rDDYP

I 7A

x6ti
Ii
I 7-hydroxyprogesterone C 3 H CHQ androstenedione 3 CH testosterone CHOH 3 5 J SD ft YPi7AI H 1 7B

CIR Panel Book Page 94

progesterone :0

Figure 1. Testosterone biosynthetic pathway. As provided in Clark and Cochrum (2007)

43

SCCP/1192/08

Scientific Committee on Consumer Products SCCP

OPINION ON

Triclosan
COLIPA n P32

The SCCP adopted this opinion at its 19th plenary of 21 January 2009

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SCCP/1192/08 Opinion on triclosan

About the Scientific Committees Three independent non-food Scientific Committees provide the Commission with the scientific advice it needs when preparing policy and proposals relating to consumer safety, public health and the environment. The Committees also draw the Commission's attention to the new or emerging problems which may pose an actual or potential threat. They are: the Scientific Committee on Consumer Products (SCCP), the Scientific Committee on Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) and are made up of external experts. In addition, the Commission relies upon the work of the European Food Safety Authority (EFSA), the European Medicines Evaluation Agency (EMEA), the European Centre for Disease prevention and Control (ECDC) and the European Chemicals Agency (ECHA). SCCP Questions concerning the safety of consumer products (non-food products intended for the consumer). In particular, the Committee addresses questions related to the safety and allergenic properties of cosmetic products and ingredients with respect to their impact on consumer health, toys, textiles, clothing, personal care products, domestic products such as detergents and consumer services such as tattooing. Scientific Committee members Claire Chambers, Gisela Degen, Ruta Dubakiene, Bozena Jazwiec-Kanyion, Vassilios Kapoulas, Jean Krutmann, Carola Lidn, Jean-Paul Marty, Thomas Platzek, Suresh Chandra Rastogi, Jean Revuz, Vera Rogiers, Tore Sanner, Gnter Speit, Jacqueline Van Engelen, Ian R. White Contact European Commission Health & Consumer Protection DG Directorate C: Public Health and Risk Assessment Unit C7 - Risk Assessment Office: B232 B-1049 Brussels Sanco-Sc6-Secretariat@ec.europa.eu

European Commission 2009 (ISSN) The opinions of the Scientific Committees present the views of the independent scientists who are members of the committees. They do not necessarily reflect the views of the European Commission. The opinions are published by the European Commission in their original language only. http://ec.europa.eu/health/ph_risk/risk_en.htm

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SCCP/1192/08 Opinion on triclosan

ACKNOWLEDGMENTS

Dr. C. Chambers Prof. G. Degen Dr. B. Jazwiec-Kanyion Prof. V. Kapoulas Prof. J.-P. Marty Prof. T. Platzek Dr. S.C. Rastogi Prof. J. Revuz Prof. V. Rogiers Prof. T. Sanner Dr. J. van Engelen Dr. I.R. White

(rapporteur)

(chairman)

Keywords:

SCCP, scientific opinion, preservative, triclosan, P32, directive 76/768/ECC, CAS 3380-34-5, EINECS 222-182-2

Opinion to be cited as: SCCP (Scientific Committee on Consumer Products), Opinion on triclosan, 21 January 2009

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SCCP/1192/08 Opinion on triclosan

TABLE OF CONTENTS

ACKNOWLEDGMENTS

...

1.

BACKGROUND

2.

TERMS OF REFERENCE

3.

OPINION

4.

CONCLUSION 123

5.

MINORITY OPINION 123

6.

REFERENCES 123

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1. BACKGROUND Triclosan (CAS 3380-34-5) with the chemical name 5-chloro-2-(2,4-dichlorophenoxy)phenol or 2,4,4'-trichloro-2'-hydroxy-diphenyl ether has a long history of use as a preservative in cosmetic products. It is currently regulated in Annex VI, entry 25 with a maximum concentration of 0.3%. An opinion on triclosan (SCCP/1040/06) was adopted by the SCCP at the 9th plenary meeting of 10 October 2006 with the following conclusions to the request: 1. "On the basis of the available data, the SCCP is of the opinion that there is presently no evidence of clinical resistance and cross-resistance occurring from the use of triclosan in cosmetic products. Information is required on consumer exposure to triclosan from all sources, including cosmetic products. 2. For a toxicological assessment of the safe use of triclosan, the SCCP requires a dossier to be submitted in which data is provided to all relevant exposure and toxicological end-points and conforming to currently accepted standards. This should be regarded as a matter of urgency because triclosan has been identified in human milk of some European populations." The dossier provided by Industry consists of an update on the bacterial resistance issue (submission III) and of a toxicological dossier for triclosan (submission IV). Furthermore the Norwegian authority on cosmetics has earlier this year submitted a report "Risk assessment on the use of triclosan in cosmetics; Development of antimicrobial resistance in bacteria II".

2. TERMS OF REFERENCE 1. Does SCCP consider a continued use of triclosan as a preservative in cosmetic products as safe for the consumer at the current concentration limit of maximum 0.3% taking into account the provided toxicological data? Does SCCP consider a continued use of triclosan as a preservative in cosmetic products as safe taking into account the new provided documentation of resistance development by certain micro-organisms and cross-resistance?

2.

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3. OPINION This opinion only addresses possible toxicological effects of triclosan on human health (question 1 in Terms of Reference). It does not cover aspects of potential resistance induction in micro-organisms by triclosan, which will be treated in a separate opinion. 3.1. Chemical and Physical Specifications 3.1.1. 3.1.1.1. Chemical identity Primary name and/or INCI name

Triclosan (INCI) 3.1.1.2. Chemical names

2,4,4-trichloro-2-hydroxy-diphenylether 3.1.1.3. Trade names and abbreviations

Irgasan DP300, Irgasan PG60, Irgacare MP, Irgacare CF100, Irgacide LP10 Triclosan is also referred to as Irgasan, DP300, FAT 80023), CH 3565, and GP 41-353 in a number of toxicology studies 3.1.1.4. CAS: EINECS: 3.1.1.5. CAS / EINECS number 3380-34-5 222-182-2 Structural formula
CI O OH

CI

CI

3.1.1.6. Formula: 3.1.2.

Empirical formula C12H7Cl3O2 Physical form

White crystalline powder 3.1.3. Molecular weight 289.5

Molecular weight:

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3.1.4.

Purity, composition and substance codes

Identification: IR Spectroscopy The purity of batches of triclosan used in personal care products since the 1970s has been as described in the following table:
Purity Specifications for triclosan since 1970 Test Point Effective from 1970 99.0 100.0% Effective from 26.09.1985 99% min Effective from 1.1.1990 99% min Effective from 31.12.1994 99.0-100% Effective from 26.6.2000 97.0-103.0% Effective from 06.11.2003 97.0 103.0%

Triclosan Active Substance1


1

Analysis by gas chromatography

The purity of different batches of triclosan used in the toxicology studies is described in the following table.
Purity of triclosan Batches Used in Toxicology Studies Triclosan Batch Number/Information FAT 80023/A Mischung 652 FAT 80023/B CH3565, Mg 120 FAT 80023 P4-11-210 , Package Nr. RP68118 FAT 80'023 BA Irgasan DP300, Batch #5.2.0211.0 Lot No. S 15155 T01 = Unilever No. S15155 T01 FAT 80023/H 5/0/0194/0, Batch No. EN46856.02 FAT 80023/Q Batch No. EN 91390.76 Triclosan R&D name: GP41353 PBS 5357.0, Lot No. 800187 C-P sample No 39317 Lot 19851206, Irgacare MP C-P sample No. 38328 Lot 19851206, Irgacare MP FAT 80023/R Batch No. EN 275927.26 FAT 80023/S Batch No. 505017 Lot 020750A7 P&G No. D1063.01 D1063.02R
1 2

Purity1 99.3% 99.3 99.9 99 99% Within specifications2 99.6 100.3 101 101 99.5% 99.5% 99.8 99.7

Analysis using gas chromatography A compiled analytical report is not available, but data shows the batch was within specifications.

Water content: 0.1%

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3.1.5.

Impurities / accompanying contaminants


0.1% 0.5% 10 mg/kg 10 mg/kg <0.001 g/kg <0.001 g/kg 0.5 mg/kg 0.25 mg/kg 0.25 mg/kg 0.5 mg/kg 0.1% 1 mg/kg 2 mg/kg 10 mg/kg 10 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg 2 mg/kg 20 mg/kg

Individual related compound (Gas Chromatography) Total related compounds (Gas Chromatography) 2,4 Dichlorophenol Sum of 3- and 4-Chlorophenol 2,3,7,8 Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzo-furan 2,8-Dichloroldibenzo-p-dioxin 1,3,7-Trichlorodibenzo-p-dioxin 2,8-Dichlorodibenzo-furan 2,4,8-Trichlorodibenzo-furan Ash Mercury Arsenic Antimony Lead Cadmium Nickel Copper Chromium Sum of heavy metals as lead sulfide precipitation

3.1.6.

Solubility
Solubility of triclosan in some selected solvents and chemicals

Solvent Distilled water (20C) Distilled water (50C) 1 N caustic soda 1 N sodium carbonate 1 N ammonium hydroxide Triethanolamine Acetone Ethanol 70% or 95% Isopropanol Propylene glycol Polyethylene glycol Methyl cellosolve (Union Carbide Corp.) Ethyl cellosolve (Union Carbide Corp.) Dipropylene glycol Glycerine n-Hexane Petroleum jelly (white, USP) Tween 20 (ICI America Inc.)

Solubility at 25C (g triclosan/100 g solvent) 0.001 0.004 31.7 0.40 0.30 >100 >100 >100 >100 >100 >100 >100 >100 ~40 0.15 8.5 ~0.5 >100

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Solubility of triclosan in some selected solvents and chemicals Solvent Tween 80 (ICI America Inc.) Triton X-100 (Rohm & Haas) Olive oil Castor oil Solubility at 25C (g triclosan/100 g solvent) >100 >100 ~60 ~90

3.1.7.

Partition coefficient (Log Pow)

Log Po/w: 4.8 3.1.8. Additional physical and chemical specifications 57 1C / 1.55 0.04 g/cm3 4 x 10-6 mmHg (20C) / 8.14 (20C) /

Melting point: Boiling point: Relative density: Vapour pressure: Viscosity: pKa: Refractive index: UV_Vis spectrum (200-800 nm): 3.1.9.

Homogeneity and Stability

The stability of triclosan under normal storage conditions (ambient temperature) has been assessed with a batch produced in 1973 and re-analyzed 4 and 9 years after manufacturing. The study showed that triclosan does not decompose under normal storage conditions; the quality has remained constant over the 9 years of storage.
Storage time Appearance Content active substance
1

Starting time (1973) White, fine crystalline 99%

After 4 years (1977) White, fine crystalline 99.7%

After 9 years (1982) White, fine crystalline 99.5%

Irgasan Dp 300, Batch No. EN 30142 storage conditions: ambient temperature

General Comments to physico-chemical characterisation Stability of triclosan in marketed products is not reported.

3.2. Function and uses Triclosan is an antibacterial ingredient that has been used in consumer products for over 30 years. It is widely used as a non-ionic antibacterial agent in personal care products (e.g., bar and liquid soaps, deodorants (Danish EPA, AR5), skin-care products, foot-care products oral care products, and make-up products). Triclosan was approved in 1986 by the European Community Cosmetic Directive for preservative in cosmetics products at concentrations up to 0.3%. Triclosan was evaluated also by SCF [SCF, 2000, AR8] and EFSA [EFSA, 2004, AR6] for use in food contact materials and classified in SCF_List 3 with a restriction of 5 mg/kg of food.

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Other fields of applications are textiles (i.e., sport wear) and plastic materials (i.e., plastic containers, brushes), with a triclosan concentration of up to 0.3%. In the EU, about 85% of the total volume of triclosan is used in personal care products, compared to 5% for textiles and 10% for plastics and food contact materials. The quantity used within the EC reached approximately 450 tons (as 100% active) in the year 2006. At or near typical usage levels, triclosan appears to intercalate into the membrane of bacteria, resulting in destabilised structure/function. This level of exposure results in disruption of nutrient uptake, inhibition of amino acid incorporation, inhibition of uracil incorporation, as well as membrane lysis. 3.3. Toxicological Evaluation A number of the animal toxicology studies were conducted prior to the publication of GLP standards and the establishment of OECD guidelines for the conduct of such studies; of these studies, many were still considered relevant, based on comparability to OECD guidelines. However, other non-GLP/non-OECD animal studies provided limited supportive information. The pivotal repeated-dose, sub-chronic and chronic studies were conducted pursuant to GLP regulations and generally followed the OECD guidelines. These pivotal studies are described in more detail. 3.3.1. Acute toxicity

A number of acute toxicity studies have been conducted for triclosan using the oral, dermal, intraperitoneal or intravenous routes of administration in mice, rats, rabbits, and dogs. The study designs were not always consistent with OECD guidelines for acute toxicity studies and there were no GLP compliance statements. A number of the acute toxicity studies were conducted prior to the establishment of GLP regulations. 3.3.1.1. Acute oral toxicity

Triclosan is not acutely toxic via the oral route of administration, with high oral intubation LD50 values in the range of 3,750 to 5,000 mg/kg bw in mice and rats, and an oral capsule LD50 value of greater than 5,000 mg/kg bw in dogs. Ref.: 1 3.3.1.2. Acute dermal toxicity

The dermal LD50 value for triclosan was reported to be greater than the high dose of 9,300 mg/kg bw tested in rabbits. These data indicate that triclosan is not acutely toxic via the dermal route of administration. Ref.: 1 3.3.1.3. Acute inhalation toxicity

No acute inhalation toxicity studies were available. 3.3.1.4. Additional acute toxicity studies

Mice and rats administered triclosan intravenously at 10, 20 and 30 (rats only) mg/kg bw in a vehicle solution of triethyleneglycol/water (1/2) showed signs of slight cramps, exopthalmos (mice only), mydriasis (rats only), dyspnoea, and ventral decubitus, with recovery by 24 to 48 h after dosing. Ref.: 2, 3

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Mice in the intraperitoneal studies showed signs of general lethargy, lassitude, close huddling, lack of response to tactile stimuli and no increase in respiration rate and hyperactivity with death occurring between 6 and 72 hours. Ref.: 4, 5 Data from the study by Kanetoshi et al. (1992) were also mentioned in a review by Bhargava and Leonard (1996). Additional References Bhargava, Leonard 1996 Table 1: summary of LD50 values from toxicity studies in mice, rats, rabbits, and dogs
Route of Administration Intravenous LD50 (mg/kg) 19 Reference; GLP and OECD Status Walther, 1968a (2); Predates GLP and OECD Mouse Oral Gavage 4,350 DeSalva et al., 1989 (1); Not reported, but some studies likely predate GLP and OECD Mouse (CD-1) (Female) Intraperitoneal 184 Miller et al., 1982 (4); GLP: not reported OECD: no comparable guidelines Mouse (ddy) (Male) Intraperitoneal 1,090 Kanetoshi et al., 1992 (5); GLP: not reported OECD: no comparable guidelines Rat (Wister CFE) Intravenous 29 Walther, 1968b (3); Predates GLP and OECD Rat Oral Gavage 3,750 to 5,000 DeSalva et al., 1989 (1); Not reported, but some studies likely predate GLP and OECD Rabbit Dermal >9,300 DeSalva et al., 1989 (1); Not reported, but some studies likely predate GLP and OECD Dog Oral Capsule >5,000 DeSalva et al., 1989 (1); Not reported, but some studies likely predate GLP and OECD

Species Mouse (NMRI)

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3.3.2 3.3.2.1. Table 2:


Species (Strain)

Irritation and corrosivity Skin Irritation Findings from Non-GLP Skin Irritation Studies with triclosan
Application Details Major Findings Reference, GLP and OECD Status Thomann and Maurer, 1978 (7) Predates GLP and OECD Erythema reactions were observed at 24 h after the start of exposure [3/6 positive responses in intact skin (mean score: 2.5); 5/6 positive in abraded skin (mean score 2.8)]. Oedema reaction at 24 h was positive in 1/6 animals. Erythema reactions improved at 72 h (mean scores: 1.3 and 2.5 in intact and abraded skin, respectively). Triclosan did not induce corrosion effects. Triclosan was classified as a moderate irritant based on an overall score of 3.58 (irritance scores of greater than 6 would have been considered severe). Sachsse and Ullmann, 1975 (6) Predates GLP and OECD

Guinea pig (Pirbright white)

0.1 mL single application, test site: 2 cm2 area on the shaven back, concentrations of 0.1, 0.5, 1.0, and 5.0% (duration of exposure and occlusion status unknown) Triclosan-soaked 2.5 cm2 gauze patches with occlusive dressings applied to shaved intact skin or abraded skin sites on the backs and flanks of the rabbits for 24 h (concentration of triclosan unknown).

Erythema reactions were only observed in the highest dose group (4/10 positive responses) at 24 hours post-application. No positive responses were reported at 48 hours.

Rabbit (Russian)

The results of the guinea pig study suggests that triclosan is not a skin irritant at concentrations below 5%, while both studies show that the erythema reactions are reversible. Skin irritation data were also available from 14-day repeated-dose and 90-day sub-chronic dermal toxicity studies of triclosan in the rat, mouse, and newborn monkey (Tables 10, 14 and 15). In the 14-day mouse and rat studies, conducted as dose range finding studies for longer-term repeated dose toxicology studies [Burns, 1997a (8); Burns, 1996 (9); Burns, 1997b (10)], erythema and scaling were observed in the mouse at doses of 1.5 mg/animal/day and higher (applied as solutions of 1.5 to 6%). Erythema, oedema, fissuring, eschar, alopecia, thickening and discoloration of the test site were noted in the rat at doses of 3.0 and 6.0 mg/animal/day (applied as solutions of 1% and 2% in 0.3 mL acetone). These studies show that repeated topical application of triclosan (up to 6%) resulted in moderate to severe skin irritation in the rat and mouse. In the 90-day subchronic toxicity study in rats, reversible skin irritation was observed starting at the lowest dose tested of 10 mg/kg bw/day (a concentration of approximately 0.5% triclosan in propylene glycol applied in a volume of 2 mL/kg body weight) [Trimmer, 1994 (11)]. In an early non-GLP study, dermatitis was observed in weanling dogs treated for 90 days with 200 mg/kg body weight/day, but not in dogs treated with the lower doses of 20 or 2 mg/kg bw/day [Dorner, 1973 (12)]. Limited and selective reporting of findings made the interpretation of the data from this dog study unreliable. In an early non-GLP study in newborn Rhesus monkeys bathed using a 15 mL of a 0.1% soap solution containing triclosan, no signs of dermal irritation were observed after 90 days of daily bathing [Hazleton Laboratories, 1979a (13)]. Additional skin irritation data are available from studies in humans (Section 3.3.11.3-1).

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3.3.2.2. Table 3:
Species (Strain) Hamster (Syrian golden)

Mucous membrane Irritation Findings from a Mucosal Irritation Study with Triclosan in Hamsters
Application Details Major Findings Reference; GLP and OECD Status Baert et al., 1996 (14); GLP: not specified

1.5% sodium lauryl sulphate (SLS), 1.5% SLS and 0.3% triclosan, or 0.3% triclosan in paste, 1 cm2 area of the right cheek pouch, 4 applications for 15 seconds at 24-hour intervals.

The histological structure of the mucosa of the triclosan-treated animals (Group 3) was similar to that of the control specimens. Structural changes, including basal hyperplasia, acanthosis, hypergranulosis, and orthokeratotic hyperkeratosis, were present in animals treated with SLS, or with SLS and triclosan. No signs of inflammation were observed in the subepithelial connective tissue.

The results of this study show that the application of triclosan in a paste (0.3% triclosan) does not result in mucosal irritation in the hamster cheek pouch. Table 4:
Species (Strain) Rabbit (New Zealand White)

Findings from a GLP Eye Irritation Study with Triclosan


Application Details Major Findings Reference, GLP and OECD Status Ullmann, 1980 (15); GLP: comparable OECD 405 consistent

0.1 g of pure solid triclosan was instilled into the conjunctival sac of the left eye (control: right eye). Examinations were on Days 1, 2, 3, 4, and 7.

Based on mean irritation scores in the cornea, iris, and conjunctiva, triclosan was found to cause slight primary eye irritation when applied to the rabbit eye mucosa.

Table 5:
Species (Strain) Rabbit (strain not reported)

Findings from a Non-GLP Eye Irritation Study with Triclosan


Application Details Major Findings Reference; GLP and OECD Status Lyman and Furia, 1969 (16); Predates GLP and OECD

Triclosan was applied either in gum Arabic (1, 2, 5, 10, or 20% concentrations) into the conjunctival folds (observations up to 24 h), or as an undiluted substance (observations up to 11 days) without rinsing, or with rinsing after 2 or 4 seconds of exposure.

Triclosan at concentrations of 1, 2, 5, and 10% produced only slight to moderate reddening of the conjunctiva as observed 2 hours after exposure, with recovery within 24 hours. Triclosan at a 20% concentration caused reddening and slight swelling of the conjunctiva as observed 24 hours after the start of exposure. The undiluted (pure) triclosan caused fully reversible eye irritation effects, as observed in rinse experiments, with a return to normal eye state between 7 and 11 days after exposure.

The results of eye irritation studies in rabbits showed that triclosan causes slight eye irritation when tested in its undiluted form. Triclosan did not produce either severe irritation or corrosive effects. Minimal to slight irritation effects to the cornea, iris, and conjunctivae were observed in tests using pure triclosan, resulting in an overall irritation score that indicated slight primary eye irritation [Ullmann, 1980 (15)]. Similarly, when tested at concentrations of 1, 2, 5, and 10% in gum Arabic, triclosan was shown to cause only

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reddening of the conjunctivae as observed at 2 hours, which was fully reversed within 24 hours after the start of exposure [Lyman and Furia, 1969 (16)]. Triclosan at the high concentration of 20% caused slight to pronounced reddening and slight swelling of the conjunctiva that was still observed after 24 hours. Summary of Irritation/Corrosivity Data Triclosan was a skin irritant at a level of 5% but not at 1% in the guinea pig skin irritation study. Repeated topical application of 1 to 6% triclosan for 14 days in dermal toxicity dose range-finding studies resulted in moderate to severe skin irritation in the rat and mouse. Lower concentrations were without effect in these studies. However, all of the doses in the 14- and 90-day dermal toxicity studies are considerably higher than exposures expected from the use of triclosan-containing personal care products. Effects were also observed in a skin toxicity study of 90 days duration in the rat at a concentration of 0.5%. In summary, the irritation/corrosivity data from either irritation studies in the hamster, guinea pig, and rabbit, or skin toxicity studies conducted in the mouse, rat, monkey, and dog suggest that triclosan may cause slight reversible skin irritation at concentrations of 0.5 to 5% under experimental conditions. Triclosan was not an irritant to mucous membranes in the hamster cheek pouch assay at a level of 0.3%. Triclosan at concentrations of 1 to 10% produced only slight, reversible irritation in the rabbit eye. 3.3.3. Table 6:
Species (Strain) Guinea pig, Hartley (albino)

Skin sensitisation Findings from Sensitisation Studies with Triclosan


Application Details Major Findings Reference; GLP and OECD Status Toxicological Resources, 1974 (17); Predates GLP and OECD

Guinea pig Buehler test. N=5 treated animals, 6 control animals. 1% triclosan in a cream/gel (occluded dermal application for both induction and challenge). Induction: 9 5-hour exposures on backs of animals. No adjuvant. Challenge: 14 to 21 days after induction. Split Adjuvant method. N=20 animals/treated or control group. Induction: triclosan (10% in petrolatum) applied 3 times. Complete Freund's adjuvant administered intradermally between 2nd and 3rd induction doses. Challenge: triclosan (3% in petrolatum) applied once 13 days after induction.

Induction: Slight primary irritation was observed after the first few treatments but was alleviated with regular wash-off procedures. Challenge: Treated sites showed slight irritation (redness) 24 and 48 hours after the challenge. Previously untreated sites did not show any significant oedema or erythema after challenge. Skin contact sensitization did not occur.

Guinea pig, Hartley (albino)

Induction: Slight erythema without oedema or vesiculation was observed in 7 of 20 triclosantreated animals. Erythema disappeared 1 or 2 days after the last application. Challenge: Bright pink and moderately elevated reaction in 1 of 20 animals at 24 and 48 hours post-challenge. At 72 hours, erythema was still present but without oedema. There were no reactions in any of the control group animals. The authors concluded that triclosan has a very low sensitisation index.

Lachapelle and Tennstedt, 1979 (18); Predates GLP and OECD

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Species (Strain) Guinea pig, Pirbright white

Application Details

Major Findings

Reference; GLP and OECD Status Maurer et al., 1979 (19); Predates GLP and OECD

Modified Maximisation test (Optimization). N=10/sex/group. Induction: 0.1% triclosan in propylene glycol, 6 intracutaneous injections over 3 weeks. (Complete Freund's adjuvant was used during the 2nd and 3rd weeks.) 1st challenge: 14 days after last induction, intracutaneous (0.1% triclosan in 40% propylene glycol). 2nd challenge: 14 days after 1st induction, occluded patch (0.1% triclosan in petrolatum). Guinea pig Buehler test. N=10/group. Induction: 25% triclosan initially (reduced to 10%, then 2% (topically applied 3 times / week for 3 weeks). No adjuvant. Challenge: 5% triclosan in propylene glycol, considered the highest non-irritating concentration, applied to a nave site. Positive control: DNCB treatment.

Induction: Erythema results were not reported for the induction phase. Challenge: The incidence of positive reactions was similar for triclosan-treated and control animals (4/20 after first challenge compared to 4/19 in the vehicle control group; 3/20 after second challenge compared to 1/19 in the vehicle control group). The investigators concluded that triclosan showed no skin-sensitising potential in this study.

Guinea pig (Hartley albino)

GLP-compliant. Conducted according to OECD Guideline No. 406. Skin reactions scoring >0.5 were considered to be positive (maximum score: 3). Induction: very faint to severe erythema, depending on the dose level (note that doses were continually reduced to reduce irritation). Challenge: After challenge, triclosan treatment induced weak, non-confluent reactions of very weak erythema (scores of 0.5) in 6/10 animals (not considered to indicate sensitisation). There were no scores greater than 0.5 among triclosantreated animals. Negative controls showed similar faint erythema reactions in 2/5 animals (scores of 0.5). Positive controls showed strong reactions (scores of 1 to 3) in 10/10 animals (mean erythema scores of 1.8). The investigators concluded that triclosan is not a contact sensitizer.

Wnorowski, 1994 (20); GLP: compliant OECD: No.406 consistent

Triclosan as tested in 4 studies was found not to cause skin sensitisation. Both the 1974 and the 1978 studies used methods that were similar to those recommended by current OECD guidelines for sensitisation studies, and the more recent 1994 study was GLPcompliant and followed OECD (No. 406). In a small Buehler test in guinea pigs, slight irritation was observed during the induction period with 1% triclosan, attributed to build-up of the cream formulation on the test site [Toxicological Resources, 1974 (17)]. The study investigators concluded that no sensitisation was observed, as previously untreated sites did not show any significant oedema or redness following the challenge dose. Slight irritation (erythema without oedema) also was observed during induction in a much larger sensitisation study using the split adjuvant method in guinea pigs [Lachapelle and Tennstedt, 1979 (18)]. In this study, a positive sensitisation reaction following challenge was observed in only 1 of 20 animals. Thus, these authors also concluded that triclosan has a very low sensitisation index. In the third study, a modified maximisation test in guinea pigs, there was no significant difference in sensitisation reactions following challenge in animals treated with 0.1% triclosan in propylene glycol compared to animals treated with the vehicle alone [Maurer et al., 1979 (19)]. In the GLP study, a Buehler assay conducted following OECD guidelines, triclosan showed no evidence of sensitisation potential, in contrast to the positive control substance [Wnorowski, 1994 (20)]. Altogether, the results of these studies suggest that triclosan is not a sensitising agent as tested in the guinea pig.

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Summary of Sensitisation Data Triclosan has been tested in skin sensitisation studies in guinea pigs, with both adjuvant and non-adjuvant methods used. In summary, the results of these GLP and non-GLP studies indicate that there is no evidence for sensitisation with triclosan in various formulations and concentrations (up to 10% in petrolatum) in the guinea pig. Results of related studies in Humans are reported in section 3.3.11. 3.3.4. Dermal / percutaneous absorption

In Vitro Studies of Dermal/Percutaneous Absorption A single in vitro percutaneous absorption study with triclosan was conducted in rat skin [Moss et al., 2000 (21)]. The main findings in this study are provided in Table 7. After 24 hours, 58% of the applied dose remained on the skin surface and in the stratum corneum (33 and 25%, respectively) and 41.2% of the applied dose was recovered in the receptor fluid and in the epidermis and dermis (23 and 18.2%, respectively). Thus, it can be considered that 41.2% of the applied dose was absorbed percutaneously within 24 hours (penetration through the stratum corneum into deeper layers of the skin is considered to represent absorption). Triclosan was primarily absorbed through the skin as the parent compound, with some glucuronide and sulphate conjugates being detected in the receptor fluid. Glucuronidation was the primary route of metabolism of triclosan in rat skin. Table 7:
Method Diffusion cell system using dorsal skin. Seven L of 64.5 mM [3H]-triclosan in an ethanol-water (9:1, v/v) solution (0.48 Mbq) was applied to the exposed skin surface (0.64 cm2).

Findings from an In Vitro Percutaneous Absorption Study for Triclosan in Rat Skin
Major Findings After 24 hours, approximately 23% of the applied dose appeared in the receptor fluid, and 33%, 25%, and 18.2% remained on the skin surface, stratum corneum, and epidermis and dermis, respectively. Of the radioactivity in the receptor fluid at 24 hours, 17.3% of the applied dose was present as triclosan, 4.1% as triclosan glucuronide, and 0.9% as triclosan sulfate. Of the radioactivity in the skin at 24 hours, 13% of the dose was recovered as triclosan, 1.4% as triclosan glucuronide, and 1.6% as triclosan sulfate. Reference; GLP Status Moss et al., 2000 (21); GLP: not specified

In Vivo Studies of Dermal/Percutaneous Absorption The main findings in the in vivo percutaneous absorption studies for triclosan are provided in Table 8. Table 8:
Species Mouse

Findings from In Vivo Percutaneous Absorption Studies for Triclosan


Method Single application of liquid soaps. Test site: 1 cm x 3 cm area on the back. 22.54 to 25.49 g triclosan/cm2 skin. Major Findings Triclosan deposition on hairless mouse skin was 0.98 0.11 and 1.16 0.13 g/cm2 skin, following application of approximately 22.54 and 25.49 g triclosan/cm2 skin. The percent of the applied dose deposited on the skin was 4.425 0.617 and 4.809 1.236, respectively. Reference; GLP Status Demetrulias, 1985 (22); GLP: not specified

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Species Mouse

Method 0.05 mL of [3H]Irgasan DP300 (1.6 mg, 10 Ci) in olive oil was applied to a 5 cm2 area of the shaved back.

Major Findings Maximum tissue levels (at 12 or 18 hours) were highest in the gallbladder (402 57 g/g), followed by liver (13.4 3.5 g/g), fat (10.0 3.5 g/g), lung (7.5 2.9 g/g), blood (6.5 1.9 g/g), and kidney (4.3 0.8 g/g). Maximal levels in the testes, heart, and spleen were in the range of 1.1 to 1.8 g/g. Low levels were detected in the brain (0.2 to 0.5 g/g). Toxicokinetic data from a dose range finding repeated dose dermal irritation study. Two pooled plasma samples per treatment group (5 mice/sex/pooled sample). Mean plasma triclosan levels in males: 32.5, 63.3, 98.8, 148.2, and 173.3 g/mL at doses of 0.3, 0.6, 1.5, 3.0, and 6.0 mg/animal/d, respectively. Females: 63.2, 124.8, 144.0, 124.0, 295.2 g/mL for same doses. Note that absorbed amount as percentage of applied dose was not calculated. Toxicokinetic data from a dose range finding repeated dose dermal irritation study. Two pooled plasma samples per treatment group (5 mice/sex/pooled sample). Mean plasma triclosan levels in males: 7.4, 22.2, 38.6, 75.4, and 72.6 g/mL at doses of 0.3, 0.6, 1.5, 3.0, and 6.0 mg/animal/d, respectively. Females: 9.2, 33.0, 47.0, 101.8, 112.1 g/mL for same doses. Note that absorbed amount as percentage of applied dose was not calculated. Toxicokinetic data from a dose range finding repeated dose dermal irritation study. Plasma samples were taken from 10 rats/sex/treatment group. Mean plasma triclosan levels in males: 1.0, 2.1, 6.6, 14.1, and 31.6 g/mL at doses of 0.3, 0.6, 1.5, 3.0, and 6.0 mg/rat/day, respectively. Females: 1.2, 2.4, 5.2, 9.2, and 18.1 g/mL. Note that absorbed amount as percentage of applied dose was not calculated. Ethanol solution: 27.6% of the applied dose was absorbed within 48 hours. Blood levels were in the range of 0.07 to 0.30 g/mL with Tmax at 6 hours. Shampoo: Penetration of [H3]-triclosan was dependent on concentration and independent of duration of contact (5, 10, or 20 minutes). Absorption after 48 hours was in the range of 2.8 to 4.1% of the applied dose. Deodorant: Report notes difficulty in administering a standard, accurate, known dose, so refers to data from the application using an ethanol solution as vehicle. After 24 hours, 0.88% of radioactivity was in the urine, 11.84% in the faeces, 0.02% in the blood, 26.13% on the skin surface, 4.31% in the skin, 0.24% in the cage wash, 7.72% in the carcass, 36.33% in the stratum corneum, and 1.38% on the skin cover. Recovery of radioactivity was 90.46% of the dose. The applied dose remained primarily on the adhesive pad (~64%). Absorbed triclosan was primarily excreted in the faeces. 0.5%, 14.66%, 0.1%, 5.5%, 7.2% of the applied dose was recovered in the urine, faeces, blood, skin, and carcass/tissues, respectively.

Reference; GLP Status Kanetoshi et al., 1992 (5); GLP: not specified

Mouse (CD-1)

0, 0.3, 0.6, 1.5, 3.0, or 6.0 mg/animal/d in acetone for 14 days, applied to 2x3 cm2 area on the dorsal area once daily.

Burns, 1997a (8) GLP: compliant

Mouse (CD-1)

0, 0.3, 0.6, 1.5, 3.0, or 6.0 mg/animal/d in propylene glycol for 14 days, applied to 2x3 cm2 area on the dorsal area once daily.

Burns, 1996 (9) GLP: compliant

Rat (Crl:CDBR)

0, 0.3, 0.6, 1.5, 3.0, or 6.0 mg/animal/d in acetone for 14 days, in a volume of 300 L, applied to 2x3 cm2 on the dorsal area once daily.

Burns, 1997b (10) GLP: compliant

Rat

Triclosan in an ethanol solution, in shampoo, or in an aerosol deodorant was applied to 7.5 cm2 clipped dorsal skin

Black and Howes, 1975 (23); Predates GLP

Rat

Triclosan in an ethanolwater (9:1, v/v) solution (6.92 Mbq) was applied to a 9.6 cm2 circular area on the shaved backs of the rats. Single application of triclosan (4 mg/cm2, 400 mg/kg, 10 Ci/rat) to the shaven back

Moss et al., 2000 (21); GLP: not specified Hong et al., 1976 (24); Predates GLP

Rat

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Species Rat

Method Single application of triclosan solution (ethanol, acetone), in cream, or in Vaseline to the neck

Major Findings Solutions within 96 hours, 2.8 to 3.7% and 69 to 89% of the applied dose were excreted in the urine and the faeces, respectively. Cream within 48 hours, 1% and 22% of the applied dose were excreted in the urine and faeces, respectively. Vaseline within 144 hours, 13% and 60% of the applied dose were excreted in the urine and faeces, respectively. Solutions 47 to 53% and 38% of the applied dose excreted in the urine and faeces, respectively, independent of dose. Creams 29 to 48% of the applied dose was excreted in urine, inversely dependent on quantity of cream applied per unit area. 2% excreted in faeces. Soap solutions 2 to 10% of the applied dose was absorbed (radioactivity measured in urine, faeces, skin, expired air, and organs and tissues). Trace amounts of triclosan (i.e., lower than the detection limit) were detected in some of the blood and faecal samples, but in most cases none was present. The amount of triclosan in the urine was in the range 0.1 to 3.8 g (no difference between animals with intact or abraded skin).

Reference; GLP Status Ciba-Geigy, 1976b (25); Predates GLP

Rabbit

Single or repeated (5x) application of triclosan in solution (propylene glycol, DMSO, nickethamide, or hexane), in cream, or in soap solution (site not reported) Urine-soaked diapers containing 6.4 to 26.9 g triclosan/g were applied to intact or abraded skin (dorsal and flank) and reapplied twice at 4-hour intervals, then left on overnight Single application or repeated application (twice daily for 5 days) of soap suspension or non-soap detergent suspension was applied to a 20 cm2 area (40 cm2 area in 1 experiment) on the clipped skin of the back for 2 minutes.

Ciba-Geigy, 1976b (25); Predates GLP

Rabbit

Ciba-Geigy, 1977b (26); Predates GLP

Guinea pig

Triclosan remained primarily on the stratum corneum, and small amounts penetrated into the epidermis, dermis, hair follicles, and sebaceous glands. Repeated application did not increase localization in any area of the skin. Increasing concentrations of triclosan resulted in increasing triclosan deposition throughout the skin depth. Recovery of triclosan in rinse water accounted for 80-90% (single application) and 95% (repeated application mean) of the applied activity. Blood levels were in the range of 0.002 to 0.006 g/mL and 0.014 to 0.027 g/mL after a single application (40 cm2 vs. 20 cm2). The blood level was 0.019 g/mL following repeated applications. Levels of triclosan in the tissues were extremely low (ng/g range). Triclosan was excreted mainly in the urine with relatively small amounts in the faeces. Peak blood levels (6-8 hrs post-dose) were 0.7 to 2.7% (mean = 1.4%) of the applied dose. Daily urinary excretion of free triclosan and conjugates was in the range of 1 to 4% (mean = 2%) of the applied dose. Blood levels were 130 and 165 ppb on Day 7 and 14, respectively.

Black et al., 1975 (27); Predates GLP

Dog

1.3 to 5.0 mL mouthrinse, 15 minutes daily for 7 days Triclosan in water (200 mg/kg), once daily for 2 weeks, nuchal skin.

Lin et al., 1994 (28); GLP: not specified Hong et al., 1976 (24); Predates GLP

Dog

Monkey

2 Rhesus monkeys, 3 days old, were washed once with a soap solution containing triclosan (1 mg/mL).

Blood levels reached a plateau by 8 to 12 hours and remained up to 24 hours after treatment. Levels of conjugated triclosan (glucuronide and sulfate) in the blood were in the range of 0.25 to 0.68 ppm. No free, unconjugated triclosan was detected. Triclosan sulfate levels in the blood increased, while triclosan glucuronide levels decreased with time post-treatment.

Parkes, 1978a (29); Predates GLP

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Species Monkey

Method 10 monkeys/group were washed daily for 90 days with 15 mL of a soap solution (0.1% triclosan) (size of application area not reported). 5/group were retained for a 30day recovery period.

Major Findings Levels of conjugated triclosan (glucuronide and sulfate) in the blood ranged from 0.17 to 0.97 ppm. No free, unconjugated triclosan was detected. Triclosan glucuronide predominated in the initial samples (Day 1 or 2). Triclosan sulfate predominated in all subsequent blood samples (8090% of total present after 90 days). Urinary concentrations of triclosan were in the range of 0.3 to 4.8 ppm (primarily the glucuronide conjugate). Faecal concentrations of triclosan were in the range of <0.1 to 10.5 ppm (primarily the unconjugated form). Small amounts of triclosan (<0.1 to 1.9 ppm) were detected in tissues from treated animals. Following the 30-day recovery period, triclosan was detected only in skin samples.

Reference; GLP Status Hazleton Laboratories, 1979b (30); Predates GLP

Deposition and absorption of triclosan were investigated in 4 mouse studies. In a skin deposition study, the triclosan residue on mouse skin was approximately 1 g/cm2 (4.4 to 4.8% of the applied dose) following application of liquid soap containing [14C]-triclosan In a percutaneous (22.5 to 25.5 g/cm2) for 10 minutes [Demetrulias, 1985 (22)]. absorption study in the mouse, the maximal radioactivity in tissues (observed at 12 or 18 hours) following absorption of [3H]-triclosan was in the range of 14 to 67% of the maxima obtained following oral administration (g/g range) [Kanetoshi et al., 1992 (5)]. The absorption of triclosan was assessed in two 14-day repeated dose dermal toxicity studies that showed dose-dependent increases in plasma triclosan levels following application of triclosan in propylene glycol and in acetone vehicles, respectively [Burns, 1996 (9), 1997a (8)]. Triclosan in plasma was detected at the lowest doses used in the studies (0.3 mg/mouse/ day, or 12 mg/kg bw/day in a 25 g mouse, giving plasma levels of 7.4 and 32.5 g/mL using propylene glycol and acetone vehicles, respectively). These results indicate that triclosan was readily absorbed through the skin and distributed to tissues in the mouse. Four percutaneous absorption studies were conducted in the rat. The results of these studies show that the extent of percutaneous absorption of triclosan is dependent on the vehicle used for application. The extent of triclosan absorption was in the range of 23 to 28% of the applied dose either in ethanol, ethanol/water, soap suspension, or a cream formulation. Greater absorption was observed with triclosan in an aqueous solution or in Vaseline, while lower absorption was observed with triclosan in shampoo. These data are shown in Table 9. In addition to the absorption data from these four studies, plasma data from rats that received 14 days of repeated dermal applications of triclosan in acetone showed dose-dependent increases in plasma triclosan levels starting at the lowest dose used in the study (0.3 mg/rat/day, or 1.2 mg/kg bw/day in a 250 g rat, giving plasma levels of 1.0 g/mL) [Burns, 1997b (10)]. Taken together, these data indicate that triclosan is readily absorbed through the skin of rats. Table 9:
Vehicle Pure ethanol Shampoo Aerosol deodorant1 Solution (ethanol, acetone) Cream Vaseline Ethanol/water (9:1)

Percutaneous Absorption of Triclosan in the Rat


Time Point (hours) 48 48 48 96 48 144 24 % Dose Absorbed 28 3 to 4 52 93 23 73 21 Reference Black and Howes, 1975 (23) Black and Howes, 1975 (23) Black and Howes, 1975 (23) Ciba-Geigy, 1976b (25) Ciba-Geigy, 1976b (25) Ciba-Geigy, 1976b (25) Moss et al., 2000 (21)

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Soap suspension
1

72

28

Hong et al., 1976 (24)

The determination of absorption following aerosol application was reported to be difficult by the authors of this study due to the lack of a standardized application of the spray. As such, the value of 52% should be interpreted cautiously.

The percutaneous absorption of triclosan was also investigated in the guinea pig [Black et al., 1975 (27)]. Autoradiography of skin after a single application of a soap suspension containing radiolabelled triclosan ([3H] or [14C]) showed that triclosan was absorbed with about 20% of the applied dose being absorbed percutaneously even after repeat applications of the soap solution. The results of a percutaneous absorption study with diapers washed in a solution containing triclosan suggest that the absorption of triclosan through the skin was very low in rabbits [Ciba-Geigy, 1977b (26)]. It should be noted that in this study triclosan was not applied directly to the skin and that the amount of triclosan in the skin was not determined. In another percutaneous absorption study in rabbits, the extent of absorption of triclosan was dependent on the type of formulation (solutions: >85% of the applied dose was absorbed; creams: 29 to 48% was absorbed; soap solutions: 2 to 10% was absorbed), as was observed in studies with rats [Ciba-Geigy, 1976b (25)]. In a small percutaneous absorption study in dogs (n=3), triclosan in water (200 mg/kg) was applied to the shaven nuchal skin of dogs once daily for 2 weeks [Hong et al., 1976 (24)]. Triclosan blood levels were 130 and 165 ppb (ng/mL) on Day 7 and 14, respectively. The total extent of absorption could not be determined, as levels in the urine, faeces, tissues, and expired air, were not reported. Another study in the dog investigated the absorption of triclosan through the oral mucosa [Lin et al., 1994 (28)]. In this study, peak blood levels, which occurred within 6 to 8 hours, represented 0.7 to 2.7% (mean = 1.4%) of the applied dose. Daily urinary excretion was in the range of 1 to 4% (mean = 2%) of the applied dose. There were no apparent sex differences in plasma concentrations or urinary excretion of triclosan in dogs. Again, the total extent of absorption was not determined. The percutaneous absorption of unlabelled triclosan was investigated in a pilot study and a 90-day study with infant Rhesus monkeys [Parkes, 1978a (29); Hazleton Laboratories, 1979b (30)]. In the pilot study, triclosan was detected in all blood samples following a single dermal exposure to a soap solution containing triclosan (1 mg/mL, 0.1%), with blood levels detected up to 24 hours, and peak levels observed at 8 to 12 hours. In the 90-day study, only the glucuronide and sulfate conjugates were detected in blood samples, the glucuronide predominating in the early blood samples (Days 1 to 2), and triclosan sulfate predominating in all subsequent blood samples (samples were taken daily for the 90-day duration of the study). Triclosan was excreted in the urine primarily as the glucuronide conjugate, but was excreted in the faeces primarily in the free or unconjugated form. Low levels of triclosan were detected in tissues. The results of this monkey study indicate that triclosan was absorbed percutaneously following 90 days of daily washing with 15 mL of soap (1 mg triclosan/mL) and that the proportion of plasma glucuronide and sulphate conjugates altered following chronic administration. Summary of Dermal/Percutaneous Absorption Data In summary, data from the percutaneous absorption studies conducted with triclosan indicate that it was relatively well absorbed through the skin in all species tested. The extent of absorption was dependent on the formulation in which it was delivered (e.g., greater absorption was observed following application of triclosan in solution than in a cream or Vaseline formulation) and the duration of time that the applied dose remained on the skin (e.g., lather/rinse off vs. apply/leave on). In the rat, the extent of percutaneous absorption was approximately 23 to 28% of the applied dose of triclosan in ethanol,

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ethanol/ water, soap suspension, or a cream formulation (although higher absorption was observed in some studies). 3.3.5. Repeated dose toxicity

A number of repeated dose toxicity studies have been conducted for triclosan in mice, rats, hamsters, rabbits, dogs, and primates. 3.3.5.1. Repeated dose (14 days) dermal toxicity studies

Three short-term dermal toxicity studies were conducted in mice and rats for the purposes of establishing dose ranges for larger studies of longer durations. The major findings from these GLP dose range finding studies are presented in Table 10. Triclosan was tested in 2 mouse studies using different vehicles (acetone and propylene glycol). Using a similar study design, rats were administered triclosan in an acetone vehicle. In the 2 mouse studies, triclosan was administered at dose levels of 0, 0.3, 0.6, 1.5, 3.0, or 6.0 mg/animal/day for 14 days in acetone [Ref.: 8] or in propylene glycol [Ref.: 9]. These dermally-applied doses correspond to systemic doses of approximately 12, 24, 60, 120, or 240 mg/kg body weight/day for a 25 g mouse (triclosan concentrations of 0.3, 0.6, 1.5, 3, and 6 % in 0.1 mL application volume). The triclosan was applied to a 2 x 3 cm2 hairless area on the dorsal side of the animal. Appropriate untreated and vehicle controls were used. As these were dose range finding studies, although GLP-compliant, they were not entirely compliant with OECD guidelines for dermal studies (clinical chemistry, haematology, or urinalysis investigations were not conducted, and macroscopic and microscopic evaluations were limited). Both mouse studies showed similar dose-related dermal and liver findings such as dermal irritation, increased liver weight, coagulative necrosis and centrilobular hypertrophy. Dose-related dermal findings started at 1.5 mg / animal / day (60 mg/kg bw/d) for triclosan in propylene glycol and at 3.0 mg / animal / day for triclosan in acetone vehicle. Liver effects were observed in animals treated with dermal doses of 1.5 mg / animal / day ( 60 mg/kg bw/d). The NOAEL is 24 mg/kg bw/d. Ref.: 8, 9 In rats, a similar dose range finding study was conducted using an acetone vehicle. Dermally-applied doses of 0.3, 0.6, 1.5, 3.0, or 6.0 mg/day in the rat study correspond to systemic doses of approximately 1.2, 2.4, 6, 12, or 24 mg/kg body weight/day for a 250 g rat (triclosan concentrations of 0.1, 0.2, 0.5, 1, and 2 % in 0.3 mL application volume). Skin irritation such as erythema was observed at 6.0 mg/day, with findings in one female animal at 1.5 mg/day considered to be incidental. Other related signs of skin irritation such as eschar, and oedema were also noted. Histopathology of the erythema, scaling, and eschar showed acanthosis and hyperkeratosis at the highest dose. Gross pathology revealed dark areas of the liver in a few treated animals (not dose-related); however, no change in organ weight was observed and no histopathology was associated with the gross pathology findings. Ref.: 10 In summary, these 3 rodent dermal studies revealed a similar pattern of toxicity with respect to dose-related dermal irritation and hyperkeratosis at the site of application. In addition, liver-related effects such as increased organ weight associated with centrilobular hypertrophy were observed in both mouse studies but not in the rat, indicating a species difference in response to triclosan. The NOAEL was determined to be 0.6 mg/day (24 mg/kg body weight/day) in both mouse studies, based on liver effects observed at doses of 1.5 mg / animal / day. The NOAEL in the rat study was determined to be 3.0 mg / animal / day (12 mg/kg bw/day), based on dermal irritation at the highest dose of 6.0 mg / animal / day.

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Table 10: Findings from GLP Short-Term Repeated Dose Dermal Toxicity Studies for Triclosan
Species (Strain) Mouse (CD-1) Dosing Regimen and Duration 0, 0.3, 0.6, 1.5, 3.0, or 6.0 mg/animal/d in acetone for 14 days, in a volume of 100 L, applied to 2x3 cm2 area on the dorsal area once daily (triclosan concentrations of 0, 0.3, 0.6, 1.5, 3, and 6%) 0, 0.3, 0.6, 1.5, 3.0, or 6.0 mg/animal/d in propylene glycol for 14 days, in a volume of 100 L, applied to 2x3 cm2 area on the dorsal area once daily (triclosan concentrations of 0, 0.3, 0.6, 1.5, 3, and 6%) 0, 0.3, 0.6, 1.5, 3.0, or 6.0 mg/animal/d in acetone for 14 days, in a volume of 300 L, applied to 2x3 cm2 on the dorsal area once daily (triclosan concentrations of 0.1, 0.2, 0.5, 1, and 2%) Major Findings Reference; GLP and OECD Status Burns, 1997a (8) GLP: compliant OECD: comparable

Dermal irritation such as erythema was observed at the site of application. Oedema, fissuring, eschar was observed at the 2 highest doses which correlated with hyperkeratosis. Acanthosis was also observed in males at 1.5 mg/d. The liver showed dose-related increase in absolute and relative liver weight in females at 1.5 and in both sexes at 3.0 and 6.0 mg/d. This increase associated with centrilobular hypertrophy. Mononuclear infiltrate was also observed but only at the high dose. The NOAEL is considered to be 0.6 mg/d. This corresponds to 24 mg/kg bw per day

Mouse (CD-1)

Dose-related dermal irritation was observed at the site of application starting at 1.5 mg/d. Dose-related increases in absolute and relative liver weights in both sexes at 6.0 mg/d and in males at 0.3, 1.5 and 3.0 mg/d. Centrilobular hypertrophy at doses 1.5 mg/d. The statistical increase in male liver weights at 0.3 mg/d was not correlated with any histopathology changes. The NOAEL is considered to be 0.6 mg/d. This corresponds to 24 mg/kg bw per day

Burns, 1996 (9); GLP: compliant OECD: comparable

Rat (Crl:CDBR)

Dose-related skin irritation such as erythema was observed at 6.0 mg/d, with findings in 1 animal at 1.5 mg/d considered incidental. Other related signs of skin irritation such as eschar, oedema were also noted. Histopathology of the erythema, scaling and eschar showed acanthosis and hyperkeratosis at the highest dose. Gross pathology revealed dark areas of the liver noted in a few treated animals (not dose-related); however, no change in organ weight was observed and no histopathology accompanied this gross finding. The NOAEL was estimated by investigators to be 3.0 mg/animal/d equivalent to 12 mg/kg bw per day.

Burns, 1997b (10); GLP: compliant OECD: comparable

3.3.5.2.

Repeated dose (21 days) inhalation toxicity

The inhalation toxicity of triclosan after 14 days of repeated dose administration was evaluated in the rat. This study was performed prior to GLP regulations and the establishment of OECD guidelines In this study, groups of 10 male and 10 female rats were initially exposed to triclosan at concentrations ranging from 50 to 1,300 mg/m3. Following the first day, due to the occurrence of deaths, dyspnoea and general clinical signs indicative of poor health in the treated animals, the test concentrations were reduced to 0, 50, 115, or 301 mg/m3 for dosing on Days 2-15, i.e., the remainder of the study. Although there were 12 unscheduled deaths related to high-dose level exposure, 11 of the 12 rats died during Day 1 as a result of the very high initial doses. Necropsy revealed congestion, inflammatory changes in mucous membranes and nasal cavity. In the remaining animals, dose-related increases in leukocyte count and alterations in serum chemistry such as glutamic-pyruvic transaminase, alkaline phosphatase were

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observed. Slight focal inflammation of the mucous membranes was observed in high-dose animals at the end of the study. The NOAEL for inhaled triclosan was determined in this study to be 50 mg/m3, the lowest dose tested. Ref.: 32 3.3.5.3. Repeated dose (28 days) oral toxicity study in mice

A GLP-compliant 28-day repeated dose oral toxicity study of triclosan was conducted in the mouse, using OECD Guideline 407 for the design of the study. In this mouse study, the significant findings were in the liver, which showed reversible hepatocellular hypertrophy and focal necrosis in mice that received the high dose (136 and 169 mg/kg body weight/day in males and females, respectively). Ref.: 31 A brief summary of the major findings from the repeated dose oral toxicity study is presented in the table below. Table 11: Findings from Short-Term Repeated Dose Oral Toxicity Studies for Triclosan
Species (Strain) Dosing Regimen and Duration Major Findings Reference; GLP and OECD Status Ciba-Geigy, 1987 (31); GLP: compliant OECD: No.407 consistent

Mouse (MAGf)

0, 50, or 1,000 ppm in the diet (0, 6.5, or 136 mg/kg bw/d in males and 0, 8.3, or 169 mg/kg bw/d in females) for 28 days with 14 days recovery

Slight, but significant reversible decreases in erythroid parameters. Biochemistry showed significant increases (2to 3-fold) in liver function enzymes in plasma (high-dose animals); changes were almost completely reversed by the end of 14-d recovery. Slight changes in urea (high-dose animals) and creatinine (high-dose females) were not fully reversed. No macroscopic findings, but histopathological examination showed liver changes in high-dose animals, including hepatocyte hypertrophy, hepatocyte necrosis (focal) bordered by macrophages in some cases. Some Kupffer cells in the area contained a pigment that was assumed to be iron. These changes were reversed in 14-day recovery period. Low-dose animals showed no histological changes or changes in haematology or blood chemistry (except for a slight, not significant increase in liver function enzymes in males of the low-dose group). Electron microscopy of selected livers of high-dose animals showed reversible proliferation of smooth endoplasmic reticulum and increase in number and/or size of peroxisomes. No NOAEL was determined by investigators; however, it should be noted that no adverse effects were observed at the low dose (50 ppm).

Comment A reversible decrease in phosphate was observed in females of both doses and liver enzymes were slightly increased but not significant in low dose males. 3.3.5.4. Sub-chronic (90 days) oral toxicity studies

The safety of triclosan has been evaluated in sub-chronic oral toxicity studies in mice, rats, hamsters, rabbits, dogs, and baboons, using either dietary administration or capsules. Test species were evaluated for clinical observations, body weight, body weight gain, food and water consumption, haematological, clinical chemistry, ophthalmological and urinalysis parameters, macroscopic observations, and microscopic findings. In addition, these investigations included a wide range of dose levels for triclosan. Pivotal studies conducted in mice, rats and hamsters were conducted pursuant to GLP and generally followed OECD guidelines. Findings from these studies are presented in Table 12 (GLP- and OECDcompliant studies). Rabbit, dog, and baboon studies were conducted prior to the

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establishment of GLP standards and OECD guidelines. Findings from these studies are presented in Table 13 (non-GLP and/or non-OECD compliant studies). In the pivotal studies conducted in compliance with GLP and following OECD guidelines for 90-day (sub-chronic) oral toxicology studies, the highest doses tested were 900 mg/kg body weight/day in the mouse, 600 mg/kg bw/day in the rat, and 900 mg/kg bw/day in the hamster. Subchronic oral toxicity study in mice In the GLP mouse study, CD-1 mice (15/sex/group) were administered triclosan via the diet at dose levels corresponding to 0, 25, 75, 200, 350, 750, or 900 mg/kg bw/day for 13 weeks. Additional satellite groups (10/sex/group), for interim sacrifice at 7 weeks, were administered 0, 25, 350, and 900 mg/kg body weight/day. This study contained a GLP and OECD compliance statement and was well designed and conducted. The results of this study indicated that the liver was the primary target organ in mice. Treatment-related increases in alkaline phosphatase were observed in females and males, at 25 mg/kg bw/day and at 200 mg/kg body weight/day, respectively. Decreased erythrocyte count and haemoglobin were noted at 25 mg/kg bw/d (male) and all animals at 75 mg/kg bw/d. Other changes noted included decreased total cholesterol in all animals at 25 mg/kg bw/day and higher and increased alanine aminotransferase (males at 350 mg/kg body weight/day and above, females at 750 mg/kg body weight/day and above). Increased gamma glutamyltransferase was observed in both males and females at 750 mg/kg body weight/day. Dose-related increases in absolute and relative liver weights were observed in both males and females starting at 75 mg/kg bw/day. Gross and histopathology findings in the liver included enlarged dark or thickened lobes, with histomorphologic centrilobular hepatocellular hypertrophy, vacuolization, pigment accumulations, necrosis, and/or inflammation. These hepatic changes were noted in males at 75 mg/kg bw/day and in both males and females at all higher doses. In the more severe cases of hepatocellular hypertrophy, hepatocytes were individualized, although the overall hepatic architecture was still intact. In addition to liver findings, increased extramedullary haematopoiesis was observed in the spleen of animals at 750 and 900 mg/kg bw/day and, at doses of 200 mg/kg body weight/day and higher, hyperplasia in male glandular stomachs and inflammation in female kidneys occurred. No histomorphologic alterations were observed in males at 25 mg/kg bw/day or in females at 25 or 75 mg/kg bw/day. A NOAEL was not established from this study since treatmentrelated changes in haematology parameters, increased alkaline phosphatase, and decreased cholesterol were observed at the low dose. Ref.: 33 Subchronic oral toxicity study in rats In a 90-day GLP toxicity study in rats that served as a dose range-finding study for a 2-year oral carcinogenicity study, triclosan (FAT 80023) was administered to Sprague-Dawley rats (25/sex/group) via the diet at concentrations of 0, 1000, 3000, or 6000 ppm (approximately 0, 100, 300, or 600 mg/kg bw/day based on calculations of mean body weight, food consumption and target dose level. No test article-related mortality occurred. Decreased body weight along with gradually decreased food consumption was observed at the high-dose groups. Diet spillage at study initiation occurred at all treatment groups. Treatment-related effects on erythrocyte parameters RBC, haemoglobin and haematocrit were observed in mid- and high-dose groups. Increased ketones were found in high-dose males and decreased triglycerides in high-dose males and females. Interim necropsy findings, conducted at 45 days, revealed increased liver weight changes in males (mid- and high-dose) and females (high-dose only). At terminal necropsy, absolute liver weight for high-dose males and relative liver weight for both male and female dose groups were increased. Kidneys weights were increased in males at the highest dose and spleen weights were decreased in mid-and high-dose males. Histopathologic examination revealed mild hepatic centrilobular cytomegaly and fatty methomorphosis in mid- and high-dose males. These changes were also common to female rats, but occurred at a lower frequency. No

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histomorphologic changes were observed in the spleen. The low dose was not associated with any treatment-related findings; thus, the NOAEL was determined to be 1,000 ppm (~100 mg/kg body weight/day). Ref.: 34 Subchronic oral toxicity study in hamsters In the 13-week GLP hamster study, triclosan (FAT 80023) was administered to Syrian Golden Hamsters (15/sex/treatment group; 20/sex/control, 10/sex/group for interim sacrifice at 7 weeks) via the diet at doses of 0, 75, 200, 350, 750, or 900 mg/kg body weight/day. Treatment was not associated with any mortality or clinical signs. Decreased body weight gain was observed at 750 and 900 mg/kg body weight/day. Food consumption was decreased in males at 900 mg/kg body weight/day and females at 350 mg/kg body weight/day and above. Water consumption was increased at all groups given 200 mg/kg body weight/day and higher. Increased coagulation times, changes in red cell morphology and red cell indices indicated microcytic type anaemia in high-dose animals (750 and 900 mg/kg body weight/day). Clinical chemistry disturbances of liver and kidney function were observed at doses of 750 mg/kg body weight/day and 900 mg/kg body weight/day. Biologically significant clinical chemistry changes noted in alkaline phosphatase and alanine aminotransferase indicated possible liver toxicity; however, organ weight determinations and macroscopic and microscopic examination revealed no corresponding findings. The main target organ toxicity in hamsters was dose-related nephrotoxicity (tubular casts, tubular basophilia, tubular dilation). Although the LOEL may be estimated to be 200 mg/kg body weight/day, study investigators determined the NOEL to be 75 mg/kg body weight/day due to increased water consumption and some changes in urinalysis parameters. Ref.: 35 Comment of the SCCP The NOAEL is set at 200 mg/kg bw/d based on nephrotoxicity indicated by microscopic findings and polyuria, haemoglobinuria and haematouria.

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Table 12: Findings from GLP Sub-Chronic Oral Toxicity Studies for Triclosan
Species (Strain) Dosing Regimen and Duration 0, 25, 75, 200, 350, 750, 900 mg/kg bw/d for 13 weeks via dietary admixture. Interim sacrifice at 7 weeks. Major Findings Reference; GLP and OECD Status Trutter, 1993 (33); GLP: compliant OECD: No.408 consistent

Mouse (CD-1)

Conducted pursuant to OECD Guideline No. 408. Clinical signs at highest dose. Decreased food consumption and decreased body weight at 750 mg/kg bw/d. Increased alkaline phosphatase in females at 25 mg/kg bw/d and males at 200 mg/kg bw/d. Decreased total cholesterol at 25 mg/kg bw/d, increased alanine aminotransferase (males at 350 mg/kg bw/d, females at 750 mg/kg bw/d). GGT increased 750 mg/kg; however, not statistically significant. Decreased erythrocyte count and haemoglobin at 25 mg/kg bw/d (male) and all animals at 75 mg/kg bw/d. Increased liver weight and liver to body weight ratio at doses of 75 mg/kg bw/d in the main study and at doses of 350 mg/kg bw/d at interim sacrifice. Decreased kidney weights were observed in males at 350 mg/kg bw/d and females at 900 mg/kg bw/d. Enlarged dark/thickened lobes correlated with histomorphologic centrilobular hepatocellular hypertrophy, vacuolization, pigment accumulations, necrosis and/or inflammation noted in males at 75 mg/kg bw/d and all animals at 200, 350, 750 mg/kg bw/d. Extramedullary haematopoiesis was observed in the spleen of higher dose (750 mg/kg) animals. No histomorphologic alterations were observed in males at 25 mg/kg bw/d and in females at 75 mg/kg bw/d. A NOAEL could not be determined. Conducted pursuant to OECD Guideline No. 408. High doses were accompanied by decreased body weight with gradual decreased food consumption and diet spillage at initiation for all treatments. Treatment-related effects on erythrocyte parameters were observed. Interim necropsy revealed increased liver weight changes in males (mid- and high-dose) and females (high dose only). At terminal necropsy, liver weight for high-dose males and relative liver weight for both male and female dose groups were increased. Histopathologic examination revealed mild hepatic centrilobular cytomegaly and fatty methomorphosis in high and mid-dose males. These changes were also common to female rats; however, at a lower frequency. The NOAEL was considered to be 1,000 ppm (~100 mg/kg bw/d).

Rat (SpragueDawley)

0, 1,000, 3,000, or 6,000 ppm via diet (~ 0, 100, 300, or 600 mg/kg bw/d) for 90 days via dietary admixture. Interim sacrifice at Day 45.

Litton Bionetics, 1983 (34); GLP: compliant OECD: No.408 consistent

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Species (Strain)

Dosing Regimen and Duration 0, 75, 200, 350, 750 or 900 mg/kg bw/d for 13 weeks via dietary admixture. Interim sacrifice at Week 7.

Major Findings

Reference; GLP and OECD Status Schmid et al., 1994 (35); GLP: compliant OECD: No.408 consistent

Hamster (Syrian Golden)

Conducted pursuant to OECD Guideline No. 408. Decreased body weight gains were observed at 750 mg/kg bw/d. Food consumption was decreased in males at 900 mg/kg bw/d and females at 350 mg/kg bw/d. Water consumption was increased at all groups given 200 mg/kg bw/d and higher. Increased coagulation times, changes in red cell morphology and red cell indices in high-dose animals (750 mg/kg bw/d). Slightly increased alanine aminotransferase levels in females at the highest dose, and in both sexes increased cholesterol at the highest dose. At 750 mg/kg bw/d, decreased levels of alkaline phosphatase were observed. At 900 mg/kg bw/d, decreased gamma glutamyltransferase was observed. Urinalysis revealed polyuria, haemoglobinuria and haematouria in all animals given 350 mg/kg bw/d. Interim and terminal necropsy revealed no change in any major organ weight in females. In males, increased absolute and relative kidney weights at 750 mg/kg bw/d after 13 weeks and 900 mg/kg bw/d at the interim and terminal necropsy. Associated with these findings was tan discolour and/or granulated kidneys. Histopathology showed nephrotoxicity (e.g., tubular casts, tubular basophilia, tubular dilation) which was dose-related with respect to incidence and severity; microscopic changes were noted starting at the dose of 350 mg/kg bw/d. Stomach inflammation (gastritis, glandular erosions) was also observed at 750 mg/kg bw/d. Based on changes noted in water consumption, which were not considered adverse, the LOEL was determined to be 200 mg/kg bw/d; study investigators determined the NOEL to be 75 mg/kg bw/d. At 750 and 900 mg/kg/day, the kidneys were identified as a target organ based on macroscopic, histopathologic and clinical findings. Clinical chemistry changes also indicated liver effects; however, no microscopic findings were observed. The red blood cell also showed treatmentrelated effects. At 350 mg/kg/day, microscopic findings of nephrotoxicity were observed. The NOEL was determined by study investigators to be 75 mg/kg bw/day.

Subchronic oral toxicity study in rabbits The results of 2 studies in rabbits were inconsistent; in 1 study, triclosan was reported to be well-tolerated, with no treatment-related effects up to the dose level of 125 mg/kg bw/day, whereas in a second study, the NOAEL was determined to be 3 mg/kg bw/day, as animals given doses of 30 or 150 mg/kg bw/day showed pulmonary infections. However, it should be noted that study investigators stated that the relationship of the lung findings to triclosan was unclear. Ref.: 36, 37 Subchronic oral toxicity study in dogs, study 1 Inconsistency also was found in a comparison of 2 sub-chronic dog studies, the first of which showed haematology, clinical chemistry, macroscopic and microscopic findings at all doses tested, including some effects at the lowest dose tested of 25 mg/kg bw/day. As a result, no NOAEL was determined for the study. Ref.: 38

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Subchronic oral toxicity study in dogs, study 2 In a subsequent study, where doses were lowered to 0, 5, 12.5, or 25 mg/kg body weight/day, there were no treatment-related findings at any dose, even though the highest dose tested in the second study was the same as the lowest dose tested in the first study. Ref.: 39 Subacute & subchronic oral toxicity study in baboons In baboon studies 2 20 animals per dose level were administered 0, 1, 3, 10, 30 and 100 mg/kg via oral gelatine capsules for 4 or 13 weeks (6 animals 3 mg/kg bw/d), respectively. Symptoms such as agitation, anger and aggression were observed in the one female receiving 100 mg/kg. No other clinical signs were observed in other treatment groups. No treatment-related changes were found in ophthalmoscopic examinations and in water consumption, haematology and body weight. Findings were similar among terminal histopathological examination of treated groups from 4 weeks and 13 weeks of treatment. Evidence of chronic interstitial pneumonitis was seen throughout all animals. Lymphocytic infiltration was seen in the liver and large intestines. These findings were not different among control and treated animals. Ref.: 40 Long term (1 year) oral toxicity study in baboons A total of 56 animals (3/sex/dose (main study) plus 2/sex/dose (interim, 6 months) plus 2/sex/dose (recovery group, 4 weeks after dosing) were used. Animals were dosed orally, once daily, with prefilled capsules with 0, 30, 100, or 300 mg/kg bw/d triclosan. The controls received 1 capsule containing 600 mg lactose + 0.5% magnesium stearate per day. Vomiting was observed in some mid and high dose animals accompanied by an increased incidence of low food intake. Deterioration of condition and abdominal pain was observed in High Dose animals. Incidence of diarrhoea was increased in Mid Dose animals and greatly increased in High Dose animals. No significant effects in Males were observed except at 39 and 52 weeks, when decreased WBC noted in Mid Dose and High Dose groups. Females showed slight decreases in erythroid parameters at all time points for High Dose group, but only significant up to 26 weeks. Also decreased WBC was observed in High Dose Females at 52 weeks (not significant). Decrease in absolute brain weight and increases in kidney and liver weights (relative to BW) in High Dose animals were significant when Male and Female data combined (data at termination). NOEL is estimated to be 30 mg/kg bw/d based on the absence of any effect, including diarrhoea, in baboons at the low dose level in this study. Ref.: 41 Comment In baboon studies conducted at doses up to 300 mg/kg bw/d, both 4/13-week and 1-year investigations showed time-dependent haematology findings. Incidental clinical chemistry changes were observed; however, there was no evidence of hepatic or renal injury accompanying these findings [Ref. 40; 41]. Clinical signs observed in the longer-term study were not observed in the 4/13-week study. Significant differences in study design as well as quality of investigation likely contributed to discrepancies between these studies.

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Table 13: Findings from Non-GLP, Non-OECD Sub-Chronic Oral Toxicity Studies for Triclosan
Species (Strain) Dosing Regimen and Duration 0, 12.5, 25, 62.5 or 125 mg/kg bw/d for 90 days via dietary admixture. Major Findings Reference; GLP and OECD Status Leuschner et al., 1970a (36); Predates GLP and OECD OECD: comparable Paterson, 1969 (37); Predates GLP and OECD OECD: comparable

Rabbit (New Zealand White)

Conducted prior to OECD guidelines, but considered similar in design. Terminal examination revealed no organ weight changes or gross pathology findings. Histopathology examination found no differences in high-dose animals compared to control animals. Incidental microscopic changes noted in high-dose animals included granulomatous infiltrations in lungs in 1 female and 2 males and nephrosis in 1 high-dose female. Overall, triclosan was well tolerated at all doses. No NOAEL was determined in this study, in which the highest dose used did not produce treatment-related effects. Conducted prior to OECD guidelines, but considered similar in design. Dose-related mortality was observed at 30 and 150 mg/kg bw/d. At doses of 30 and 150 mg/kg bw/d, neutrophilia and lymphopenia were observed on various observation days but were not consistent throughout the study. The lung (which was associated with macroscopic and microscopic changes) appeared to be the target organ. Pulmonary infection was observed in 3/6 rabbits at 30 mg/kg bw/d and 3/6 rabbits from 150 mg/kg bw/d. Perirenal abscess in 1 rabbit was observed at 30 mg/kg bw/d. Limited organ weight determinations showed no treatment-related findings. Gross macroscopic findings in the lung corresponded with histopathologic lung lesions, oedema in 30 and 150 mg/kg bw/d treated animals and lung necrosis in 2 high-dose animals. No such histomorphology changes were noted in control or 3 mg/kg bw/d animals. Based on findings of neutrophilia and lymphopenia at 30 mg/kg bw/d and absence of histomorphologic alterations, 3 mg/kg bw/d was determined to be the NOAEL. However, the study authors stated that the relationship of the lung lesions, infection, oedema and sometimes necrosis observed at the 2 highest doses to test article administration is unclear. Dosing accidents or regurgitation with resultant pulmonary infection was suggested. Conducted prior to OECD guidelines, but considered similar in design, with the exception that clinical pathology evaluations were limited. Seven unscheduled deaths (1-25 mg/kg bw/d, 2-100 mg/kg bw/d and 4-200 mg/kg bw/d). Severity of diarrhoea was dose-related. Haematology evaluations revealed decreased haemoglobin, PCV and red blood cells and increased ESR and reticulocytes in dogs at 50 mg/kg bw/d as well as during interim evaluation in moribund dogs. Increased alkaline phosphatase in all animals at doses of 50 mg/kg bw/d. Additionally, high SGOT and SGPT were noted in these animals. Bile salts, polymorphonuclear leukocytes were observed in dogs at 25 mg/kg bw/d. Terminal organ weight determinations revealed increased liver, pancreas and adrenal weights for 100 and 200 mg/kg bw/d animals. Bile retention, necrosis, pathological fat and unusual Kupffer cell activation were observed histopathologically in the liver. In animals which showed severe liver damage, the bone marrow was hyperplastic. In 2 dogs given 200 mg/kg bw/d and one dog at 100 mg/kg bw/d, focal interstitial nephritis was observed. Convoluted epithelium of the kidney was observed in 2 dogs given 25 mg/kg bw/d but not observed at any other dose. Based on the findings in this study, no NOAEL was determined.

Rabbit (Albino)

0, 3, 30, 150 mg/kg bw/d for 13 weeks via oral gavage.

Dog (Beagle)

0, 25, 50, 100 or 200 mg/kg bw/d via gelatine capsules for 91 days.

Paterson, 1967 (38); Predates GLP and OECD OECD: comparable

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Species (Strain)

Dosing Regimen and Duration 0, 5, 12.5 or 25 mg/kg bw/d via diet for 90 days.

Major Findings

Reference; GLP and OECD Status Leuschner et al., 1970b (39); Predates GLP and OECD OECD: comparable Noel et al., 1969 (40); Predates GLP and OECD OECD: comparable

Dog (Beagle)

Conducted prior to OECD guidelines, but considered similar in design. Occasional pasty to thin faeces were noted in all groups. Haematology, clinical chemistry and urinalysis evaluations were limited to high-dose group animals. Macroscopic evaluations found no gross pathology for any organ. Organ weight measurements showed no changes in relative or absolute weights. Histopathologic evaluation of all major organs from all animals showed no difference among treated and control animals. No NOAEL was determined in this study, in which the highest dose used did not produce treatment-related effects. Conducted prior to OECD guidelines, but considered comparable in design (but limited in number of parameters evaluated). Blood chemistry revealed no haematology changes. Urinalysis showed no changes. Clinical chemistry changes included high plasma urea levels in 2 animals at 30 mg/kg bw/d, 2 animals at 3 mg/kg bw/d, 1 control baboon and increased SGPT in individual male baboons at 1 mg/kg bw/d and 3 mg/kg bw/d. Terminal necropsy after 4 weeks and after 13 weeks showed no organ weight differences among treated and controls. No gross macroscopic findings were observed. After 4 weeks of treatment, minor changes such as dark nodules in the large intestine wall at 10 mg/kg bw/d. Adhesion in lung surface and rib cage, slight thickening of capsule of the liver in 10 mg/kg bw/d female and 1 mg/kg bw/d male, and control male and female were observed. Limited conclusions can be derived from this study due to insufficient number of animals evaluated. A NOAEL was not determined for this study. Conducted prior to OECD guidelines, but considered similar in design (but limited in number of parameters evaluated). Vomiting and diarrhoea occurred at mid and high-doses. Haematology parameters showed decreased white blood cells in males at Week 29 and 52 in mid and high-dose groups. In females, slight decreases in erythroid parameters at all time points were observed for the high-dose group (statistically significant only up to Week 26). Incidental changes in potassium and sodium were observed. Slight changes, both increases and decreases in SGOT and AP were observed; however, not considered treatment-related. No differences were found in urinalysis. At necropsy, decreased absolute brain and increases in kidney and liver weights were observed in high-dose animals. Histopathologic examination found similar histology in both treated and control animals. The NOEL was determined to be 30 mg/kg bw/d.

Baboons (papio cynocephalus and anubis)

4-week dosing: 0, 1, 10, 30, or 100 mg/kg bw/d via gelatine capsules. 13-week dosing: 0 or 3 mg/kg bw/d via gelatine capsules.

Baboons (papio)

0, 30, 100, or 300 mg/kg bw/d via capsules daily for 1 year. Necropsies were scheduled at 6 months, 12 months, and 13 months (28 days post-treatment).

Ciba-Geigy, 1975a (41); Predates GLP and OECD OECD: comparable

3.3.5.5.

Sub-Chronic Dermal Toxicity

The sub-chronic dermal toxicity of triclosan has been investigated in rats, dogs, and monkeys. The studies conducted in weanling dogs and newborn Rhesus monkeys were not GLP-compliant and not conducted pursuant to OECD guidelines. The pertinent details from these studies are summarized in Tables 14 (GLP rat study) and 15 (non-GLP, non-OECD studies). Sub-Chronic Dermal Toxicity (90 d) in rats The rat study was conducted according to both GLP and OECD guidelines (Crl:CDBR (VAF/Plus) strain). IRGASAN DP300 (0, 10, 40, 80 mg/kg bw/d) diluted in propylene glycol was applied to clipped, unabraded dorsal surface of each animal (approx. 10% of total body

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surface) at dose volume of 2.0 mL/kg daily (approximately 0.5 to 4.0% triclosan). The appropriate dose was applied under a gauze pad and covered. Daily contact exposure was for at least 6 hours. Dermal observations of erythema and/or showed oedema in all treated groups. No treatment-related changes were found in ophthalmoscopic examinations and in water consumption and body weight. Occult blood was observed in the urine of high-dose and satellite male rats and to a lesser extent in mid-dose males and females. Isolated changes were observed in erythrocyte parameters in high dose animals. Small but statistically significant changes were observed in some serum chemistry parameters. NOAEL = 80 mg/kg/day (excluding dermal irritation) Ref.: 11 Sub-Chronic Dermal Toxicity (90 d) in dogs Weanling dogs exposed to triclosan through dermal application for 90 days of doses ranging from 2 to 200 mg/kg bw/day in a non-GLP study showed no toxicity except for dermal irritation at the highest dose tested. Ref.: 12 Sub-Chronic Dermal Toxicity (90 d) in monkeys The major findings from a 90-day bathing study conducted in newborn Rhesus monkeys showed that repeated exposure to triclosan (0.1% in a soap solution, 5 min exposure) was well-tolerated. No treatment-related toxicities were observed. Ref.: 13 Table 14: Findings from GLP Sub-Chronic Dermal Toxicity Studies for Triclosan
Species (Strain) Dosing Regimen and Duration Major Findings Reference; GLP and OECD Status Trimmer, 1994 (11); GLP: compliant OECD: compliant

Rat (Crl: CDBR VAF/ Plus strain)

0, 10, 40, or 80 mg/kg bw/d in propylene glycol applied under gauze for at least 6 hours (90 days). Triclosan was applied to approx. 10% of total body surface in a volume of 2 mL/kg bw. Approximate triclosan test concentrations were ~0, 0.5, 2, and 4% for a 0.25 kg animal. A recovery group was included at the high dose.

Conducted pursuant to OECD Guideline No. 411. Dermal erythema and/or oedema was observed in all treatment groups. Haematology parameters showed isolated changes in erythrocyte parameters in high-dose animals; however, these findings were within expected range. Similarly, clinical chemistry findings showed small but statistically significant changes in serum chemistry. Occult blood in urine was observed in the high dose and satellite male rats and to a lesser extent in mid-dose females and males. Histopathology examinations observed eschar and desquamation, hyperplasia/hyperkeratosis of epidermis, dermal inflammation and focal necrosis observed at all doses. Reversal of the dermal effects was seen during the 28-day recovery period. Microscopic changes in the urinary bladder of 3 males were observed. Coagulative necrosis of hepatocytes was also observed. Both findings lacked a dose-response. With respect to general toxicity, the NOAEL was determined to be 80 mg/kg bw/d (the highest dose tested). No NOAEL for dermal toxicity was determined.

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Table 15: Findings from Non-GLP, Non-OECD Sub-Chronic Dermal Toxicity Studies for Triclosan
Species (Strain) Dosing Regimen and Duration Major Findings Reference; GLP and OECD Status Dorner, 1973 (12); Predates GLP and OECD

Dog (Beagle weanling)

0, 2, 20, or 200 mg/kg bw/d applied to a shaved area of the neck for 90 days (12.5% triclosan added to 50% corn-starch vehicle w/vol; size of application area not reported.) 0.1% soap solution, lathered for 5 minutes and then washed for 90 days (size of application area not reported). Recovery group: 30 days without bathing.

Conducted prior to OECD guidelines and missing a number of study parameters. Upper respiratory disease during the study up to Day 25. Diarrhoea in all animals was not attributed to the test substance. Dermal irritation (dermatitis) was observed at the high dose and was dose-dependent. There were no treatment-related biochemical or haematological changes. Microscopic examination revealed no histomorphology changes in the skin. Test substance was reported to show little toxic effect aside from dermal irritation (pathology data not reported).

Monkey (Rhesus, newborn)

Conducted prior to OECD guidelines and not comparable to current OECD guidelines (e.g., exposure method). Mild anaemia, red blood cell and haemoglobin values showed variations between animals, but was not considered treatment-related. These changes were attributed to frequent sampling. Limited clinical chemistry parameters were evaluated due to limitations in blood sampling; however, of the parameters measured, no consistent changes were noted. Termination examination showed no organ weight changes. Histopathologic evaluation revealed similar findings of focal adrenal mineralisation, pulmonary infiltration, and low-grade pneumonia in both treated and control animals. Hepatocellular vacuolar changes and hepatic extramedullary haematopoiesis were observed in both treatment and control animals. No histological changes were observed in skin sections taken for examination. No differences were noted between animals sacrificed after 90 days of treatment and recovery animals. This 0.1% triclosan soap solution was well-tolerated under the conditions of this study.

Hazleton Labs, 1979a (13); Predates GLP and OECD OECD: no applicable guidelines

3.3.5.6.

Chronic (> 1 year) Toxicity

Chronic toxicity was assessed in the carcinogenicity studies with rats, mice and hamsters (See also 3.3.7). Long Term Toxicity/Carcinogenicity study 18 Months (Mouse) Animals were dosed daily via the diet for 544-552 days in total. Dietary admixtures were mixed weekly for 13 weeks, then every 4 weeks; amounts of triclosan were adjusted using most recent weekly body weight and feed consumption data. 0, 10, 30, 100, or 200 mg/kg bw/d in the diet (control, low dose, low mid dose, high mid dose, or high dose). A significant decrease in survival was observed in High Mid Dose males and high dose females whereas high dose males the decrease was slight but not significant. No adverse effects on Body weight, food consumption and urine parameters attributable to the test substance were observed. In biochemistry significant changes at 18 months were increased (230-560%) liver function enzymes in high Mid Dose and High Dose Males and Females; decreased (75-90%) cholesterol in all treated Males and Females; decreased bilirubin (up to 67%) in Females (all doses except Low Dose). In haematology: small (<15%) but significant dose-related changes in erythroid parameters (Males and Females). Increased % reticulocytes (males) and platelets (males and females; 25-37%, dose-related) significant at 18 months. Increased WBC, neutrophils, & lymphocytes at higher doses in

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Males (6 months) were not considered indicative of test substance toxicity by the study authors. No changes were recorded at low Dose. Dose-related, significant increases in liver weights were observed in males and females at low Mid Dose, high Mid dose and High Dose. Increase in incidence of hepatic nodules/masses and/or discolorations in Males and Females of high Mid Dose and High Dose groups compared to control. Slight increase in testicular germinal epithelium degeneration/atrophy was observed at High Dose (only controls and High Dose examined). No treatment-related histopathological changes other than in liver. Hepatocyte hypertrophy was present in all animals except Low Dose Females. Brown pigment in hepatocytes in higher dose rats was found to be lipofuscin and iron. The LOAEL was 10 mg/kg bw/d based on liver changes. This dose level was considered as NOAEL based on haematotoxicity when excepting the target organ liver. Ref.: 66 Long Term Toxicity/Carcinogenicity study 104-week (Rat) Animals were dosed daily via the diet at concentrations of 0, 300 (Low Dose), 1,000 (Mid Dose), 3,000 (High Dose) ppm in 104-week study. An extra group of rats was given a toxic dose of 6,000 ppm and killed at 52 weeks. Doses were calculated weekly based on food intake and weekly mean BW. At 52 weeks, calculated doses were 0, 12, 40, and 127 mg/kg bw/d (Males) and 0, 17, 56, and 190 mg/kg bw/d (Females), respectively. The dose of 6,000 ppm gave doses of 247 (Males) or 422 (Females) mg/kg bw/d. No treatment-related effects on mortality were observed. Body weight was significantly decreased in High Dose Females up to week 52. Food Consumption was generally significantly increased in High Dose Males. In biochemistry transient changes in protein, BUN, glucose, bilirubin, triglyceride and liver enzymes were found. In haematology slight (4-10%) but significant changes in erythroid parameters (red blood cell count in Males and Females), including in Low Dose Males rats at week 104 were recorded. Also increases in mean corpuscular haemoglobin concentration was observed in females at mid and high dose and in males in addition at low dose. Slightly increased altered red cell morphology was observed in High Dose Males. Increased clotting time (High Dose Males), decreased WBC (33%) in Females was measured at 104 weeks. Decreases in % monocytes were transient (High Dose Females) but significant at 104 weeks (High Dose Males). At 52 weeks slight relative weight decreases at High Dose were observed: liver, brain, kidneys (Males) and heart, ovaries and spleen (Females). At 104 weeks increased absolute adrenal weight (Mid Dose Males) and decreased brain weight (High Dose Males); increased absolute and relative ovary weight (High Dose Females), decreased relative spleen weight (High Dose Females), and decreased absolute and relative spleen weights (Mid Dose Females). Morphology revealed hepatocyte hypertrophy and hepatocytic inclusions (hyaline-staining, round-shaped) in Male rats: significant High Dose (13 weeks), 6,000 ppm (52 weeks). No other histological lesions were considered to be treatment-related. The study authors considered the NOAEL as ~48 mg/kg/d for both sexes, combined (~56 mg/kg bw/d in Females and ~40 mg/kg bw/d in Males). Ref.: 67 Comment SCCP considers the NOAEL as 12 mg/kg bw/d due to haematoxicity and decreased absolute and relative spleen weights (Mid Dose Females). Long Term Toxicity/Carcinogenicity study 95 Weeks (Hamster) The animals were administered 0, 12, 75, or 250 mg/kg bw/d FAT 80023/S. in the diet for 90 weeks. Survival was significantly decreased in males and was generally poor (38-58%) in females at 90 weeks. Body weight gain was significantly decreased in all high-dose hamsters and was accompanied by a slight (3%), but significant decrease in food

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consumption in high-dose females. Biochemical changes observed at termination included increases (<50%) in blood urea nitrogen in high-dose hamsters and in triglycerides in midand high-dose males. Haematological changes observed included slight (<15%), but significant decreases (but not dose-related) in erythroid parameters in mid- and high-dose animals, increased white blood cells in high-dose animals, and increased lymphocytes in high-dose females at termination. At termination, renal nephropathy was observed in animals of all dose groups, with increased incidence and severity at the high-dose level. In addition, males showed atypical hyperplasia in the stomachs (fundic region), along with spermatozoa and germ cell effects at the high dose. One high-dose female showed atypical hyperplasia in the fundic region that was considered to be treatment-related. High-dose females showed distended gastric glands and a few treated females of all doses showed benign papillomas of the non-glandular region of the stomach (not discussed in report). Hepatic effects were few, with only rarified hepatocytes reported in a few male animals (6/60 in high dose vs. 3/121 in controls). The NOAEL was set as 75 mg/kg bw/d. Ref.: 68 Comment on setting a NOAEL In the Table below the derived NOAELs from subchronic and chronic studies in different species were compiled. EPA in its recent evaluation selected the NOAEL of the baboon study (30 mg/kg bw/d) for risk assessment based on clinical signs of toxicity which are presumably due to oral treatment. This might not be relevant for cosmetic uses. The applicant in its safety evaluation used the NOAEL of the 95-week study in hamsters as this species was judged to be the most relevant to humans based on pharmacokinetics (75 mg/kg bw/d). Alternatively as a more conservative value, the NOEL of the 104-week rat study ( 48 mg/kg bw/d for both sexes) was used. SCCP considers the NOAEL of this long term toxicity study in rats as 12 - 17 mg/kg bw/d ( 14.5 mg/kg bw/d) due to haematoxicity and decreased absolute and relative spleen weights. Haematoxicity was also detected in the 13-week subchronic oral toxicity studies in mice and rats, in hamsters only at higher doses and in the 1-year toxicity study in baboons. This was further confirmed by changes in haematology parameters in the long term studies in mice and hamsters. Interestingly, also in the 13-week subchronic dermal toxicity study in rats changes in erythrocytes parameters were observed. The SCCP will use the NOAEL of 12 mg/kg bw/d of the long term toxicity study in rats for risk assessment.
Subchronic oral toxicity study in mice Subchronic oral toxicity study in rats Subchronic oral toxicity study in hamsters Long term (1 year) oral toxicity study in baboons Long Term Toxicity / Carcinogenicity study 18 Months (Mouse) Long Term Toxicity / Carcinogenicity study 104-week (Rat) Long Term Toxicity / Carcinogenicity study 95 Weeks (Hamster) A NOAEL was not established from this study since treatment-related changes in haematology parameters, increased alkaline phosphatase, and decreased cholesterol were observed at the low dose 25 mg/kg body weight/day. The low dose was not associated with any treatment-related findings; thus, the NOAEL was determined to be 1,000 ppm (~100 mg/kg body weight/day). The NOAEL is set at 200 mg/kg bw/d based on nephrotoxicity indicated by microscopic findings and polyuria, haemoglobinuria and haematouria. NOEL is estimated to be 30 mg/kg bw/d based on the absence of any effect, including diarrhoea, in baboons at the low dose level in this study. The LOAEL was 10 mg/kg bw/d based on liver changes. This dose level was considered as NOAEL based on haematotoxicity when excepting the target organ liver. The study authors considered the NOAEL as 48 mg/kg/d for both sexes, combined ( 56 mg/kg bw/d in Females and 40 mg/kg bw/d in Males). SCCP considers the NOAEL as 12 mg/kg bw/d due to haematoxicity and decreased absolute and relative spleen weights (Mid Dose Females). The NOAEL was set as 75 mg/kg bw/d.

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3.3.6. 3.3.6.1.

Mutagenicity / Genotoxicity Mutagenicity / Genotoxicity in vitro

Bacterial gene mutation assay Study 1 Guidelines: Species/Strain: Replicates: Test substance: Solvent: Batch: Purity: Concentrations: Treatment: GLP: Date: / Salmonella typhimurium TA92, TA98, TA100, TA1535, TA1537 triplicates in a single test FAT 80 023/A DMSO / / 0.1, 0.3, 0.9, 2.7 and 8.1 g/ml without metabolic activation Additionally 24.3 and 72.9 g/ml without metabolic activation for TA92 0.1, 0.3, 0.9, 2.7, 8.1, 24.3 and 72.9 g/ml with metabolic activation Plate incorporation method with 48 h incubation time / May 1978

The Ames-test was performed with the bacterial tester strains Salmonella typhimurium TA92, TA98, TA100, TA1535 and TA1537 with and without S9-mix. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Toxicity was evaluated on the basis of a reduction in the number of spontaneous revertant colonies. The experiment was performed with the direct plate incorporation method. Justified negative and positive controls were concurrently tested. Results The test compound did not induce an increase in the number of revertant colonies in any strain at any concentration tested both in the presence or absence of metabolic activation. Conclusion Under the experimental conditions used FAT 80 023/A was not mutagenic in the gene mutation tests in bacteria both in the absence and the presence of S9 metabolic activation. Ref.: 42 Comment The test is performed before the implementations of OECD guidelines. Batch number and purity were not reported. The test has only limited value. Study 2 Guideline: Strain: Replicates: OECD 471 Salmonella typhimurium TA98, TA100, TA1535 and TA1537 3 replicates in 2 individual experiments both in the presence and absence of S9-mix. Test substance: Triclosan [Irgasan DP 300; 2,4,4-trichloro-2-hydroxy diphenyl] Solvent: DMSO Batch: S 15155 TO1 Purity: > 99% Concentrations: 0.015, 0.05, 0.15, 0.5 and 1.5 g/plate both without and with S9-mix Metabolic activation: Experiment 1: 3% and 10% Experiment 2: 10% and 30%

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Treatment: GLP: Date:

pre-incubation method with 60 minutes incubation and a selection period of 72 h. In compliance March September 1988

Triclosan was investigated for the induction of gene mutations in Salmonella typhimurium (Ames test). Test concentrations were based on the results of a preliminary toxicity study with concentrations up to the (nowadays) prescribed maximum concentration of 5000 g/plate. Toxicity was evaluated on the basis of a substantial reduction in the number of micro-colonies in the background bacterial lawn. The experiments were performed with the pre-incubation method. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Negative and positive controls were in accordance with the OECD guideline. Results In the preliminary toxicity study triclosan was toxic towards the tester strains doses and consequently 1.5 g/plate was chosen as the top dose level. experiments toxicity was reported at the top dose level. No substantial revertant colony numbers in any of the tester strains were observed at any tested either in the absence or presence of metabolic activation. at the higher In the main increases in of the doses

Conclusion Under the experimental conditions used triclosan was not genotoxic (mutagenic) in the gene mutation tests in bacteria both in the absence and the presence of metabolic activation. Ref.: 43 Study 3 Guideline: Strain: Replicates: Test substance: Solvent: Batch: Purity: Concentrations: OECD 471 Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 triplicates both in the presence and absence of S9-mix TA100: triplicates in 2 retests in the absence of S9-mix 39316 DMSO CC# 14663-09 / Experiment 1: 0.00167, 0.005, 0.0167, 0.05, 0.1 and 0.167 g/plate without S9-mix 0.05, 0.167, 0.5, 1.67, 2.5 and 5 g/plate with S9mix Experiment 2 and 3: 0.000167, 0.0005, 0.00167, 0.005, 0.01, 0.0167, 0.0333, 0.05, 0.1 and 0.167 g/plate without S9-mix for TA100 only. direct plate incorporation with 48 incubation without and with S9-mix In compliance March 1993

Treatment: GLP: Date:

Test compound 39316 was investigated for the induction of gene mutations in Salmonella typhimurium (Ames test). Test concentrations were based on the results of a preliminary toxicity pre-screen with concentrations up to the prescribed maximum concentration of 5000 g/plate evaluating the growth of the background lawn and/or frequency of spontaneous revertants. The experiments were performed with the direct plate incorporation method. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Negative and positive controls were in accordance with the OECD guideline.

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Results Toxicity was reported at the higher doses tested. However, due to excessive toxicity for strain TA100 without S9-mix only three acceptable dose levels remained. In a retest a statistical significant but not dose dependent increase in revertant colonies was observed. This finding was not confirmed in a second retest. Biologically relevant, dose dependent increases in the number of revertants were also not observed in the other strains at any of the doses tested both in the absence or presence of metabolic activation. Conclusion Under the experimental conditions used test compound 39316 was not genotoxic (mutagenic) in the gene mutation tests in bacteria both in the absence and the presence of S9 metabolic activation. Ref.: 44 Comment The test was not repeated. Purity of test substance 39316 (triclosan) was not reported.

Mutagenicity test with Saccharomyces cerevisiae Study 1 Guideline: Strain: Replicates: Test substance: Solvent: Batch: Purity: Concentrations: Treatment: GLP: Date: / Saccharomyces cerevisiae MP-1 triplicates FAT 80 023/A DMSO / / 10, 20, 30, 40, 50, 60 and 200 mg/l 3.5 h treatment, followed by an expression period of 4-6 days for interand intragenic recombinants or 8 days for cycloheximide resistance. / November 1978

FAT 80 023/A was investigated for the induction of gene mutations in a mutagenicity test with Saccharomyces cerevisiae. Test concentrations were based on the results of a preliminary toxicity study. 4-nitroquinoline-N-oxide served as positive control. For the determination of inter- and intragenic recombinants yeast cells were cultured on normal and tryptophan free agar, for the determination of cycloheximide resistance, an indication of forward mutations, on cycloheximide agar. Results In the main experiment no colonies were found at the highest dose as a result of the inhibitory effect of FAT 80 023/A on the growth of the yeast cells. Treatment with FAT 80 023/A did not result in a significant increase in the incidence of intergenic recombinants nor in an increase in cycloheximide resistance. The occasional increases in intragenic recombinants in the mid doses of 30 and 40 mg/l were not dose dependent and can be attributed to variation inherent in the test system Conclusion Under the experimental conditions used FAT 80 023/A was not mutagenic in this mutagenicity test in yeast. Ref.: 45 Comment

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The test is performed before the implementations of OECD guidelines. Batch number and purity of FAT 80 023/A (triclosan) were not reported. The test has only limited value.

Study 2 Guideline: Strain: Replicates: Test substance: Solvent: Batch: Purity: Concentration: Treatment: GLP: Date: / Saccharomyces cerevisiae MP-1 20 replicates in 3 experiments Irgasan DP 300 [5-chloro-2-(2,4-dichlorophenoxy)-phenol] DMSO / 99.7% isomer pure 0.2 mg/ml 3.5 h treatment, followed by an expression period of 4 days for interand intragenic recombinants or 8 days for actidione resistance. / June 1978

Irgasan DP 300 was investigated for the induction of gene mutations in a mutagenicity test with Saccharomyces cerevisiae. Only one test concentration was used. For the determination of inter- and intragenic recombinants yeast cells were cultured on normal and tryptophan free agar, for the determination of actidione resistance, an indication of forward mutations, on actidione agar. A positive control was not included. Results The results demonstrated that Irgasan DP 300 had an effect in the mutation and intergenic recombination system but not on the intragenic recombination system. Conclusion Under the experimental conditions used Irgasan DP 300 was mutagenic in this mutagenicity test in yeast. Ref.: 46 Comment The test is performed before the implementations of OECD guidelines. Batch number was not reported. Only one dose was tested. The test has only very limited value.

In vitro gene mutation assay with Mouse Lymphoma cells Study 1 Guideline: Species/strain: Replicates: Test substance: Solvent: Batch: Purity: Concentrations: Treatment: GLP: Date: / Mouse lymphoma cell line L5178Y/tk+/Duplicates, two independent tests FAT 80 023/A 0.05 N NaOH / / 15.8 g/ml (18h treatment) and 28.9 g/ml (4 h treatment) without metabolic activation 4 or 18 h treatment and an expression period of 3 days. The selection period was not mentioned (probably 12 days) / May 1978

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FAT 80 023/A was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Test concentrations were based on the results of a toxicity test on cell survival. The concentration required to produce 80% cell-kill was calculated. In the main test, cells were treated for 4 or 18 followed by an expression period of 3 days to fix the DNA damage into mutations. The incidence of mutants was determined with methothrexate, cytosine arabinoside and thymidine as antimetabolites. Negative and positive controls were not included. Results Following treatment with FAT 80 023/A an increase in the mutant frequency was not observed at both dose levels tested in the absence of S9-mix. Conclusion Under the experimental conditions used FAT 80 023/A was not mutagenic in mouse lymphoma cells in vitro. Ref.: 49 Comment The test is performed before the implementations of OECD guidelines. Toxicity was not measured in the main experiment. Batch number and purity of FAT 80 023/A (triclosan) were not reported. The test has only limited value. Study 2 Guideline: Cells: Replicates: Test substance: Solvent: Batch: Purity: Concentrations: / L5178Y mouse lymphoma cells duplicate cultures in 2 independent experiments Triclosan DMSO S15155 TO1 > 99% Experiment 1: 5.0, 7.5, 10.0, 15.0, and 20.0 g/ml without S9-mix 3.5, 7.5, 10.0 and 15.0 g/ml with S9-mix Experiment 2: 2.5, 5.0, 7.5, 10.0 and 15.0 g/ml without S9-mix 1.0, 5.0, 7.5, 10.0 and 15.0 g/ml with S9-mix 3 h treatment without and with S9-mix; expression period 48 h and selection period of 12 days In compliance September 1988

Treatment GLP: Date:

Triclosan was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Test concentrations were based on the results of a preliminary toxicity test considering suspension growth. In the main test, cells were treated for 3 h in the absence or presence of S9-mix followed by an expression period of 48 h to fix the DNA damage into a stable tk mutation. Liver S9-mix fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Toxicity was measured in the main experiments as mean % survival relative to the solvent control cultures. Negative and positive controls were included. Results In experiment 2 without S9-mix and in experiment 1 with S9-mix the highest dose tested showed the appropriate level of toxicity (10-20% adjusted relative total growth after the highest dose). In the experiment 1 without S9-mix and in experiment 2 with S9-mix excessive toxicity was seen at the highest dose but the first analysable doses did show the appropriate level of toxicity.

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A more or less dose dependent increase in the mutant frequency was found in all experiments. However, the increase in mutant frequency was either less than 2-fold the background mutant frequency or for concentrations with a mutant frequency higher than the 2-fold back ground level the relative survival exceeded the 10%. Conclusion Under the experimental conditions used, triclosan was considered not mutagenic in the mouse lymphoma assay at the tk locus. Ref.: 50 Comments The test was not performed according the OECD guideline. In vitro unscheduled DNA synthesis test Study 1 Guideline: Species/strain: Replicates: Test substance: Solvent: Batch: Purity: Concentrations: Treatment: GLP: Date: / primary rat hepatocytes triplicate subcultures Triclosan DMSO S15155 TO1 / Experiment 1: 0.6, 1.3, 2.5, 5, 10, 20, 40 and 80 g/ml Experiment 2: 0.16, 0.3, 0.6, 1.3, 2.5, 5, 10 and 20 g/ml 18 - 20 h treatment In compliance May - September 1988

Triclosan was investigated for the induction of unscheduled DNA synthesis (UDS) in primary hepatocytes of rats. Hepatocytes were isolated from male Fischer F344 rats. Hepatocytes were exposed simultaneously to triclosan and 10 Ci/ml 63H-thymidine for 18 - 20 h. After treatment cultures were divided in 3 subcultures for quantification of UDS whereas a fourth subculture was used to determine the toxicity of each treatment. Evaluation of autoradiography was done 7 days after exposure. UDS was measured by counting nuclear grains and subtracting the average number of grains in 3 nuclear-sized areas adjacent to each nucleus; this value is referred to as net nuclear grain count. Unscheduled synthesis was determined in 50 randomly selected hepatocytes. Negative and positive controls were included. Results In experiment 1 at concentrations above 10 g/ml no viable cells were seen at the end of the exposure period. In experiment 2 at 10 g/ml only 14% survival was observed at the end of exposure whereas at 20 g/ml no viable cells were seen at all. Although autoradiography was performed on all treated cultures, the autoradiographs of 20, 40 and 80 g/ml of experiment 1 and of 20 g/ml of experiment 2 were not scored due to excessive toxicity. At none of the remaining dose levels in both experiments neither an increased net nuclear grain count, nor a notable increase in cells with 6 or more nuclear grains per cells, nor a notable increase in cells with 20 or more nuclear grains per cells (cells in repair) was observed. Conclusion Under the experimental conditions used triclosan did not induce unscheduled DNA synthesis in primary rat hepatocytes and, consequently, is not genotoxic in the in vitro UDS test.

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Ref.: 52 Comment The test was not performed according the OECD guideline. Purity was not reported. The test has only limited value. Study 2 Guideline: Species/strain: Replicates: Test substance: Solvent: Batch: Purity: Concentrations: Treatment: GLP: Date: / primary rat hepatocytes triplicate cultures 39317 DMSO / / 0.25, 0.5, 1 and 2.5 g/ml 18 - 20 h treatment In compliance March - April 1993

39317 was investigated for the induction of unscheduled DNA synthesis (UDS) in primary hepatocytes of rats. Hepatocytes were isolated from male Fischer F344 rats. Hepatocytes, grown on cover slips, were exposed simultaneously to 39317 and 10 Ci/ml 3H-thymidine (specific activity 50 - 80 Ci/mM) for 18 - 20 h. Test concentrations were based on the results of a preliminary screen on precipitation of 39317. Evaluation of autoradiography was done 7 days after exposure. Coverslips from each dose were pre-screened for toxicity by visual inspection under a microscope. UDS evidenced as a net increase of grains over the nucleus was quantified by determining the nuclear and cytoplasmic grain counts. Cytoplasmic grain count was performed in 3 nuclear-sized areas adjacent to each nucleus. Unscheduled synthesis was determined in 150 nuclei per dose point. Negative and positive controls were included. Results Analytical results of the test article performed by the sponsor indicated that the actual concentrations were 0.3, 0.6, 1.18 and 3 g/ml. At none of the dose levels an increased net nuclear grain count nor an increase in cells with 5 or more nuclear grains per cells (cells in repair) was observed. Conclusion Under the experimental conditions used 39317 did not induce unscheduled DNA synthesis in primary rat hepatocytes and, consequently, is not genotoxic in the in vitro UDS test. Ref.: 53 Comment The test was not performed according the OECD guideline. The test was not repeated. Batch number and purity were not reported. However, stability and purity were reported as the responsibility of the sponsor. In vitro chromosome aberration test Study 1 Guideline: Species/strain: Replicates: OECD 473 (1983) Chinese hamster ovary (CHO-K1 BH4) cells duplicates in a single test

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Test substance: Solvent: Batch: Purity: Concentrations: Treatment: GLP: Date:

Triclosan DMSO S15155 TO1 > 99% 0.1, 0.3, 0.5 and 1.0 g/ml without metabolic activation 4.8, 9.5, 19 and 30 g/ml with metabolic activation 24 h treatment without S9-mix. 6 h treatment with harvest time 24 h after start of treatment with S9mix in compliance April August 1988

Triclosan has been investigated in the absence and presence of metabolic activation for the induction of chromosomal aberrations in CHO cells. Test concentrations were chosen on the basis of the results of a preliminary toxicity test measuring cell death and decline in mitotic index. In the absence of S9 cells were treated for 24 h and immediately harvested; in the presence of S9 cells were treated for 6 h and harvested 24 h after the start of treatment. Two hours before harvest, each culture was treated with colchicine solution (final concentration 0.25 g/ml) to block cells at metaphase of mitosis. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Chromosome (metaphase) preparations were stained with 10% Giemsa and examined microscopically for chromosomal aberrations. Negative and positive controls were in accordance with the OECD guideline. Results In both the absence and the presence of a metabolic activation system, triclosan did not cause a statistical increase in cells with chromosome aberrations. Conclusion Under the experimental conditions used triclosan did not show evidence for a genotoxic (clastogenic) activity in CHO cells in vitro. Ref: 47 Comment It is not known whether in the main test the cells were sufficiently exposed since the mitotic index was not determined. Yet, in the experiment with metabolic activation a dose dependent increase was seen with a 4-fold increase in the highest dose compared to the untreated control. Therefore the SCCP consider this test as equivocal. The test was not repeated. The results of this test can only be used as supportive evidence. Study 2 Guideline: Species/strain: Test substance: Solvent: Batch: Purity: Concentrations: Treatment: GLP: Date: OECD 473, 1983 Chinese hamster V79 cells Duplicates in a single experiment FAT 80023/Q ethanol EN 91390.76 / 0.1, 1.0 and 3.0 g/ml without and with S9-mix 4 hours with harvest times 7 (high dose only), 18 and 28 h (high dose only) after the start of treatment. in compliance February December 1990

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FAT 80023/Q was investigated in the absence and presence of metabolic activation for the induction of chromosomal aberrations in V79 cells. Test concentrations were chosen on the basis of the results of a pre-experiment for toxicity test measuring reduction in plating efficiency. Cells were treated for 4 h. Harvest times were 7 h (high dose only), 18 h or 28 h (high dose only) after the beginning of treatment. Liver S9 fraction from Aroclor 1254induced rats was used as exogenous metabolic activation system. Two hours (7 h harvest time) or 2.5 h (18 and 28 h harvest times) before harvest, each culture was treated with colchicine solution (final concentration 0.2 g/ml) to block cells at metaphase of mitosis. Chromosome (metaphase) preparations were stained with Giemsa and examined microscopically for chromosomal aberrations. Negative and positive controls were in accordance with the OECD guideline. Results In the cytogenetic experiment, the mitotic index was reduced after treatment with the highest concentration at each fixation interval, except at interval 18 and 28 h in the presence of S9-mix. At the harvest time 7 h (both without and with S9) and 28 h (with S9) no increase in cells with chromosomal aberrations was observed. A dose dependent and biologically relevant increase in cells with chromosomal aberrations was found at the harvest times of 18 h (both without and with S9-mix) and 28 h (without S9-mix). Conclusion Under the experimental conditions used FAT 80023/Q induced an increase in the number of aberrant cells and, consequently, is mutagenic (clastogenic) in V79 cells in vitro. Ref: 48 Comment Exposure of the cells (measured as a decrease in the mitotic index) was not determined in the main test. In the protocol, it is mentioned that at harvest time 28 h only the high dose (3.0 g/ml) is tested. However, in the table with the result at harvest time 28 h without S9mix, (table 22 of the study report) the test article concentration is reported as 1.0 g/ml. The test was not repeated. Purity of FAT 80 023/A (triclosan) was reported as cf. Analytical Certificate in sponsors file. Summary: genotoxicity/mutagenicity of triclosan in vitro
strain/cell type test compound concentration S9 result quality ref

Bacterial gene mutation assay TA92, TA98, TA100, TA1535, TA1537 TA98, TA100, TA1535, TA1537 TA98, TA100, TA1535, TA1537, TA1538 FAT 80 023/A 0.1-8.1-(72.9) g/plate 0.1-72.9 g/plate triclosan 39316 0.015-1.5 g/plate 0.000167-0.167 g/plate 0.05-5 g/plate Yeast gene mutation assay S. cerevisiae MP-1 S. cerevisiae MP-1 FAT 80 023/A Irgasan DP 300 10-200 mg/l 0.2 mg/ml negative mutagenic limited value limited value 45 46 + /+ + negative negative negative negative negative limited value appropriate sufficient 42

43 44

Gene mutation assay in mammalian cells L5178Y/tk+/- mouse lymphoma cells FAT 80 023/A 15.8-28.9 g/ml negative limited value 49

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strain/cell type L5178Y/tk+/- mouse lymphoma cells

test compound triclosan

concentration 2.5-20 g/ml 1-15 g/ml

S9 +

result negative negative

quality appropriate

ref 50

Unscheduled DNA Synthesis test primary rat hepatocytes primary rat hepatocytes Chromosome aberration test CHO cells triclosan 0.1-1 g/ml 4.8-30 g/ml V79 cells FAT 80023/Q 0.1-3 g/ml + /+ negative negative mutagenic supportive evidence appropriate 47 triclosan 38317 0.16-80 g/ml 0.25-2.5 g/ml negative negative limited value sufficient 52 53

48

3.3.6.2.

Mutagenicity / Genotoxicity in vivo

Host mediated assay with S. typhimurium in mice Guideline: Species: Bacteria: Group sizes: Test substance: Solvent: Batch: Purity: Dose levels: Route: Cell removal: GLP: Date: / albino NMRI mice Salmonella typhimurium TA98, TA100, TA1535 and TA1537 6 mice/group FAT 80 023/A 2% CMC / / 50, 100, 200 and 400 mg/kg bw oral gavage 1 h after the injection of the bacteria / March 1979

FAT 80 023/A was investigated for the induction of gene mutations in the host mediated assay with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. After being fasted for 16 h the mice were treated orally by gavage 2 h, 1 h and immediately before bacteria were injected. The bacteria were injected in the lateral vein of the tail. One h after the injection of the bacteria the mice were killed, the livers removed and homogenised. The homogenate was centrifuged and the centrifugate was resuspended in saline. Five plates with 0.2 ml of the undiluted samples were used for the determination of the mutant count. The total bacterial count present in the animals was determined for each dosage or control group as a whole. Dilutions of 10-5 and 10-6 of the pooled samples were spread on 5 and 4 NB (nutrient brooth) plates respectively. The mutant rate is calculated from the mutant count and the total bacterial count. The mutation factor is the ratio of the mean mutation rate for each dosage group to the mean mutation rate of the control group. Negative and positive controls were not included. Results At the tested doses 50, 100, 200 and 400 mg/kg the mutation rates in comparison with those of the control were not significantly increased in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 Conclusion Under the experimental conditions used FAT 80 023/A was not mutagenic in this host mediated assay with S. typhimurium in mice.

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Ref.: 54 Comment The test is performed before the implementations of OECD guidelines. Batch number and purity of FAT 80 023/A (triclosan) were not reported. The test has only limited value. Host mediated assay with mouse lymphoma cells in mice Guideline: Species: Cell type: Group sizes: Test substance: Solvent: Batch no: Purity: Dose levels: Route: Cell removal: GLP: Date: / DBA/2f/Bom mice Mouse lymphoma cell line L5178Y/tk+/6 mice/group FAT 80 023/A 0.05 N NaOH / / 1313 mg/kg bw oral gavage 6 days after cell inoculation and 3 days after compound administration / May 1978

FAT 80 023/A was investigated for the induction of gene mutations in the host mediated assay. Test concentrations were based on the results of a toxicity test on cell survival. The mice were inoculated intraperitoneally with 106 cells per animal. FAT 80 023/A was administered orally. Six days after cell inoculation and 3 days after FAT 80 023/A administration, the cells were removed from the peritoneal fluid under a-septic precautions. The cells were seeded and cultured to fix the DNA damage into mutations. The incidence of mutants was determined with methothrexate, cytosine arabinoside and thymidine as antimetabolites. Negative and positive controls were not included. Results At 1313 mg/kg bw the target cell count was reduced with 50%. There was no increase in the number of mutant colonies in comparison with the control. Conclusion Under the experimental conditions used FAT 80 023/A was not mutagenic the host mediated assay with mouse lymphoma cells. Ref.: 49 Comment The test is performed before the implementations of OECD guidelines. Batch number and purity of FAT 80 023/A (triclosan) were not reported. The test has only limited value. Mouse spot test Study 1 Guideline: Species/strain: Group size: Test substance: Batch: Purity: Dose level: / T stock males and C57BL/6JHan females / Irgasan DP 300 / / 50 mg/kg bw

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Route: intraperitoneal injection Day of administration:day 10 of pregnancy Vehicle: Hanks balanced salt solution GLP: / Date: August 1978 Irgasan DP 300 has been investigated for the induction of mutations in the mouse spot test. Female C57BL/6JHan mice were exposed by ip injection to a single dose of 50 mg/kg bw Irgasan DP 300 at day 10 of pregnancy. Between 2 and 5 weeks of age the F1 animals were analysed for the occurrence of spots. Results The examination was rendered difficult because with the exception of some midventral white spots all spots consisted of a more or less large mixture of mutant and non-mutant hairs. The frequency of colour spots in mice in the test and control groups clearly show that Irgasan DP 300 in a dose of 50 mg/kg is active in the spot test. Compared to other compounds the authors consider Irgasan DP 300 as a mutagen of only medium effectiveness. Conclusion Under the experimental conditions used Irgasan DP 300 was mutagenic in the mouse spot test. Ref.: 63 Comment The test is performed before the implementations of OECD guidelines. Group size, batch number and purity were not reported. The test has only limited value. Study 2 Guideline: Species/strain: Group size: Test substance: Batch: Purity: Dose level: Day of administration: Vehicle: Scoring for mutations: GLP: Date: / male T stock and female C57BL/E mice / Irgasan DP 300 (triclosan) / 99.7% 0, 1, 2, 4, 8 and 25 mg/kg bw day 9.25 or 10.25 of pregnancy 60% methanol day 12 after birth / March - October 1979

Triclosan has been investigated for the induction of somatic mutations in the mouse spot test. Female C57B1/E mice were mated with T stock males. On days 9.25 or 10.25 post fertilisation triclosan was administered by ip injection. At 12 days after birth the offspring was scored for coat colour spots. A positive control was not included. Results Triclosan when injected in 60% methanol killed 12 out of 41 treated females of the 25 mg/kg group. In the other treatment groups all animals survived. Prenatal survival was clearly reduced by 25 mg/kg triclosan in both the 9.25 and 10.25-day treated groups. Level of exposure below 25 mg/kg produced no obvious effects on prenatal survival. Postnatal survival was severely reduced after 25 mg/kg and slightly but significantly reduced after 8

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mg/kg in both the 9.25 and 10.25-day treated groups and significantly reduced after 4 mg/kg (average exposure 3.2 mg/kg) only in the 10.25-day treated group. Within the 9.25 day group the incidence of recessive spots and midventral white spots is not increased by triclosan treatment. Within the 10.25 day set triclosan has a clear effect on the incidence of midventral white spots at 25 mg/kg but no effect on the incidence of recessive spots in comparison to the control group. However, 25 mg/kg was markedly toxic both to the mothers and the animals exposed in utero. Conclusion Under the conditions of this test, triclosan did not induce somatic mutations at non/sub toxic concentrations and, consequently, triclosan is not mutagenic in this mouse spot test. Ref.: 64 Comment The experiment is conducted before the development of the OECD guidelines. The batch nr was not reported. The test can be used as supportive evidence. Bone marrow chromosome aberration test in Chinese hamsters (Cricetulus griseus) Study 1 Guideline: Species: Group sizes: Test substance: Batch: Purity: Dose levels: Vehicle: Treatment: Sacrifice times: GLP: Date: / Female Chinese hamsters (Cricetulus griseus) 4 females/group GP 41 353 (triclosan) 3 / 150, 300 and 600 mg/kg bw 0.5 % CMC oral by gavage, daily application on 2 consecutive days, 24 apart. 6 h after the last treatment / 1973

Triclosan has been investigated for the induction of chromosome aberrations in bone marrow cells of Chinese hamsters. Chinese hamsters were exposed twice by oral gavage 24 h apart on two consecutive days. The highest dose 600 mg/kg bw is approximately 1/3 of the LD50. Two h after the second treatment the animals were injected intraperitoneally with 10 mg colcemid/kg. Bone marrow cells were collected 6 h after the last treatment. Bone marrow preparations were stained with acetic-orcein and examined microscopically for chromosome aberrations. Cyclophosphamide was used as positive control. Results In comparison to the vehicle control there was no biologically relevant or statistically significant enhancement in the number of cells with chromosome aberrations with any dose level tested. Conclusion Under the experimental conditions used triclosan is not genotoxic (clastogenic) in bone marrow cells of Chinese hamsters. Ref.: 55 Comment

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The test is performed before the implementations of OECD guidelines. Purity was not reported. There were no indications of toxicity and thus sufficient exposure is not proven. The test has only limited value. Study 2 Guideline: Species: Group sizes: Test substance: Batch: Purity: Dose levels: Vehicle: Treatment: Sacrifice times: GLP: Date: / Female Chinese hamsters (Cricetulus griseus) 6 male and 6 females/group FAT 80 023/A 652 / 75, 150, 300 and 600 mg/kg bw 0.7 % CMC oral by gavage, thrice weekly for twelve weeks. 6 h after the last treatment / February 1979

FAT 80 023/A has been investigated for the induction of chromosome aberrations in bone marrow cells of Chinese hamsters after long term treatment. Chinese hamsters were exposed thrice weekly by oral gavage for 12 weeks. The highest dose 600 mg/kg bw is approximately 1/3 of the LD50. Two h after the last treatment the animals were injected intraperitoneally with 10 mg colcemid/kg. Bone marrow cells were collected 6 h after the last treatment. Bone marrow preparations were stained with acetic-orcein and examined microscopically for chromosome aberrations. The solvent was used as negative control; a positive control was not included. Results Of the 12 animals treated with 600 mg/kg bw 5 died within the first week, additionally 3 by the 3rd, 4th and 9th week. One of the animals in the low dose group died at the end of the experiment. In comparison to the vehicle control animals, long term treatment with FAT 80 023/A did not result in a biologically relevant or statistically significant enhancement in the number of cells with chromosome aberrations with any dose level tested. Conclusion Under the experimental conditions used FAT 80 023/A is not genotoxic (clastogenic) in bone marrow cells of Chinese hamsters. Ref.: 56 Comment The test is performed before the implementations of OECD guidelines. Long term treatment is not a routinely performed protocol. Purity of test substance FAT 80 023/A (triclosan) was not reported. There were no data on toxicity and thus sufficient exposure is not proven. The test has only limited value. Bone marrow chromosome aberration test in rats Guideline: Species/strain: Group size: Test substance: Batch: Purity: OECD 475 (1984) Wistar rats 5 rats/sex/group FAT 80023/Q EN 91390.76 /

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Dose level: Route: Vehicle: Sacrifice times: GLP: Date:

4000 mg/kg bw oral, once 1% carboxymethylcellulose-suspension 6 h, 24 h and 48 h. In compliance December 1990 April 1991

FAT 80023/Q has been investigated for the induction of chromosome aberrations in bone marrow cells of rats. The test concentration was based on a pre-experiment for toxicity measuring acute toxicity. In the main experiment rats were exposed orally to 4000 mg/kg bw, the maximum tolerated dose. From approximately 18 h before treatment with the test compound the animals were fasted. Bone marrow cells were collected 6 h, 24 h or 48 h after dosing. Two and a half h before sacrifice animals were injected intraperitoneally with the spindle inhibitor colcemid (2.0 mg/kg bw) to arrest cell in metaphase. To describe a cytotoxic effect, and thus exposure of the target cells, the mitotic index (percentage cells in mitosis) was determined. Bone marrow preparations were stained with Giemsa and examined microscopically for the mitotic index and chromosomal aberrations. Negative and positive controls (24 h sacrifice time only) were in accordance with the OECD guideline. Results FAT 80023/Q did not induce a reduction in the mitotic index and is considered not cytotoxic for bone marrow cells and thus exposure of the target cells is not proven. Exposure to 4000 mg/kg bw FAT 80023/Q did not induce a biological relevant increase in cells with chromosomal aberrations in bone marrow cells of the rat up to a sacrifice time of 48 h after treatment. Conclusion Under the experimental conditions used FAT 80023/Q was not genotoxic (clastogenic) in bone marrow cells of rats. Ref.: 57 Comment Purity of FAT 80023/Q (triclosan) was reported as see Analytical Certificate in sponsors file. Since the test substance did not induce a decrease in the mitotic index as compared to the concurrent negative control data, evidence of exposure of the target cells is lacking and thus the test has only limited value. Bone marrow micronucleus test in mice Guideline: Species/strain: Group size: Test substance: Batch: Purity: Dose level: Route: Vehicle: Sacrifice times: GLP: Date: OECD 474 (1982) CD-1 mice 5 mice/sex/group Triclosan S 15155 TO1 99% 5000 mg/kg bw oral by gavage, once 1% methylcellulose 24 h, 48 h and 72 h after dosing. In compliance March - August 1988

Triclosan has been investigated for the induction of micronuclei in bone marrow cells of mice. The test concentration was based on a preliminary toxicity test with concentrations up to the maximal prescribed dose of 5000 mg/kg bw recording cell death and signs of malreaction. In the main experiment rats were exposed orally to 5000 mg/kg bw. Following

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dosing the animals were examined regularly and any mortalities or clinical signs of reaction to the test compounds were recorded. Bone marrow cells were collected 24 h, 48 h and 72 h after dosing. Toxicity and thus exposure of the target cells was determined by measuring the ratio between polychromatic and normochromatic erythrocytes (PCE/NCE ratio). Bone marrow preparations were stained with 10% Giemsa and examined microscopically for the PCE/NCE ratio and micronuclei. Negative and positive controls were in accordance with the OECD guideline. Results In the preliminary toxicity study mice treated with the highest dose (5000 mg/kg bw) showed slight piloerection, hunched posture, waddling and ptosis. One male mouse treated with the highest dose (5000 mg/kg bw) died. In the main test the mice only showed the first 6 h after exposure piloerection and hunched posture. At the sacrifice time 24 h and 48 h but not at 72 h triclosan induced a reduction in the PCE/NCE ratio confirming exposure of the target cells. Exposure to 5000 mg/kg bw triclosan did not induce a biological relevant increase in cells with micronuclei in bone marrow cells of mice up to a sacrifice time of 72 h after treatment. Conclusion Under the experimental conditions used triclosan was not genotoxic (cacogenic and/or aneugenic) in bone marrow cells of mice. Ref.: 60 Nucleus anomaly test on somatic interphase nuclei of Chinese hamsters Study 1 Guideline: Species: Group size: Test substance: Batch: Purity: Dose level: Route: Vehicle: Sacrifice times: GLP: Date: / Chinese hamsters (Cricetulus griseus) 3 rats/sex/group GP 41 353 (triclosan) Mg. 3 / 150, 300 and 600 mg/kg bw oral, twice 24 h apart 0.5 % carboxymethylcellulose solution 24 h after the last dose. / May 1974

Triclosan has been investigated for the occurrence of nucleus anomalies in interphase nuclei of bone marrow cells of Chinese hamsters (Cricetulus griseus). Chinese hamsters were exposed twice by oral gavage 24 h apart on two consecutive days. The highest dose 600 mg/kg bw is approximately 1/3 of the LD50. Bone marrow cells were collected 24 h after the last treatment. Bone marrow preparations were stained with undiluted May-Grnwald and subsequently with 5% Giemsa and examined microscopically for nuclear anomalies. As anomalies were registered: single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, bizarre forms of nuclei, polyploid cells and necrobiotic cells. Cyclophosphamide was used as positive control. Results In all triclosan exposed groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. Conclusion

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Under the experimental conditions used triclosan was not genotoxic in bone marrow cells of Chinese hamsters. Ref.: 58 Comment The test is performed before the implementations of OECD guidelines. The nuclear anomaly test is not a routinely performed test. Purity was not reported. The test has only limited value Study 2 Guideline: Species: Group size: Test substance: Batch: Purity: Dose level: Route: Vehicle: Sacrifice times: GLP: Date: / Chinese hamsters (Cricetulus griseus) 6 rats/sex/group FAT 80 023/A 652 / 75, 150, 300 and 600 mg/kg bw oral, thrice weekly for 12 weeks 0.7 % carboxymethylcellulose solution 6 h after the last dose. / August 1978

FAT 80023/A has been investigated for the occurrence of nucleus anomalies in interphase nuclei of bone marrow cells of Chinese hamsters (Cricetulus griseus) after long term treatment. Chinese hamsters were exposed thrice weekly by oral gavage for 12 weeks. The highest dose 600 mg/kg bw is approximately 1/3 of the LD50. Bone marrow cells were collected 6 h after the last treatment. Bone marrow preparations were stained with undiluted May-Grnwald and subsequently with 5% Giemsa and examined microscopically for nuclear anomalies. As anomalies were registered: single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells and polyploid cells. The solvent was used as negative control; a positive control was not included. Results The bone marrow of only 3 female and 3 male hamsters (for the highest dose only 2 male hamsters) was evaluated. Of the animals treated with 600 mg/kg bw 1 died within the 2nd week, additionally 3 by the 7th, 8th and 9th week and further 3 in the 12th week. One of the animals treated with 300 mg/kg bw died within the 5th week. In all FAT 80023/A exposed groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. Conclusion Under the experimental conditions used FAT 80 023/A was not genotoxic in bone marrow cells of Chinese hamsters. Ref.: 59 Comment The test is performed before the implementations of OECD guidelines. The nuclear anomaly test is not a routinely performed test. Purity of test substance FAT 80 023/A (triclosan) was not reported. The test has only limited value

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Sex linked recessive lethal test in Drosophila melanogaster Guideline: Species: Test substance: Batch no: Purity: Dose levels: Treatment: / Male Drosophilas melanogaster of wild type strain Karsns 60 Irgasan / / Experiment 1: 1000 ppm Irgasan in 1% sucrose Experiment 2: 1000 ppm Irgasan in Ringer solution Experiment 3: 1000 ppm in corn agar substrate Experiment 1: In glass tubes to which 0.5 ml of the sucrose solution was added for 24 h Experiment 2: Injection of 1000 ppm Irgasan Experiment 3: In ordinary vials with corn agar containing 1000 ppm Irgasan for 7 days. not in compliance 1979

GLP: Date:

Irgasan was tested in a sex linked recessive lethal assay in Drosophila melanogaster. Dose selection was based on the results of a toxicity test with adult male flies with and without pre-treatment with the inducer of the metabolic detoxication enzymes. Males were either treated with 1000 ppm Irgasan in 1% sucrose in glass tubes for 24 h, or injected with 1000 ppm Irgasan in Ringer solution or kept on vials with corn agar containing 1000 ppm Irgasan for 7 days. The flies which were treatment with Irgasan in sucrose were before treatment put for 4 h in empty tubes in order to increase the liquid consumption. Three broods of flies were studies: 0-3, 4-6, 7-10 days after treatment. New virgin females were given to the male for each brood. Flies treated with Irgasan in sucrose solution and flies injected with Irgasan in Ringer solution were analysed chemically by GLC for the determination of the Irgasan content immediately after treatment and between 1 and 4 days after. Results Both in flies treated with Irgasan in sucrose solution or injected with Irgasan in Ringer solution the concentration of Irgasan reached control values in less than less 72 h or 48 h, respectively. Apparently Irgasan is excreted very fast and will thus not accumulate in the flies. No effect of Irgasan in any of the broods within the experiments was recorded. The frequency of the lethals recorded after treatment with Irgasan is in accordance both with the parallel and the historic controls with this particular wild type strain. Conclusion Under the experimental conditions described, Irgasan did not induce an increase in sex linked recessive lethals and, consequently, Irgasan was not genotoxic in the sex linked recessive lethal test in Drosophila melanogaster. Ref.: 51 Comment The test was not performed according the OECD guideline. Batch number and purity were not reported. The test has only limited value. Dominant lethal test Guideline: Species: Group sizes: / NMRI mice 12 male mice/group

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Test substance: Solvent: Batch: Purity: Dose levels: Route: Sacrifice times: GLP: Date:

GP 41 353 2% sodium carboxymethylcellulose 3 / 750 and 1500 mg/kg bw oral gavage females were autopsied at day 14 of pregnancy / October 1971

GP 41 353 has been investigated for the induction of dominant lethals in mice. Mice were exposed by oral gavage with 750 and 1500 mg/kg bw which are equal to approximately 1/6 and 1/3 of the LD50 respectively. Each male mouse from each group was mated with 4 untreated females immediately after treatment. After 1 week the females were removed from the cages and replaced by another group of 3 untreated females. This procedure was continued for 8 consecutive weeks. This time of 8 mating periods comprises all stages of the maturation of the male germ cell. The mated females were sacrificed and autopsied on the 14th day of pregnancy. The number of alive and dead foetuses as well as early embryonic resorptions was listed. A positive control was not included. Results Until 3 days after treatment the males of the high dose group were found to display signs of intolerance as indicated by slight convulsions. The symptoms did not exert any influence on the mating performance. An increase in the number of implementations, live embryos, death embryos and early embryonic resorptions due to treatment with GP 41 353 was not observed. Conclusion: Under the conditions tested GP 41 353 did not induce an increase in dominant lethals and consequently is not genotoxic in this dominant lethal test in mice. Ref.: 65 Comment The test is performed before the implementations of OECD guidelines. Purity was not reported. The test has only limited value. Chromosome aberration test in male germinal epithelium of the mouse Study 1 Guideline: Species/strain: Group size: Test substance: Batch: Purity: Dose level: Route: Vehicle: Sacrifice times: GLP: Date: / Male NMRI mice 6 mice /group FAT 80 023/A 652 / 189, 378, 756 and 1512 mg/kg bw oral by gavage, on 5 consecutive days 2% CMC 1 day after the last dose / December 1978

FAT 80 023/A has been investigated for the induction of chromosome aberrations in male germinal epithelium cells, in particular on spermatogonia, of mice. Mice were exposed on 5 consecutive days by oral gavage. Germinal epithelium cells were collected 1 day after the

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last dose. Three h before section the mice were injected intraperitoneally with 10 mg colcemid/kg. Germinal epithelium cell preparations were stained and examined microscopically for chromosome aberrations. The solvent was used as negative control; a positive control was not included. Results In the 1512 mg/kg bw group 7 out 8 animals died in the course of the second to last treatment. The remaining animal was not included in the evaluation. In the other treatment groups all animals survived. In comparison to the vehicle control animals, FAT 80 023/A did not induce a biologically relevant or statistically significant enhancement in the number of cells with chromosome aberrations at the three remaining dose levels tested. Conclusion Under the experimental conditions used FAT 80 023/A is not genotoxic (clastogenic) in germinal epithelium cells (spermatogonia) of mice. Ref.: 61 Comment The test is performed before the implementations of OECD guidelines. Purity of test substance FAT 80 023/A (triclosan) was not reported. Raw nor summarized data on the occurrence of chromosomal aberrations were not available in the report. The test has only limited value. Study 2 Guideline: Species/strain: Group size: Test substance: Batch: Purity: Dose level: Route: Vehicle: Sacrifice times: GLP: Date: / Male NMRI mice 6 mice /group FAT 80 023/A 652 / 189, 378, 756 and 1512 mg/kg bw oral by gavage, 5 intermittently doses on days 0, 2, 3, 5 and 9 2% CMC 3 days after the last dose / February 1979

FAT 80 023/A has been investigated for the induction of chromosome aberrations in male germinal epithelium cells, in particular on spermatogonia, of mice. Mice were exposed intermittently on days 0, 2, 3, 5 and 9 by oral gavage. Germinal epithelium cells were collected 3 days after the last dose. Three h before section the mice were injected intraperitoneally with 10 mg colcemid/kg. Germinal epithelium cell preparations were stained and examined microscopically for chromosome aberrations. The solvent was used as negative control; a positive control was not included. Results In the 1512 mg/kg bw group only 4 animals survived. These animals were not included in the evaluation. In the other treatment groups all animals survived. In comparison to the vehicle control animals, FAT 80 023/A did not induce a biologically relevant or statistically significant increase in the number of cells with chromosome aberrations at the three remaining dose levels tested. Conclusion

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Under the experimental conditions used FAT 80 023/A is not genotoxic (clastogenic) in germinal epithelium cells of mice. Ref.: 62 Comment The test is performed before the implementations of OECD guidelines. Purity of test substance FAT 80 023/A (triclosan) was not reported. The test has only limited value. Summary: genotoxicity/mutagenicity of triclosan in vivo
strain/cell type test compound concentration result quality ref

Host mediated assay with S. typhimurium in mice albino NMRI mice FAT 80 023/A* 50-400 mg/kg bw negative limited value 54

Host mediated assay with Mouse lymphoma cells in mice DBA/2f/Bom mice Mouse spot test T stock x C57BL/6JHan T stock x C57BL/E Irgasan DP 300 Irgasan DP 300 50 mg/kg bw 1-25 mg/kg bw mutagenic negative limited value supportive evidence 63 64 FAT 80 023/A* 1313 mg/kg bw negative limited value 49

Bone marrow chromosome aberration test Chinese hamsters (female) Chinese hamsters (female) Wistar rats GP 41 353 FAT 80 023/A* 150-600 mg/kg bw 75-600 mg/kg bw long term exposure FAT 80023/Q
*

negative negative

limited value limited value limited value

55 56

4000 mg/kg bw

negative

57

Bone marrow micronucleus test CD-1 mice triclosan 5000 mg/kg bw negative appropriate 60

Nucleus anomaly test on somatic interphase nuclei Chinese hamsters Chinese hamsters GP 41 353 FAT 80 023/A* 150-600 mg/kg bw 75-600 mg/kg bw long term exposure Sex-linked recessive lethal test Drosophila melanogaster, strain Karsns 60 Dominant lethal test NMRI mice GP 41 353 750-1500 mg/kg bw negative limited value 65 Irgasan 1000 ppm negative limited value 51 negative negative limited value limited value 58 59

Chromosome aberration test in male germinal epithelium cells male NMRI mice male NMRI mice FAT 80 023/A* FAT 80 023/A* 189-1512 mg/kg bw 189-1512 mg/kg bw negative negative limited value limited value 61 62

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3.3.7.

Carcinogenicity

Carcinogenicity studies of 18 months, 104 weeks, and 90 to 95 weeks in duration have been conducted in the rat, mouse, and hamster to assess chronic exposure effects of triclosan (Pharmaco LSR, 1995 (66); Ciba-Geigy, 1986 (67); Huntingdon Life Sciences, 1999 (68)]. The three life-time bioassays were designed according to OECD guidelines Nos. 451 and/or 453, and GLP compliant. Triclosan showed no tumourigenic effects in the rat and the hamster studies. The mouse oncogenicity study showed both hepatic adenomas and hepatocellular carcinomas following an 18-month exposure to triclosan. Descriptions of the rat, mouse, and hamster data are found in the table below. Table 16: Findings from GLP Carcinogenicity Studies for Triclosan
Species (Strain) Dosing Regimen Duration of Treatment 18 months Major Findings1 Reference; GLP and OECD Status Pharmaco LSR, 1995 (66); GLPcompliant OECD: No. 451 consistent

Mouse (CD-1)

Oral (diet) doses of 0, 10, 30, 100, or 200 mg/kg bw/d 50/sex/ dose + 20/sex/ dose (6-month interim)

Decreased survival occurred in males (100 mg/kg bw/d) and high-dose females (a smaller decrease in high-dose males was not significant); therefore, only the survival in females was considered to be affected by triclosan. Increased plasma levels of liver function enzymes were observed in higher dose mice, and decreased cholesterol was measured at all doses. There were no biochemical or pathological indications of kidney effects. Increased incidences of hepatic nodules or masses or discolorations were observed in higher dose animals. No histopathological changes other than in liver (hepatocellular hypertrophy in all animals except lowdose females) were attributed to triclosan treatment. Data show dose-dependent increases in liver adenomas and carcinomas, with increased incidence in males vs. females. Incidences of mice bearing at least 1 hepatic tumour (i.e., combined adenoma and carcinoma data) were: 6, 10, 17, 32, 42 in males and 0, 1, 3, 6, 20 in females at doses of 0, 10, 30, 100, and 200 mg/kg bw/d, respectively. In summary, effects in the liver, serum cholesterol decreases (males and females) and hepatocellular hypertrophy (males only) were observed at the lowest dose of 10 mg/kg bw/d; investigators concluded that there was no NOEL based on liver changes at all dose levels. Increased food intake was found in high-dose males. Mean body weight decreases were observed to be transient during the study except for consistent significant decreases of up to 10% in high-dose females. Sporadic changes such as slight changes in protein, glucose, bilirubin, triglyceride, and blood urea nitrogen found in the first 52 weeks of the study had disappeared by 78-104 weeks. Slight changes in erythroid parameters were sporadic. Decreases in monocytes and white blood cells in F and increased clotting time in males was observed at 104 weeks. Absolute adrenal weights were increased in mid-dose males; absolute brain weights were decreased in high-dose males; absolute and relative ovary weights were increased in high-dose females; absolute and relative spleen weights were decreased in mid-dose females, and relative spleen weights were decreased in high-dose females at 104 weeks. Hepatocyte hypertrophy and hepatocytic inclusions (hyaline-staining) were occasionally noted in male rats at early time points but were not seen at 104

Rat (SpragueDawley

Oral (diet) doses of 0, 12, 40, or 127 mg/kg bw/d in males and 0, 17, 56, or 190 mg/kg bw/d in females 60/sex/ dose + 10/sex/ dose or 20/sex/ control group (52-week interim) 20/sex in additional high-dose

104 weeks

Ciba-Geigy, 1986 (67); GLPcompliant OECD: No. 453 consistent

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Species (Strain)

Dosing Regimen

Duration of Treatment

Major Findings1

Reference; GLP and OECD Status

group (247 or 422 mg/kg bw/d in males and females) at 52 weeks

weeks. No other histological lesions, either neoplastic or non-neoplastic, were observed that were considered to be treatment-related. Specifically, there were no treatment-related tumours, including hepatic tumours, in any of the treated rats examined histologically at 52 or 104 weeks. Animals in the additional high-dose group killed at 52 weeks (247 or 422 mg/kg bw/d in males and females, respectively) showed similar types of toxicity as the main study animals, excepting that the severity or incidences of events were increased. No tumours were observed in these animals. In summary, the results show that triclosan is not tumourigenic in a 2-year study in rats at doses of up to 127 mg/kg bw/d in males and 190 mg/kg bw/d in females. A NOEL of 1,000 ppm was determined (48 mg/kg bw/d for females and males combined). Females: 90 weeks Males: 95 weeks Survival was significantly decreased in males and was generally poor (38-58%) in females at 90 weeks. Body weight gain was significantly decreased in all high-dose hamsters and was accompanied by a slight (3%), but significant decrease in food consumption in high-dose females. Biochemical changes observed at termination included increases (<50%) in blood urea nitrogen in high-dose hamsters and in triglycerides in mid- and high-dose males. Haematological changes observed included slight (<15%), but significant decreases in erythroid parameters in mid- and high-dose animals, increased white blood cells in high-dose animals, and increased lymphocytes in high-dose females at termination. Significant observations at 52 weeks (interim) were mainly renal changes in high-dose animals. At termination, renal nephropathy was observed in animals of all dose groups, with increased incidence and severity at the high-dose level. In addition, males showed atypical hyperplasia in the stomachs (fundic region), along with spermatozoa and germ cell effects at the high dose. One high-dose female showed atypical hyperplasia in the fundic region that was considered to be treatment-related. High-dose females showed distended gastric glands and a few treated females of all doses showed benign papillomas of the non-glandular region of the stomach (not discussed in report). Hepatic effects were few, with only rarified hepatocytes reported in a few male animals (6/60 in high dose vs. 3/121 in controls). There were no tumours considered to be treatment-related. In summary, triclosan had little to no effect in hamsters at 12 and 75 mg/kg bw/d, and toxic effects at 250 mg/kg bw/d that resulted in the general deterioration of highdose males after Week 80. Overall results show that triclosan is not tumourigenic in hamsters at doses of up to 250 mg/kg bw/d. However, survival in females at 90 weeks was poor. NOAEL=75 mg/kg bw/d. Huntingdon Life Sciences, 1999 (68); GLPcompliant OECD: No. 451 consistent

Hamster (Syrian)

Oral (diet) doses of 0, 12, 75, or 250 mg/kg bw/d 60/sex/ dose + 10/sex/ dose (52week interim)

Findings reported in table are significant compared to controls unless otherwise noted in text.

Doses tested in the 3 carcinogenicity assays were comparable, ranging from 10 to 200 mg/kg body weight/day in mice, 12 to 127 mg/kg body weight/day (for males) and 17 to 190 mg/kg body weight/day (for females) in rats, and 12 to 250 mg/kg body weight/day in hamsters. Survival was not altered by triclosan treatment in the rat study, whereas decreased survival was observed in both the hamster and the mouse studies. In general, neither biochemistry/clinical chemistry nor haematology analyses showed any serious

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effects that could be attributed to triclosan treatment, except in the case of liver-related changes in the mouse study. Rats showed no changes in liver function enzyme activity in plasma, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which would be indicative of hepatic damage. Liver effects in rats in the carcinogenicity study were limited to increased incidences of hepatocyte hypertrophy and hyaline-staining inclusions in high-dose males at early time points in the study. These findings were not observed in animals at the termination of the study. As with rats, hamsters showed no alterations in liver function enzymes. The effects of triclosan in hamster liver, as observed in the carcinogenicity study, were limited to a slight decrease in organ weight in high-dose females. This finding was attributed to the decreased body weight gain at this dose level. In addition to the organ weight effects, slight, but statistically significant, decreases in erythroid parameters and in triglycerides were measured in mid-dose females. The only notable histopathological finding was an increased incidence of rarified hepatocytes in a few high-dose males. In contrast to observations in rats and hamsters, liver effects in the mouse included increased liver function enzymes in plasma; decreased blood cholesterol levels; significant increases in liver weights; increased incidences of hepatic nodules, discolorations, or masses; hepatocellular hypertrophy; and, increased incidences of both hepatocellular adenomas and hepatocellular carcinomas, depending on dose level. Signs of liver effects, increases in cholesterol (both sexes) and hypertrophy (males only) were seen at the lowest dose of 10 mg/kg body weight/day. Hypertrophy was seen in mice of both sexes at 30 mg/kg body weight/day, and increases in liver function enzyme levels were seen in mice of both sexes at doses of 100 and 200 mg/kg body weight/day. Increases in numbers of hepatic tumours were observed at doses of 30 mg/kg body weight/day and higher. Summary and NOAEL Values from the Rodent Carcinogenicity Studies Three rodent lifetime bioassays have been conducted to evaluate the carcinogenic potential of triclosan. Triclosan produced hepatic effects and hepatic tumours in mice, but little evidence of toxicity and no tumours in rats. Hamsters showed increased liver toxicity relative to the rat, but no tumours. No NOAEL could be determined for the mouse, based on findings of liver effects at all doses (effects of hepatocyte hypertrophy and decreased plasma cholesterol). Increased incidences of liver tumours were observed at doses of 30 mg/kg body weight/day and higher in mice. It should be noted that triclosan is a peroxisome proliferator in mouse liver. The NOEL for the rat study was determined by study investigators to be the mid-dose of 1,000 ppm, or ~48 mg/kg body weight/day for males and females combined, based on the finding of hepatocyte changes in the high-dose group of 3,000 ppm (~127 mg/kg body weight/day in males and 190 mg/kg body weight/day in females). Although absolute adrenal weights were elevated at the 1,000 ppm dose in rats, this change was not considered to be adverse for a number of reasons, including a lack of change in relative adrenal weights, a lack of change in adrenal weights in the high-dose group, and the absence of accompanying histopathological changes in the adrenal glands. The NOAEL for the hamster study was determined to be the mid-dose of 75 mg/kg body weight/day. Although the values for certain haematologic parameters were significantly altered at doses of 12 and 75 mg/kg body weight/day, the erythroid changes were slight (on the order of 5 to 10%), and not considered to be adverse effects per se. An increase in triglycerides (40 to 50%) was not considered to be an adverse effect, as the increase was not accompanied by any changes in liver weight or histopathology. The NOAEL values for the three rodent lifetime cancer bioassays are summarized in Table 17.

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Table 17: Summary of NOAEL Values from GLP Carcinogenicity Studies for Triclosan in Rodents
Species Mouse NOAEL (mg/kg bw/day) 10 (this value is a LOEL for tumour formation only) ~48 (NOAEL)1 Comment No overall NOAEL due to hepatotoxic effects observed at the lowest dose tested. There were no tumours observed in other tissues. NOAEL based on systemic toxicity. There was no evidence of tumour formation, including in liver. There was no evidence of hepatotoxicity. NOAEL based on systemic toxicity. There was no evidence of tumour formation, including in liver.

Rat

Hamster
1

75

1,000 ppm ~48 mg/kg body weight/day for males and females, combined.

Comment According to the EU classification system, triclosan is not considered classifiable as a carcinogen. It should be noted that triclosan is a peroxisome proliferator in mouse liver. 3.3.8. Reproductive toxicity

The reproductive and developmental toxicology of triclosan has been investigated in teratology studies in the mouse, rat, and rabbit, and a two-generation reproductive toxicity study in the rat. Based on international guidelines (ICH, 2005), effects on fertility and reproduction and perinatal development should be evaluated in rodents (typically, the rat), and teratology studies be conducted in both rodent and non-rodent species (typically, the rat and the rabbit). The available studies for triclosan meet these recommendations. 3.3.8.1. Two generation reproduction toxicity

One GLP-compliant two-generation study was conducted in rats, providing both fertility and reproduction and post-natal data [Morseth, 1988 (69)]. The study was consistent with OECD guidelines (Two-Generation Reproductive Toxicity Study), with the exception that the coagulating gland was not preserved, and with ICH (Fertility and Early Embryonic Development, Stages A and B; Pre- and Post-Natal Development, ICH Stages D, E, and F) guidelines, with the exception that sperm counts and viability assessments, and specific tests for physical, sensory, reflexes and behaviour, were not conducted. In addition to the post-natal data provided in the two-generation rat study, the GLP-quality study in Colworth Wistar rats provided post-natal data [Denning et al., 1992 (74)]. This study was generally consistent with international guidelines, with the major exceptions being those listed above [i.e., as for the study by Morseth, 1988 (69)], that maturation and fertility were not assessed, and that relatively few animals (5 per treatment group) were allowed to deliver and rear offspring. The fertility, reproduction, and post-natal data from these studies are summarized in the table below. Table 18: Findings from the Reproductive and Developmental Toxicity Studies that Examined Fertility and Reproduction and/or Post-Natal Parameters
Species (Strain) Dosing Regimen (mg/kg bw/d) 0, 300, 1,000, or 3,000 ppm (Doses of ~17, 56, and 176 mg/kg bw/d in males and ~23, Major Findings1 Reference, GLP and OECD Status Morseth, 1988 (69) GLPcompliant

Charles River CD (SD)Br rats

Fertility and Reproduction: F0 Generation: There were no remarkable effects on F0 treated rats at any dose, including no effects on mating, pregnancy, duration of gestation, and parturition. F1 Generation: There were no treatment-related effects on reproduction indices.

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Species (Strain)

Dosing Regimen (mg/kg bw/d) 73, and 229 mg/kg bw/d in females, based on food consumption and body weight data after 10 weeks of dosing. Both males and females were dosed starting 10 weeks prior to mating. The rats received the appropriate control or test diet throughout the growth, mating, gestation, and lactation phases, or until terminal sacrifice.

Major Findings1

Reference, GLP and OECD Status OECD: No. 416 consistent

Post-natal Parameters: F1 Generation: Post-natal survival from Day 0 to Day 4 (i.e., viability index) was slightly reduced in the high-dose group (90, 94, 96, and 82% in the control, 300, 1,000, and 3,000 ppm groups, respectively). Adjusted mean body weights of pups in the high-dose group were generally lower than controls throughout the Day 0 to 21 lactation period. With regard to development, mean body weights of high-dose pups were lower than controls during Weeks 0 to 12. Mean body weights were slightly decreased in lowdose group females vs. controls during Weeks 4-8 and 11. Overall, the most common findings were dilated pelvises of the kidney that showed a slight increase in incidence at the high dose in pups, but no differences between treatment groups in adult animals. F2 Generation: The mean number of live F2 pups on Day 0, viability index (survival to Day 4), and survival to weaning were slightly lower in high-dose animals vs. controls. Adjusted mean body weights of high-dose pups were slightly, but significantly lower than controls on Day 0. There were no other remarkable treatment-related findings in the F2 pups, or of mature F2 offspring. Although study investigators considered the data to be somewhat equivocal, they determined the Overall NOAEL for the study to be 1,000 ppm (~65 mg/kg bw/d for males and females, combined), based on evidence of slightly reduced survival and pup body weights at the high dose. However, the data in the study indicate that a Fertility and Reproduction NOEL of 3,000 ppm (~203 mg/kg bw/day for males and females combined) would be appropriate based on the absence of treatment-related effect in both the F0 and F1 generations. The Foetal and Post-Natal NOAEL would be 65 mg/kg bw/day (based on pup body weights). [Maternal and Foetal Data are presented in Table 19] Post-natal Parameters: Survival and development of pups from birth to weaning was comparable to controls. There was no significant increase in the number of pups with anomalies. Study investigators did not determine NOEL values for this study. However, the data indicate that a Foetal and Post-Natal NOEL of 300 mg/kg bw/d would be appropriate, based on the lack of foetal and pup effects at this dose.

Rat (Colworth Wistar)

Dams received 0, 30, 100, 300 mg/kg bw/day) via oral gavage, in corn oil on Days 6-15 of gestation

Denning et al., 1992 (70); GLPcompliant2 OECD: comparable

1 2

Statistically and biologically significant findings have been outlined. Based on the presence of a statement of Quality Assurance, the study by Denning et al., [1992 (74)] in Colworth Wistar rats was assumed to contain GLP-quality data.

3.3.8.1.1

Fertility and Reproduction

Fertility and reproduction parameters of mating, pregnancy, duration of gestation, and parturition were assessed in the two-generation rat study [Morseth, 1988 (69)]. Based on the absence of effects in F0 and F1 generation rats, the NOAEL (NOEL) for fertility and reproduction was determined to be 3,000 ppm (~203 mg/kg body weight/day using male and female doses, combined), the highest dose tested. 3.3.8.1.2 Post-Natal Parameters

In the two-generation study that examined post-natal development in rats, the primary findings were a slight decrease in mean foetal body weights and a slight decrease in the Day 0 to Day 4 survival in the F1 and F2 generations at the high dose of 3,000 ppm (~203 mg/kg body weight/day) [Morseth, 1988 (69)]. Thus, the foetal and post-natal NOEL for this study was determined to be 1,000 ppm (~65 mg/kg body weight/day using

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combined male and female doses) for triclosan administered in the diet. Results from the study in Colworth Wistar rats showed no effect of triclosan on the survival and development of pups from birth to weaning [Denning et al., 1992 (70)]. In this study, the post-natal NOEL was determined to be 300 mg/kg body weight/day for triclosan administered via oral gavage. In summary, taking into account both of the studies containing post-natal data, the lowest post-natal NOAEL is considered to be 65 mg/kg body weight/day, the post-natal NOEL from the two-generation study using dietary administration of triclosan. 3.3.8.2. Teratogenicity

One-Generation Reproduction Toxicology: Developmental Toxicity Studies 3.3.8.2.1 GLP Studies

GLP-compliant teratology studies were conducted in mice, rats, and in rabbits using the oral route of administration. All 3 of the main studies were conducted along OECD guidelines. One preliminary range-finding study was conducted for each species, in addition to the definitive investigations. NOEL or NOAEL values were determined for each of the definitive studies in each species. Brief summaries of the pertinent findings from the GLP teratology studies are presented in table 19. Where appropriate, findings of foetal effects were classified either as foetal variations (an alteration that may occur at a relatively high frequency and/or represent a (reversible) retardation or acceleration in development, a transitory alteration, or a permanent alteration not considered to adversely affect survival, growth, development, or functional competence in a given species or strain) or foetal malformations (permanent change/anomalies in which there is a morphologic defect of an organ, resulting from an abnormal developmental process that occur at low incidences in a given species or strain of animal) (U.S. FDA, 2001). Table 19: Findings from GLP Teratology/Developmental Toxicity (Segment 2, ICH Stage C) Studies for Triclosan
Species (Strain) Dosing Regimen (mg/kg bw/d) 0, 5, 10, 20, 40, 80, or 160 mg/kg bw/d via the diet (Days 6-15 of gestation) Major Findings1 Reference, GLP and OECD Status Argus Research Laboratories, 1992a (70) GLP-compliant OECD: not applicable for a dose range finding study

Mouse (Crl:CD1(ICR)BR)

Dose range-finding study. Maternal Parameters: Maternal body weight gain and food consumption were reduced in the 160 mg/kg bw/d group. At the doses of 80 and 160 mg/kg bw/d, absolute liver weights and relative liver weights (relative to terminal body weights and to brain weights) were increased. Foetal Parameters: Foetal body weight data were lower for the 40, 80, and 160 mg/kg bw/d groups than for the vehicle control group. Litter averages for resorptions (early and late resorptions, percentage of resorbed conceptuses and the number of dams with resorptions) were increased at 160 mg/kg bw/day. There were no other remarkable findings. Study investigators did not determine NOEL values for this study.

Mouse (Crl:CD1(ICR)BR)

0, 10, 25, 75, or 350 mg/kg bw/d in the diet (Days 6-15 of gestation)

Maternal Parameters: Body weights and body weight gains were increased in the 350 mg/kg bw/d group. Absolute liver weights and relative liver weights (relative to terminal body weights and to brain weights) were significantly increased in the 75 and 350 mg/kg bw/d dose groups. The 350 mg/kg bw/d dose caused increases in the numbers of mice with tan-coloured livers, along with one

Argus Research Laboratories, 1992b (71) GLP-compliant

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Species (Strain)

Dosing Regimen (mg/kg bw/d)

Major Findings1

Reference, GLP and OECD Status OECD: consistent with Teratogenicity guideline

mouse in the 75 mg/kg bw/d dose group. Foetal Parameters: Foetal body weight data were slightly lower for the 75 and 350 mg/kg bw/d groups compared to the vehicle control group. There were reversible delays in ossification caused by the test article in the 75 and 350 mg/kg bw/d dosage groups, including skull ossification and reductions in the average numbers of ossified forepaw phalanges and hind paw phalanges. There were no other remarkable findings. Maternal NOEL = 25 mg/kg bw/d (based on tancoloured livers and increased liver weights) Foetal NOEL = 25 mg/kg bw/d (based on decreased foetal body weights and delayed ossification) Rat (Charles River CD SpragueDawley derived) 0, 5, 10, 25, 50, or 75 mg/kg bw/d via oral gavage, in 1% carboxymethylcellulose in a 20% glycerine in water suspension (Days 6-15 of gestation) 0, 15, 50, or 150 mg/kg bw/d via oral gavage, in 1% carboxymethylcellulose in a 20% glycerine in water suspension (Days 6-15 of gestation) Dose range finding study. Maternal Parameters: The lower maternal body weights that occurred at the high dose were due to the weight loss in a single dam. There were no other remarkable findings. Foetal Parameters: Foetal body weight data were lower for all treatment groups vs. controls; however, only the 75 mg/kg bw/d group had foetal body weights outside the low range of historical control data. There were no other remarkable findings. Study investigators did not determine NOEL values for this study. Maternal Parameters: There was a slight but significant decrease in food consumption from Days 6 through 11 of gestation at the high dose (707 vs. 765 g/kg bw/d in controls). There were no other remarkable findings. Foetal Parameters: Foetal development showed retarded ossification at the high dose (cranium, vertebrae, sternebrae, metacarpals, and pelvic girdle). There were no other remarkable findings. Maternal NOEL = 50 mg/kg bw/d (based on decreased food consumption at the high dose) Foetal NOEL = 50 mg/kg bw/d (based on delayed ossification at the high dose) Maternal Parameters: At the 300 mg/kg bw/d dose level, slight maternal toxicity was manifested as transient diarrhoea, retarded body weight gain (e.g., 9.4 vs. 13.0 g for controls during the period of Days 6 to 10), and reduced food consumption (5-15% decrease vs. controls), and increased water intake (<10% increase vs. controls). There were no other remarkable findings. Foetal Parameters: There were no remarkable findings. Study investigators did not determine NOEL values for this study. However, the data indicate that a Maternal NOEL of 100 mg/kg bw/d would be appropriate, based on decreased body weights and slight diarrhoea, with a Foetal NOEL of 300 mg/kg bw/d (based on a lack of foetal effects). Rabbit (New Zealand White) 0, 5, 10, 25, 50, or 75 mg/kg bw/d via oral gavage in 1% carboxymethylcellulose in a 20% glycerine in water Dose range-finding study. Maternal Parameters: There were slight mean maternal body weight losses and lower terminal body weights of the does treated with 75 mg/kg bw/d, as well as decreased food consumption as measured on Days 6, 8-11, 13, and 16, but not on Days 7 and 18, for this dose group. There were no other remarkable findings. Foetal Parameters: There were no remarkable findings

Bio/dynamics, 1992a (72) GLP-compliant OECD: not applicable for a dose range finding study

Rat (Charles River CD SpragueDawley derived)

Bio/dynamics, 1992b (73) GLP-compliant OECD: consistent with Teratogenicity guideline

Rat (Colworth Wistar)

0, 30, 100, 300 mg/kg bw/d via oral gavage in corn oil (Days 6-15 of gestation)

Denning et al., 1992 (74); GLP-compliant2 OECD: comparable

Bio/dynamics, 1992c (77) GLP-compliant OECD: not applicable for a dose range

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Species (Strain)

Dosing Regimen (mg/kg bw/d) suspension (Days 6 to 18 of gestation)

Major Findings1

Reference, GLP and OECD Status finding study

Study investigators did not determine NOEL values for this study. Maternal Parameters: There were slight decreases in mean maternal body weight (-5.1% at Day 10 to -7.9% at Day 16, with significant decreases on Days 14 and 16) and occasional decreased body weight gains during the dosing period at the high dose, as well as decreased food consumption for this dose group (from -7.05 at Day 11 to -41.4% at Day 14). There were no other remarkable findings. Foetal Parameters: There were no remarkable findings at any of the doses tested. Maternal NOEL = 50 mg/kg bw/d (based on decreased maternal body weights and food consumption) Foetal NOEL = 150 mg/kg bw/d (based on the absence of effects at the highest dose tested)

Rabbit (New Zealand White)

0, 15, 50, or 150 mg/kg bw/d via oral gavage in 1% carboxymethylcellulose in a 20% glycerine in water suspension (Days 6-18 of gestation)

Bio/dynamics, 1992d (78) GLP-compliant OECD: consistent with Teratogenicity guideline

Being consistent with industry standards, statistical analyses were conducted for definitive, but not range-finding, studies. Statistically and biologically significant findings have been outlined. 2 Based on the presence of a statement of Quality Assurance, the study by Denning et al. [1992 (74)] in Colworth Wistar rats was assumed to contain GLP-quality data.

GLP Studies in the Mouse The potential for triclosan to induce developmental toxicity effects has been investigated in 1 range-finding and 1 definitive teratology study in mice [Argus Research Laboratories, 1992a (71); Argus Research Laboratories, 1992b (72)]. Based on the study report, the definitive study was consistent with OECD (Teratogenicity) and ICH (Embryo-foetal development, Stage C) guidelines, with the exception that the placenta was not grossly examined. This deviation would not have affected the interpretation of the results. The test article was administered via the diet; as triclosan did not affect food consumption, the calculated average dosages were comparable to the targeted dose levels in the definitive study. Doses in the definitive mouse study were 0, 10, 25, 75, or 350 mg/kg body weight/day administered in the diet on Days 6 to 15 of gestation. Maternal toxicity was observed at doses greater than 25 mg/kg body weight/day, including liver effects (i.e., increased liver weights and tan-coloured livers). Triclosan was not teratogenic in either the dose range finding or the definitive study. Foetal effects (classified as foetal variations) included slightly decreased body weights at the two higher doses that also caused maternal toxicity, as well as reversible delays in ossification at the same doses. A foetal NOEL of 25 mg/kg body weight/day was determined based upon decreases in foetal body weights and delayed ossification at higher dose levels in the definitive study. GLP Studies in the Rat In rats, triclosan has been investigated in 1 range-finding and 2 definitive teratology studies [Bio/dynamics, 1992a (73); Bio/dynamics, 1992b (74); Denning et al., 1992 (70)]. The study in Colworth Wistar rats [Denning et al., 1992 (70)], although lacking a formal statement of GLP compliance, included a statement of Quality Assurance and was, therefore, considered to contain GLP-quality data. Based on the reports, the studies were generally consistent with the OECD (Teratogenicity) and ICH (Embryo-foetal development, Stage C) guidelines, with the major exceptions being that the placenta was not grossly examined in the Bio/dynamics studies, and there were relatively few numbers of animals in the post-natal survival and development portion of the study by Denning et al. (see Section 3.8.1). Triclosan was orally administered by gavage in all of the studies, although different vehicles were used (1% carboxymethylcellulose in 20% glycerine in water suspension in the

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Bio/dynamics studies, and corn oil in the Colworth Wistar rat study). Rats were treated on Days 6 to 15 in each of the studies. Doses of 0, 15, 50, or 150 mg/kg body weight/day were administered in the definitive oral gavage study using triclosan in 1% carboxymethylcellulose [Bio/dynamics, 1992b (73)]. The dams showed evidence of maternal toxicity in the form of slight, but significant, decreases in food consumption from Days 6 to 11 of gestation in high-dose animals. There was no evidence of any teratogenic effect of triclosan. Foetal effects (foetal variations) were manifest as delayed ossification at the high dose of 150 mg/kg body weight/day, and with maternal toxicity occurring at the same dose. Based on observations of delayed ossification at the high dose, the foetal NOEL in this study was determined to be 50 mg/kg body weight/day. The maternal NOEL was also 50 mg/kg body weight/day. The 1992 study by Denning et al. was conducted in Colworth Wistar rats at doses of 0, 30, 100, or 300 mg triclosan/kg body weight/day administered via oral gavage in corn oil, and examined both embryo/foetal and post-natal parameters. The results from this study demonstrated no foetal effects in terms of anomalies, body weights, numbers of live foetuses, etc. Maternal toxicity was in the form of delayed body weight gain, and reduced food consumption, as well as transient diarrhoea, at the highest dose, resulting in a maternal NOEL of 100 mg/kg body weight/day. Based on the absence of foetal effects at any of the doses tested, the foetal NOEL for this study was determined to be 300 mg/kg body weight/day. In summary, triclosan showed no teratogenic effects at any of the doses in either of the GLP studies in rats. The only embryo or foetal toxic effect observed was classified as a foetal variation (delayed ossification) in the 1% carboxymethylcellulose vehicle study at the dose of 150 mg/kg body weight/day, a dose that was associated with evidence of maternal toxicity (significantly decreased food consumption). The findings of maternal toxicity at the high dose in each of the studies indicated that dose levels were adequately high in each case. (It should be noted that the observed decreases in food consumption followed the administration of triclosan by oral gavage, and therefore would not likely have been due to any decreased palatability of the diet.) Hepatotoxic effects were not noted in the dams in any of the studies. No embryo or foetal toxic effects were seen at any of the doses in the corn oil study. None of the studies showed evidence of any teratogenic effects of triclosan. Taking into account both of the GLP rat teratology studies, the foetal NOEL is considered to be 50 mg/kg body weight/day in rats given triclosan via oral gavage in a 1% carboxymethylcellulose vehicle, with the maternal NOEL being also 50 mg/kg body weight/day. GLP Studies in the Rabbit In rabbits, triclosan has been investigated in one range-finding and one definitive teratology study [Bio/dynamics, 1992c (75), Bio/dynamics, 1992d (76)]. Based on the reports, the studies were generally consistent with the OECD (Teratogenicity) and ICH (Embryo-foetal development, Stage C) guidelines, with the major exception being that the placenta was not grossly examined. Triclosan was orally administered by gavage in a vehicle of 1% carboxymethylcellulose in 20% glycerine in water suspension. The does were treated from Days 6 to 18 of gestation. Doses of 0, 15, 50, or 150 mg/kg body weight/day were administered in the definitive oral gavage study in rabbits [Bio/dynamics, 1992d (76)]. The does showed evidence of significant maternal toxicity in the form of decreased body weights, body weight gains, and food consumption at the high dose. There was no evidence of embryo or foetal toxicity or of teratogenic effect of triclosan. Based on the lack of foetal effects in this study, the foetal NOEL was determined to be 150 mg/kg body weight/day administered by gavage, with the maternal NOEL being 50 mg/kg body weight/day.

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In summary, triclosan showed no teratogenic and no embryotoxic effects at any of the doses in the definitive GLP study conducted in rabbits. Findings of maternal toxicity (decreases in maternal body weight gains) at the high dose indicated that dose levels were adequately high in the study. Hepatotoxic effects were not observed in any of the dams. The foetal NOEL is considered to be 150 mg/kg body weight/day in rabbits given triclosan via oral gavage, with the maternal NOEL being 50 mg/kg body weight/day. Non-GLP Studies Table 20 presents summaries of the non-GLP developmental toxicity studies for triclosan, including findings from a genotoxicity study in mice that examined embryo/foetal toxicity following triclosan administered via the intraperitoneal route of administration. Table 20: Findings from non-GLP Teratology/Developmental Toxicity (Segment 2, ICH Stage C) Studies for Triclosan
Species (Strain) Dosing Regimen (mg/kg bw/d) Single intraperitoneal injections of 0, 1, 2, 4, 8, or 25 mg/kg bw on either Day 9 or Day 10 or gestation Major Findings1 Reference, GLP and OECD Status Russell and Montgomery, 1980 (64) GLP: not specified OECD: not specified

Mouse (C57Bl/E cross, specific for the genotoxicity assay conducted)

Note that this was primarily a genotoxicity study (not a reproductive/developmental toxicity study). Maternal Parameters: Only mortality was assessed. There were deaths in the high-dose group (12/41 dams), but no deaths in the 267 animals treated at the 1, 2, 4, and 8 mg/kg doses. Foetal Parameters: The principle endpoints assessed were embryo and post-natal survival. Embryo survival (reductions in litter size) was reduced at the high dose in animals dosed on either Day 9 or Day 10. Post-natal survival was markedly reduced at the high dose. Postnatal survival was slightly decreased at the dose of 8 mg/kg bw (Day 9 or Day 10 treatment), and at the doses of 2 and 4 combined. Study investigators did not determine NOEL values for this study. It should be noted that, due to the design of the study (e.g., lack of evaluation of parameters typically assessed in reproductive toxicity studies), it was not possible to reach any clear interpretation and conclusion of the impact of these data.

Rat (Wistar)

0, 100, 200, or 400 mg/kg bw/d via oral gavage in olive oil (Days 7-17 of gestation)

Maternal Parameters: Clinical signs of piloerection, incontinence, and diarrhoea were observed at the high dose, together with a decrease in food consumption during the dosing period. Foetal Parameters: There was a significant increase in numbers of foetal deaths at the high dose of 400 mg/kg bw/d (7.05% mortality vs. 1.59% in the controls). Study investigators did not determine NOEL values for this study. However, the data indicate that Maternal and Foetal NOELs of 200 mg/kg bw/d would be appropriate, based on the absence of any significant maternal and foetal effects at this dose level.

Kawashima et al., 1987 (77) GLP: not specified OECD: not specified

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Species (Strain)

Dosing Regimen (mg/kg bw/d) 0, or 1/1000, 1/500, 1/250, 1/100, or 1/50 of the LD50 dose in a pilot study (approximately 4, 8, 16, 40, and 80 mg/kg bw/d based on the LD50 of 4 g/kg bw in rats doses in hamsters were similarly 1/1000, 1/500, 1/250, 1/100, or 1/50 of the LD50 dose). Animals were treated during gestational Days 6-15 (rats) or Days 6-10 (hamsters).

Major Findings1

Reference, GLP and OECD Status Piekacz, 1978 (78) Predates GLP and OECD

Rat (Wistar) and Hamster (Syrian)

Limited numbers and types of parameters were assessed (dams: body weight, clinical signs, mortality; foetuses: body weights, placental weights, foetal length, foetal tail length, number of foetuses, number of males, numbers of resorptions, gross abnormalities). Maternal Parameters (Rat): There were no remarkable findings. Foetal Parameters (Rat): A significant decrease in number of live foetuses and number of males was reported in the 1/1000 (lowest dose) group vs. controls. However, this was not considered to be treatment-related based on a lack of dose relationship. Also, the decrease in number of foetuses did not appear to be due to an increase in resorptions but could have reflected a decrease in ovulation or implantation rate (could not be confirmed due to lack of corpora lutea data). Maternal Parameters (Hamster): Mortality was reported at the highest dose tested (no details provided). Foetal Parameters (Hamster): Body weights were significantly decreased in the high-dose group. The significant decrease in number of live foetuses in the low dose group is not considered to be treatment-related due to the lack of a dose relationship. Also, there was no increase in number of resorptions, suggesting that there might have been a decrease in ovulation or implantation rate (could not be confirmed due to lack of corpora lutea data). The decrease in number of live foetuses in the highdose group was accompanied by maternal mortality. Study investigators did not determine NOEL values for this study. It should be noted that, due to the design of the study (e.g., lack of evaluation of parameters typically assessed in reproductive toxicity studies), it was not possible to reach any clear conclusion of the impact of these data.

Statistically and biologically significant findings have been outlined.

Non-GLP Studies in the Mouse In addition to the GLP studies in mice, limited reproductive toxicity data were found in a published, non-GLP mouse spot test genotoxicity study [Russell and Montgomery, 1980 (64)]. Since this was a genotoxicity study, and not a reproductive toxicity study, there were a large number of deviations from international guidelines for the conduct of reproductive toxicity studies: the lack of evaluation of typical parameters for evaluation in reproductive and developmental toxicity studies (e.g., external and internal examination of the foetuses, litter parameters); the dosing regimen (a single injection on gestational Days 9 or 10 vs. dosing throughout the period of organogenesis); the route of administration (the intraperitoneal route is not appropriate for reproductive toxicity testing); the choice of vehicle solution (although vehicle controls were included and no toxic vehicle effects were reported, 60% methanol is not considered to be an appropriate, non-toxic vehicle for use in a reproductive toxicity study), and the choice of statistical analytical methods (it is presumed, based on the use of the Fishers Exact test for the genotoxicity data, that the same test was used for the reproductive effects data). As such, the significance of the studys stated statistically-significant findings are questionable. As maternal deaths were reported to have occurred in the high-dose group, developmental toxicity findings at this dose level could be attributed to maternal toxicity, and not to the direct toxicity of triclosan to foetuses per se. Without data from the evaluation of appropriate reproductive toxicity study parameters, it was not possible to reach a conclusion regarding the significance, if any, of the post-natal survival data. Overall, the choice of methods used indicates that the data in this non-GLP mouse study are of limited value.

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Non-GLP Studies in the Rat and Hamster Additional reproductive and developmental toxicity data in rats were provided in two nonGLP studies [Kawashima et al., 1987 (77); Piekacz, 1978 (78)]. Although no statement of GLP compliance was included in the published report, the study by Kawashima et al. [1987 (77)] appears to have been well-conducted using methodology comparable to published guidelines for the conduct of reproductive toxicity studies. Effects of significantly increased foetal mortality were reported at the high dose of 400 mg/kg body weight/day (7.05 vs. 1.59% mortality in controls, with the accompanying finding of increased numbers of resorptions at the high dose)1. These effects could not conclusively be attributed to the direct action of triclosan, as this dose also caused maternal toxicity (diarrhoea, incontinence, piloerection, decreased food consumption). There was no evidence of teratogenic effects caused by triclosan at any of the doses tested. Although study investigators did not determine a NOEL for this study, a NOEL value of 200 mg/kg body weight/day may be considered appropriate based on the absence of any significant maternal or foetal effects at this dose. The early study by Piekacz [1978 (78)] included data from experiments conducted using both rats and hamsters. This study was not consistent with current guidelines for the conduct of reproductive toxicity studies, especially with regard to the parameters evaluated, the species used (i.e., hamsters are not conventionally used as they are considered to be a highly sensitive species for reproductive studies), the number of animals (i.e., only 10 per group), and the statistical methods used (no evidence that a necessary correction (adjustment) for multiple comparisons was included). As maternal toxicity was not evaluated, excepting mortality and body weight changes, the doses selected for the study could not be evaluated for appropriateness and, moreover, any reproductive or developmental toxicity effects observed could not be evaluated with reference to maternal effects. In this study, decreases in numbers of live foetuses were reported to occur at the low dose in rats and in both the low and high doses in hamsters. These effects in the lowdose groups were not considered to be directly treatment-related, as, with regard to both rats and hamsters, there was no dose-relationship. There was also no high-dose effect in the rats. For the low dose groups (rat and hamster), there were no increases in resorptions, indicating that the decreased number of live foetuses may have reflected decreases in implantation or ovulation rates (could not be confirmed due to lack of corpora lutea data). Foetal effects in hamsters of decreased foetal numbers at the high dose of 80 mg/kg body weight/day were accompanied by maternal toxicity effects consisting of deaths, and thus were not clearly a direct effect of triclosan. Overall, regardless of the contention that the study may have been inadequately conducted compared to current standards, the study provides little to no evidence of any treatment-related effect of triclosan on the development of rats or hamsters. As with the study by Kawashima et al. [1987 (77)], there were reported to be no teratogenic effects of triclosan at any of the doses tested. No NOEL level was determined for this study. 3.3.8.3 Studies Summary and NOAEL Values from Reproductive and Developmental Toxicology

Both GLP and non-GLP reproductive and developmental toxicology studies have investigated the effects of triclosan on fertility, development, parturition and lactation. The pivotal studies were conducted pursuant to GLP regulations and generally followed the relevant ICH and OECD guidelines. Appropriate test species (i.e., rats and rabbits) and a clinically relevant route of administration (i.e., oral) were used in the studies. Although toxicokinetic parameters were not measured in these studies, the doses achieved in these test systems reached as high as 300 mg/kg body weight/day in rats and 350 mg/kg body weight/day in
1

It should be noted that the mortality rates were 1.51 and 8.19% in the control and high-dose groups, respectively, as presented in the published paper by Kawashima et al. (1987). However, a re-calculation of the data (number of dead implants / total number of implants x 100) yielded mortality rates of 1.59 and 7.05% in the control and high-dose groups, respectively. Following appropriate chi-square statistical analysis, the high dose group data were found to be significantly different from control (p<0.05).

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mice (not a species typically used in reproductive toxicity studies, due to the high sensitivity of the species) and are considered adequate to assess the potential for triclosan to cause human reproductive toxicity. In addition, evidence of maternal toxicity was observed in all of the studies, indicating that adequately high doses of triclosan have been tested. An overall, integrated view of the foetal data together with maternal data is necessary when assessing the relevance of the findings in these reproductive toxicity studies (U.S. FDA, 2001). Taking the studies all together, there was no evidence of teratogenic effects of triclosan in any of the studies, both GLP and non-GLP, and at any of the dose levels tested in rats and rabbits. Similarly, there was no evidence of teratogenic effects in studies conducted in mice and hamsters. Furthermore, in the definitive GLP studies, the effects on developing foetuses were limited to foetal variation effects of reversible, delayed ossification in rats, there being no effects observed in rabbits. It is important to note that foetal variations were observed at doses that also produced significant maternal toxicity. In the GLP-compliant mouse study, consistent with the findings from the rat studies, there were clear indications that the foetal variations of significantly decreased body weight and delayed ossification observed at the two higher doses in the mouse study were likely to be secondary to maternal toxicity consisting of hepatic effects of tan-coloured livers and increased absolute and relative liver weights (there were no foetal effects at doses that were not maternally toxic in mice). No liver effects were observed in any of the studies in rat or rabbits using doses up to 300 mg/kg body weight/day. While there were reports of decreases in numbers of live foetuses in the non-GLP studies [Kawashima et al., 1987 (77); Russell and Montgomery, 1980 (64); Piekacz, 1978 (78)], these effects also were observed at doses of triclosan that produced significant maternal toxicity (e.g., deaths). Thus, the reported effects were unlikely to have been a direct effect of triclosan but, rather, secondary to maternal toxicity. It is considered that only findings potentially indicative of reproductive toxicity at doses that do not produce maternal toxicity are of increased concern for human reproductive or developmental toxicity. Thus, taken altogether, triclosan was not teratogenic and provided no clear evidence of direct effects on reproduction, embryo/foetal toxicity, or post-natal developmental toxicity in any of the reproductive and developmental toxicology studies. Triclosan also has been investigated for potential actions as an oestrogen disruptor, as its chemical structure resembles that of known non-steroidal estrogens (e.g., DES, bis-phenol A). In a published, non-GLP study [Foran et al., 2000 (79)], Japanese medaka fry were exposed to concentrations of up to 100 g triclosan/L for 14 days, but there was no evidence of any effect of triclosan on sex ratios in developing fish. The results of this experiment were consistent with the findings from GLP studies in mammals indicating that triclosan has no effect on sex ratios or on reproductive maturity. NOAEL (NOEL) values from the definitive GLP studies are summarized in Table 21. The developmental toxicity effects of decreased foetal body weights and delayed ossification in all of the studies were observed at doses that also caused maternal toxicity. Based on the data in Table 21, the mouse NOAEL (both maternal and foetal) could represent an overall NOAEL for reproductive and developmental toxicity effects of triclosan. However, as observed in the repeated dose studies, the mouse is uniquely sensitive, showing liver effects at low doses of triclosan, including in the dams in the teratology study. Thus the low NOAEL value for foetal effects that was determined based on the mouse study may be attributed to the sensitivity of the maternal mice to liver effects, and is not due to any direct effect of triclosan on foetuses per se. The next lowest value for an overall NOAEL for reproductive and developmental toxicity is 50 mg/kg body weight/day from the study in rats. Of note, a lack of liver effects at doses up to 300 mg/kg body weight/day was seen in studies in both rats and rabbits, indicating a consistency between these two species.

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Table 21: Summary of NOAEL1 Values from GLP Reproductive and Developmental Toxicity Studies for Triclosan
Species Route of Administration Maternal NOAEL (NOEL)1 (mg/kg bw/d) Foetal NOAEL (NOEL)1 (mg/kg bw/d) Comment

Fertility and Reproduction Effects Rat Diet 2032 No adverse effects on fertility and reproduction, based on lack of remarkable findings in F0 and F1 pups. 25 Maternal liver effects noted in 2 higher dose groups. Foetal effects limited to decreased foetal body weights and delayed ossification at doses that caused maternal toxicity. Decreases in food consumption in highdose dams. Reversible, delayed ossification in foetuses at same dose. Decreases in body weight and diarrhoea in high-dose dams. No remarkable foetal effects. Decreases in body weight and food consumption in high-dose dams. No remarkable foetal effects. Slight decreases in foetal body weights and in mean number of live pups at the highest dose.

Foetal Effects Mouse Diet 25

Rat

Oral (gavage; carboxymethylcellulose) Oral (gavage; corn oil) Oral (gavage)

50

50

Rat

100

300

Rabbit

50

150

Pre- and Post-Natal Effects Rat Diet 652

In all cases, the NOEL values were taken to be the NOAEL values for the study as only NOEL values were determined. 2 Male and female doses combined

3.3.9.

Toxicokinetics

More than 30 non-clinical pharmacokinetics and/or toxicokinetics studies investigating absorption, distribution, metabolism, and excretion of triclosan have been reviewed for this dossier. The kinetics of triclosan and its metabolites have been studied in mice, rats, hamsters, guinea pigs, rabbits, dogs, and monkeys. In these studies, the oral, intravenous, dermal, intraperitoneal, intravaginal, and intraduodenal routes of administration have been examined. In mice, rats, and hamsters, single, stand-alone studies were conducted that investigated all pharmacokinetic (PK) parameters (i.e., absorption, distribution, metabolism, excretion, ADME). Unlabelled triclosan, 14C-labelled triclosan, and 3H-labelled triclosan have been used to study the pharmacokinetics of triclosan. The pharmacokinetic data assessed included the maximum concentrations in blood and tissues (Cmax), the corresponding time required to reach the peak concentrations (Tmax), the area under the curve (AUC), the elimination halflife (t1/2), and the pattern of excretion. In general, the pharmacokinetic studies used similar methodologies to monitor and quantitate the pharmacokinetic data. Following a single or series of extraction and/or partitioning step(s), the blood and tissue samples were analysed by thin layer chromatography (TLC), gas chromatography (GC) with electron capture, high performance liquid chromatography (HPLC), and liquid scintillation counting. Whole body autoradiography was used to determine the relative location of the 14C-labelled triclosan in distribution studies.

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Three stand-alone PK/ADME studies for triclosan were conducted in mice, rats, and hamsters [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82)]. These studies were similar in design, with the exception that the rat study did not investigate the metabolism of triclosan. The remainder of the data available were generated from studies where toxicokinetics was not the primary focus. These kinetic data were derived from toxicokinetic studies conducted in conjunction with toxicology studies [Caudal et al., 1975 (83); Parkes, 1986 (84); Hohensee and Berke, 1991 (85)]. Overall, however, the three PK/ADME studies in mice, rats, and hamsters provide definitive single-dose data, with supporting evidence from the remaining studies. The pharmacokinetic data for triclosan in all species investigated, including the route of administration and the pharmacokinetic parameters assessed, are summarised in Table 22. Table 22: Kinetic Parameters Measured in Non-clinical Studies
Species Mouse Rat oral oral Route1 Parameter Measured Cmax, Tmax, T1/2, AUC Cmax, Tmax, T1/2, AUC Reference van Dijk, 1995 (81) Lin and Smith, 1990 (86), Black et al., 1975 (27), van Dijk, 1996 (82) Siddiqui and Buttar, 1979 (87) Siddiqui and Buttar, 1979 (87) Black et al., 1975 (27) van Dijk, 1994 (80) Hong et al., 1976 (24) Ciba-Geigy, 1976a (88) Stierlin, 1972a (89) Parkes, 1978b (90), Ciba-Geigy, 1976a (88), Ciba-Geigy, 1977a (91) Hazleton Laboratories, 1979b (30)

Rat Rat Guinea Pig Hamster Rabbit Dog Dog Monkey Monkey
1

i.v. i.v.g. oral oral oral oral i.v. oral dermal

T1/2 Cmax, Tmax T1/2 Cmax, Tmax, T1/2, AUC Cmax, Tmax Cmax, Tmax Cmax, Tmax Cmax, Tmax Cmax, Tmax

i.v.=intravenous; i.v.g.=intravaginal

3.3.9.1.1

Single Dose Data

PK/ADME parameters have been examined following single oral doses of 14C-labelled triclosan to mice, rats, and hamsters [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82)]. The single-dose data are summarized in Table 23. Other details from these studies are included under the appropriate heading (i.e., absorption, distribution, metabolism, excretion). Table 23: Summary of Plasma Toxicokinetics of 14C-Triclosan after Single Oral Administration at Two Dose Levels to Rodents
Plasma Levels1 Species Mouse3 Route oral Target Dose (mg/kg bw) 2 200 2 200 Rat3 oral 2 Sex M M F F M Cmax (g/g) 19.48 212.8 8.79 7.67 267.2 263.3 4.772 Tmax (hr) 4 4 1 4 2 4 1 12.6 63.9 0.086 9.9 6,322 0.742 t1/2 (hr) 9.1 11.8 8.9 AUC2 (mghr/L) 166.1 4,505 119.3 C96 (mg/L) 0.02 1.1716 0.007

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Plasma Levels1 Species Route Target Dose (mg/kg bw) Sex Cmax (g/g) 4.458 200 Hamster3 oral 2 200 2 200
1

Tmax (hr) 4 1 4 1 1 1 1

t1/2 (hr)

AUC2 (mghr/L) 3,237 178.0 6,010 79.8 4,298

C96 (mg/L)

M M M F F

153.4 183.3 7.684 359.171 7.357 384.920

10.0 29.1 32 24.5 27.0

1.25 0.181 7.784 0.071 4.556

Cmax =maximal concentrations in blood and tissues, Tmax =the time required to reach the peak concentration, AUC= the plasma area under the concentration-time curve, t1/2= the elimination half-life of the radioactivity, C96=plasma concentration at 96 hr 2 AUC values representative of time of animal sacrifice: 96 h (mice, rats); 168 h (hamsters) (Tmax) 3 Data from: van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82) 4 Animals sacrificed at 96 h (mice, rats) and 168 h (hamsters).

Triclosan is rapidly absorbed as indicated by the time to reach Cmax. Two peaks (2 x Tmax) in plasma triclosan levels were detected in mice and rats, with peak plasma concentrations occurring after 1 and 4 hours in these 2 species. Cmax values obtained following 2 versus 200 mg/kg body weight/day did not reflect the 100-fold increase in dose of triclosan. A comparison of the Cmax data for the low and high doses indicates that the process of absorption may be at least partially saturated at the high dose level and elimination may be enhanced. Triclosan appeared to have high systemic exposure following oral administration, based on urinary excretion data (i.e., high levels of radioactivity were excreted in the urine following dosing, indicating good absorption). Single Dermal Dose With respect to dermal applications of triclosan, a single-application study in newborn Rhesus monkeys using a triclosan-containing soap solution (1 mg/mL, 0.1%) resulted in a Tmax of 12 hours and a Cmax of 0.5 to 0.7 ppm (500 to 700 ng/mL) [Parkes, 1978a (29)]. In comparison to the single-exposure monkey data, rats displayed 2 peaks in plasma concentration 2 hours (0.278 ppm or 278 ng/mL) and 6 hours (0.264 ppm or 264 ng/mL) following the dermal application of triclosan in an ethanol solution to a 7.5 cm2 section of rat skin [Black and Howes, 1975 (23)]. 3.3.9.1.2 Repeated Dose Data

Repeated Dose Data from Pharmacokinetic/ADME Studies In the PK/ADME studies in mice, rats, and hamsters considered to be definitive, plasma concentrations and, in the case of rats, AUC, were determined following 13 days of triclosan administration in the diet. PK analyses were conducted on blood and tissue samples taken Plasma following a dose of 14C-labelled triclosan on Day 14 of repeated dosing. concentrations at Cmax were comparable following the bolus radiolabelled dose on Day 14 compared to the single dose in both rats and hamsters. In contrast, the Cmax level for a single oral dose of triclosan in plasma following repeated daily exposure was decreased in mice, compared to plasma levels following a single dose. These findings are summarized in Table 24. Table 24: Summary of Cmax Values Following Single and Repeated Doses of 2 mg/kg bw/day of Triclosan1

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Species Mouse Rat Hamster


1

Time (h) 4 1-2 1-2

Single Dose Cmax (g/peq/g)2 14.326 4.772 4.883

Repeated Dose Cmax (g/peq/g)1 4.977 4.491 5.003

Data from: van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82) 2 g parent equivalents per gram plasma

Data from the study in rats that examined AUC following both single and repeated doses of triclosan indicate AUC levels in plasma did not change after repeated dosing for 14 days, as shown in the table below. Table 25: Comparison of Calculated AUC Values Following Single and Repeated Doses of Triclosan in Rats
Dose (mg/kg bw/day) 2 200 Single Dose AUC (ng*hr/mL) 64,000 3,237,000 Repeated Dose AUC (ng*hr/mL) 77,400 3,581,000

Repeated Dose Toxicokinetic Data from Oral Toxicology Studies Plasma levels of triclosan were determined following 12 to 14 days of oral (dietary) administration to mice of daily doses of 10 mg/kg body weight were 22,500, 22,000, and 23,600 ng/mL on Days 12, 13, and 14 of dosing, respectively [Huntingdon Life Sciences, 1997 (92)]. The AUC value calculated for these data was 489,000 ng*hr/mL. Plasma levels of triclosan were also determined in the chronic carcinogenicity bioassays conducted in mice, rats, and hamsters [Pharmaco LSR, 1995 (66); Ciba-Geigy, 1986 (67); Huntingdon Life Sciences, 1999 (68)]. Tables 26 through 28 inclusive present the blood levels of triclosan from these 3 carcinogenicity studies. For purposes of comparison, Table 29 presents triclosan levels following at least 6 months of daily oral (dietary) doses in these studies, including dose-normalized data. These data indicate that in chronic dosing studies, plasma levels in mice were slightly higher or comparable to plasma levels in rats (based on dose-normalized data) and much higher than plasma levels in hamsters (greater than 4- to 5-fold). Table 26: Plasma Concentrations (ng/mL) of Triclosan in Mice Following Chronic Dietary Administration
Dose (mg/kg/d) Males Mean 10 30 100 200 16,800 58,900 134,600 177,600 SD 4,260 15,300 25,000 49,300 Interim (6 months) Females Mean 21,900 75,700 172,100 191,500 SD 8,220 11,500 27,200 37,700 Mean 20,600 62,400 150,200 197,200 Males SD 11,100 26,100 37,400 43,100 Termination (18 months) Females Mean 21,100 98,900 169,000 236,500 SD 7,300 21,300 69,400 59,900

Source: Pharmaco LSR, 1994 (66). Table 27: Plasma Concentrations (ng/mL) of Triclosan (FAT 80023/S) in Rats Following Chronic Dietary Administration1

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Males Dose (mg/kg/d)2 Interim (52 weeks) Mean 12-9 40-34 127-107 21,808 55,024 120,368 SD 9,008 12,656 44,464 Termination (104 weeks) Mean 10,576 43,296 86,784 SD 3,392 15,760 26,416 17-11 56-34 190-114 Dose (mg/kg/d)3

Females Interim (52 weeks) Mean 28,160 70,624 170,656 SD 12,928 22,736 32,928 Termination (104 weeks) Mean 26,496 42,048 138,560 SD 18,032 31,888 43,648

Source: Ciba-Geigy, 1986 (67). 1 Note that the original total values for triclosan (free + conjugates) were in units of ng/mL in blood, not plasma. Conversions to values in ng/mL plasma (presented in this table) were based on an average blood volume of 64 mL/kg (range: 58-70 mL/kg) and an average plasma volume of 40 mL/kg (range: 36-45 mL/kg). Source for value for rat plasma volume: McGuill and Rowan, 1989. 2 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) for males consuming dietary doses of 300, 1,000, and 3,000 ppm. Daily doses (mg/kg/d) were decreased by15-20% at 104 weeks compared to 52 weeks. 3 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) for males consuming dietary doses of 300, 1000, and 3,000 ppm. Daily doses (mg/kg/d) were decreased by 35-40% at 104 weeks compared to 52 weeks.

Table 28: Plasma Concentrations (ng/mL) of Triclosan (FAT 80023/S) in Hamsters Following Chronic Dietary Administration
Dose (mg/kg/d) Males Mean 12.5 75 250 1,411 5,310 19,390 SD 467 972 3,465 578 2,578 9,942 Interim (52 weeks) Females Mean SD 188 847 3,055 Mean 1,322 8,162 50,985 Termination (95 weeks for Males; 90 weeks for Females) Males SD 232 3,404 34,197 655 3,683 43,624 Females Mean SD 148 664 56,279

Source: Chasseaud et al., 1994 (93), located in Huntingdon Life Sciences, 1999 (68)

Table 29: Steady State Concentrations of Triclosan in Mice, Rats, and Hamsters Following Chronic Dosing
Species Mouse3 Dose (mg/kg bw/d) 100 200 Dose-Normalized Sex M F M F M F Rat4 127-1075 190-1146 Dose-Normalized Hamster
3

Interim1 (ng/mL) Mean 134,600 172,100 177,600 191,500 888-1,346 958-1,721 120,368 170,656 947 898 19,390 9,942 78 SD 25,000 27,200 49,300 3,700 --44,464 32,928 --3,565 3,055 --

Termination2 (ng/mL) Mean 150,200 169,000 197,200 236,500 986-1,502 1,1831,972 86,784 138,560 811 1,216 50,985 43,624 204 SD 37,400 69,400 43,100 59,900 --26,416 43,648 --34,197 56,279 --

M F M F M F M

250 Dose-Normalized

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F
1 2

40

--

174

--

Mice=6 months, Rats=52 weeks, Hamsters=52 weeks Mice=18 months, Rats=104 weeks, Hamsters=95 weeks (M); 90 weeks (F) Abbreviations: M=Male, F=Female 3 Plasma concentration 4 Conversions from blood concentration to plasma concentration were based on an average blood volume of 64 mL/kg (range: 58-70 mL/kg) and an average plasma volume of 40 mL/kg (range: 36-45 mL/kg). Source for rat plasma volume: McGuill and Rowan, 1989. 5 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) for males consuming dietary doses of 3,000 ppm. Daily doses (mg/kg/d) were decreased by 15-20% at 104 weeks compared to 52 weeks 6 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) for males consuming dietary doses of 3,000 ppm. Daily doses (mg/kg/d) were decreased by 35-40% at 104 weeks compared to 52 weeks.

In summary, in the 3 rodent studies presented in Tables 23 and 24, target dose levels of 2 and 200 mg/kg body weight were used in each study, and thus relevant comparisons of exposure between species can be made following single doses of triclosan. The AUC data (measured over 96 hours in mice and rats and 168 hours in hamsters) show that the mouse and hamster have much higher levels of exposure (almost 3-fold) compared to the rat. The levels of exposure in the mouse and hamster are shown to be similar. Plasma levels at 96 hours were much higher in hamsters than in mice and rats. Based on data from the rat PK/ADME study, there was no change in AUC after 14 days of repeated daily dosing. Data from chronic dosing studies showed that plasma levels in mice were slightly higher or comparable to plasma levels in rats (based on dose-normalized data) and much higher than plasma levels in hamsters (greater than 4- to 5-fold). Plasma levels in the chronic mouse study were comparable to those in the 14-day study (16,800 and 21,900 ng/mL in males and females, respectively). Repeated Dermal Doses In the 90-day study in newborn Rhesus monkeys that used a 0.1% triclosan soap solution, plasma levels ranged from 0.17 to 0.97 ppm (170 to 970 ng/mL) [Hazleton Laboratories, 1979b (30)]. The data from this study showed that plateau levels of triclosan in plasma were reached within 15 days of daily treatment. Other pharmacokinetic data (e.g., AUCs or t1/2) were not available for triclosan administered via the dermal route. 3.3.9.1.3 Bioaccumulation/Bioretention

The accumulation or retention of triclosan in organs and tissues was investigated in the definitive PK/ADME studies in rodents [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82)]. Tissue distribution data from the hamster study showed persistently higher plasma levels of triclosan compared to levels in liver, kidney, and lung following administration of either the low or high dose of 14C-labelled triclosan (2 or 200 mg/kg body weight/day) [van Dijk, 1994 (80)]. Liver, kidney, and lung contained the highest levels of triclosan following oral administration, followed by the gastrointestinal tract. Levels of triclosan in plasma, liver, kidney, and lung following single versus repeated oral doses of 14 C-labelled triclosan are shown in Table 30. A comparison of levels following single dose versus repeated shows no evidence of accumulation or retention of triclosan in liver, kidney, or lung. Taken altogether, the data indicate that triclosan is efficiently eliminated from organs/tissues of the hamster and that there is no accumulation in these tissues after repeated exposure to triclosan. Table 30: Levels of Triclosan in Plasma, Liver, Kidney, and Lung Following Single or Repeated Doses of 14C-Labelled Triclosan in Hamsters1
Organ/ Tissue 2 mg/kg bw/day Male Female 200 mg/kg bw/day Male Female

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Single Dose Plasma Liver Kidney Lung


1

Repeated Dose 0.209 0.037 0.061 0.062

Single Dose 0.093 0.017 0.019 0.027

Repeated Dose 0.089 0.015 0.016 0.028

Single Dose 16.175 3.433 3.254 4.415

Repeated Dose 7.898 1.831 1.488 2.204

Single Dose 7.139 1.361 1.249 1.915

Repeated Dose 5.206 1.212 0.938 1.470

0.317 0.068 0.138 0.091

Levels in g of parental equivalents/gram of tissue.

Additional evidence of a lack of bioaccumulation or bioretention of triclosan is provided by the plasma AUC data generated in the definitive rat PK/ADME study [van Dijk, 1996 (82)]. In this study, the AUC value calculated following an oral dose of 14C-labelled triclosan on Day 14 of a repeated triclosan exposure (diet) regimen was comparable to the AUC value calculated following a single oral dose (see Table 25). The similarity of the single dose and repeated dose AUC values suggests that the amount of triclosan absorbed is equal to that eliminated. Together with the similarity in plasma Cmax values following repeated or single doses (shown in Table 24), the data show that accumulation of triclosan is unlikely to occur with repeated exposure. It should be noted that tissue distribution data in the murine PK/ADME study showed that, at the oral dose of 200 mg/kg body weight/day for 14 days, levels of triclosan in the liver of male mice were approximately 2 times higher than levels in plasma [van Dijk, 1995 (81)]. While definitive studies examining tissue distribution over time have not been conducted for dermal application studies, measurements of plasma levels of triclosan in the 90-day study of newborn Rhesus monkeys washed with a soap solution containing 0.1% triclosan showed levels of 170 to 970 ng/mL triclosan in plasma. A comparison with Cmax plasma levels of 500 to 700 ng/mL at the Tmax of 12 hours after a single application suggests that there is no evidence of bioaccumulation/bioretention of triclosan after repeated dermal exposures. In summary, both tissue distribution and plasma AUC data in hamsters and rats, respectively, provide evidence of a lack of bioaccumulation/bioretention of triclosan. Tissue distribution data in the mouse show that increased levels of triclosan are found in mouse liver compared to plasma. 3.3.9.2 3.3.9.2.1 Absorption Oral Absorption

Overall, the data from studies in rodents and non-rodents suggest that triclosan is rapidly and highly absorbed from the gastrointestinal tract by all species, with maximum plasma concentrations typically being reached between 4 to 8 hours [van Dijk, 1995 (81); Lin and Smith, 1990 (86); Black et al., 1975 (27); Siddiqui and Buttar, 1979 (87); van Dijk, 1994 (80); Ciba-Geigy, 1976a (88); Stierlin, 1972a (89); Parkes, 1978b (90)]. A direct estimation of absorption was calculated from the comparison of AUC values between the oral and i.v. routes of administration in the rat. In this study, it was estimated that an oral dose was 70 to 80% absorbed in rats, based on i.v. and oral AUC values calculated from a 5 mg/kg body weight dose [Stierlin, 1972a (89)]. Likewise, in 1 dog study a comparison of urinary and faecal recovery following both oral and i.v. administration suggests an absorption rate of 50% in this species. Autoradiography studies in mice, rats, and hamsters, also observed high levels of radioactivity in well-perfused organs within 30 minutes of an oral administration of triclosan [Kanetoshi et al., 1988 (94); Howes et al., 1989a (95); Howes et al., 1989b (96); Lin and Smith, 1990 (86); Stierlin, 1972a (89); Howes and Moule, 1989 (97)]. Altogether, these findings suggest that triclosan is highly absorbed following oral administration, with no species-related differences.

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3.3.9.3

Distribution
14

A number of studies have been carried out to determine the distribution of triclosan in the tissues of mice, rats, hamsters, and monkeys.

C-labelled

In tissue distribution analyses conducted in mice following single and repeated doses of 2 or 200 mg/kg body weight/day of triclosan, levels of triclosan were comparable between liver and plasma [van Dijk, 1995 (81)]. However, with repeated dosing, radioactivity levels in the liver remained consistently higher than levels in plasma. In other studies in mice administered radiolabelled triclosan via the oral route, the sites with the highest levels of radioactivity were typically the stomach, GI tract, and gall bladder [Kanetoshi et al., 1988 (94); Howes et al., 1989a (96); Howes et al., 1989b (97)]. Organs/tissues that are wellperfused (e.g., heart, lung, liver, kidney) also routinely displayed detectable levels of triclosan. High levels of radioactivity were still detected in the small intestine and gall bladder (the highest of all mouse tissues with levels of 1,130 g Irgasan DP300 equivalents/g tissue) at 24 hours after a single oral dose of triclosan in ddY mice (100 Ci) [Kanetoshi et al., 1988 (94)]. Similar findings were observed following i.v. administration of triclosan in mice, with the highest levels of radioactive triclosan detected in the tissues of the liver, gut, and gall bladder [Stierlin, 1972a (89); Ciba-Geigy, 1977a (91)]. In rats administered triclosan via the oral route, the tissues with the highest levels of radioactivity included the pituitary gland, bladder, large intestine, kidneys, liver, stomach, and gingiva [Lin and Smith, 1990 (86); Stierlin, 1972a (89)]; however, after 24 hours following i.v. administration of triclosan, the only tissues with levels >1.3 g/g were found to be liver, lung, and kidney [Stierlin, 1972a (89)]. In an examination of plasma, liver, and kidney levels of triclosan following oral doses of 2 or 200 mg/kg body weight/day, plasma and liver concentrations were comparable and kidney levels lower than plasma, even after repeated high doses of triclosan [van Dijk, 1996 (82)]. In the definitive PK/ADME study in hamsters, tissue distribution analyses showed that following either single or repeated doses of 2 or 200 mg/kg body weight/day of 14C-labelled triclosan, the highest levels of triclosan were: plasma >> kidney ~liver ~ lung (see Table 30) [van Dijk, 1994 (80)]. Levels in the excretory organs kidney and liver as well as in lungs were about 3 to 6 times lower compared to plasma levels. In another study in the hamster, the highest levels of radiolabelled triclosan were in the gall bladder, stomach, and GI tract, with lower levels in the kidney and liver (no levels given) [Howes and Moule, 1989 (97)]. In monkeys washed daily with 0.1% triclosan for 90 days, triclosan species were detected in the lung (0.2 to 1.3 ppm), liver (0.1 to 0.5 ppm), kidney (0.1 to 0.9), and skin (0.6 to 1.4 ppm), confirming that triclosan distributes to the liver, lung, and kidney in primates [Hazleton Laboratories, 1979b (30)]. Overall, these data suggest that in rodent species the GI tract, kidney, liver, and gall bladder are the target organs for the disposition of triclosan. A significant contribution to this observation was postulated to result from biliary excretion leading to enterohepatic circulation. In the mouse, levels of triclosan in liver were higher than plasma following longterm repeated dosing. 3.3.9.3.1 Enterohepatic Circulation

Based on whole-body autoradiography studies that identified high levels of radioactivity in the gall bladder and GI tract, and data showing the presence of 2 peak concentrations in the plasma following single or repeated dosing, it has been suggested that mice and rats exhibit enterohepatic circulation [van Dijk, 1995 (81); van Dijk, 1996 (82); Howes et al., 1989a (94); Howes et al., 1989b (96); Kanetoshi et al., 1988 (94); Ciba-Geigy, 1977a (91);

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Stierlin, 1972a (89)]. As such, these species would experience an enhanced local exposure to triclosan in the liver and GI tract. An i.v. study was conducted to investigate the potential for the enterohepatic circulation of triclosan in rats with biliary-fistula, [Ciba-Geigy, 1975b (98)]. Following a 5 mg/kg body weight i.v. dose, 73% of the dose was detected in the bile and then readministered into the duodenum. Of this re-administered dose, 38.8% (28.4% of the original dose) was found in the bile. Thus, these findings provide evidence that triclosan was reabsorbed from the GI tract. Although not definitive, additional evidence that supports the enterohepatic circulation in mice and rats comes from excretion data. Specifically, data from a number of studies have shown that triclosan is excreted primarily via the faecal route in mice and rats, regardless of the route of administration (i.e., even following an i.v. dose) [Hazleton Laboratories, 1979b (30); Lin and Smith, 1990 (86); Howes et al., 1989b (96); van Dijk, 1995 (81); van Dijk, 1996 (82)]. These data suggest a strong GI component to the distribution of triclosan in these species, even following an intravenous dose. In contrast, although high levels of triclosan were detected in the gallbladder and GI tract of hamsters, there were no pharmacokinetic or distribution data comparable to those in mice and rats that would suggest any extensive enterohepatic circulation in this species [Howes and Moule, 1989 (97)]. 3.3.9.3.2 Plasma Protein Binding

The plasma protein binding of triclosan in human, mouse, and hamster blood was investigated in an in vitro study [Sagelsdorff and Buser, 1995 (99)]. Plasma was incubated with Irgasan DP300 at final concentrations of 3.2, 6.4, and 16 g/mL. An equilibrium dialysis method was used and the ratio of the bound to the unbound fractions was constant over the tested concentrations (i.e., no saturation of binding was observed during testing). The plasma protein binding was determined to be 98.4 to 99.2, 98.1 to 98.7, and 98.7 to 99% in humans, mice, and hamsters, respectively [Sagelsdorff and Buser, 1995 (99)]. Thus, triclosan is highly bound in the plasma, with no species differences observed. 3.3.9.4 Metabolism

The metabolism of triclosan has been studied in a number of species including mice, rats, hamsters, dogs, and monkeys. An early study in the rat showed that metabolites of triclosan occurred mainly via aromatic hydroxylation of the ortho and meta positions to the ether bond of the benzene rings and to a smaller degree by scission of the ether bond. Scission of the ether bond produces 2,4-dichlorophenol (found in faeces and urine) and 4-chlorocatechol (excreted in the urine) [Tulp et al., 1979 (100)]. Two key studies have examined the metabolic pathway of triclosan in mice [van Dijk, 1995 (81)] and hamsters [van Dijk, 1994 (80)]. In the liver and skin, triclosan is primarily metabolised to glucuronide and sulfate conjugates, as well as other non-parent metabolites. 3.3.9.4.1 Biotransformation of triclosan

Triclosan is metabolised to parent sulfate and parent glucuronide conjugate compounds in all species examined to date. However, the relative ratio of these conjugates differs among species. The specific identity of the sulfate and glucuronide metabolites has been determined in mice, rats, and hamsters using TLC and gas-chromatography mass spectrometry (GC-MS) [van Dijk, 1995 (81); van Dijk, 1996 (82); van Dijk, 1994 (80)]. In addition to the parent compound and parent conjugates (glucuronide and sulfate), several non-parent metabolites and conjugates were detected [parent (M1), parent glucuronide (M7), parent sulfate (M4), non-parent metabolites (M2 and M3) and corresponding conjugates]. The non-parent metabolites are products of phase 1 metabolism, which are subsequently conjugated (phase 2 metabolites). Following repeated dosing at target dose levels of 200 mg/kg body weight/day, there were more than 7-fold and 2-fold decreases in the levels of the non-parent metabolites in the urine of male mice (52.5 to 7.3%) [van Dijk, 1995 (81)] and hamsters (38.5 to 16.4% )

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[van Dijk, 1994 (80)], respectively. Concomitant increases in the appearance of glucuronide conjugates in the urine were observed for both species, suggesting a decrease of phase 1 metabolism with repeated doses of triclosan. As well as identifying and characterizing the triclosan metabolites in mice [van Dijk, 1995 (81)] and hamsters [van Dijk, 1994 (80)], much effort has been made in stand-alone or specific portions of studies to identify the predominant triclosan species in the urine, faeces, and plasma of mice [van Dijk, 1995 (81)], hamsters [van Dijk, 1994 (80)], and dogs [Hohensee and Berke, 1991 (85)]. These data are summarized in Table 31 below. In general, the sulfate, glucuronide, and free species are predominantly found in the plasma, urine, and faeces, respectively. Table 31: Predominant Triclosan Species in Plasma, Urine, and Faeces of Animal Species Following Single and Repeated Dosing
Species Mouse (% of radio-activity recovered) Route oral Duration single Plasma Free: 64(M) Sulfate: 73(F) Urine Low dose: Glucuronide 23% (M)2; High dose: Free 65% (M); Glucuronide 70% (F) Low dose: Free, 18-38%; High dose: Glucuronide, 63-75% Free3, 25-33% Free 43% (M); Glucuronide 32% (F) Free 2,308 (M) 57,350 (F) Faeces Free 66-96 Reference van Dijk, 1995 (81)

repeated

Sulfate 78-90

Free 68-96

i.v.

single repeated

Free 74-86 Free 68-75 Free 17,000 ng/g (M) 150,000 ng/g (F) Free 55-87 Free 57-91 Free 20-51 Free 27-65 Free 46,367 Free 114-294 Hohensee and Berke, 1991 (85) Parkes, 1978b (90) van Dijk, 1996 (82)

Rat (ng/mL)

oral

single/ repeated

Sulfate 30 d=11,880 (M) 92 d=13,300 (M)

Hamster(%)

oral

single repeated

Glucuronide 28-36 Glucuronide 24-56 Sulfate 9,688 Sulfate1 1.61-3.18

Glucuronide1 56-77 Glucuronide 60-87 Glucuronide 27-58 Glucuronide 33-56 Glucuronide 2,541 Glucuronide 21.3-78.8

van Dijk, 1994 (80)

i.v.

single repeated

Dog (ng/mL)

oral

30-day

Monkey (ppm, at 24 hr)

oral

single dose

Abbreviations: M, male; F, female 1 Single, low-dose female had 43.3% free and 12.5% glucuronide 2 Single, low-dose male had no detectable glucuronide 3 Single, low-dose female had 39% glucuronide, male: 0% glucuronide.

A preliminary study was conducted to investigate the metabolism of triclosan in monkeys and dogs administered single oral doses of radiolabelled triclosan (5 mg/kg) [Ciba-Geigy, 1976a (88)]. Blood levels of radiolabelled triclosan compounds in dogs and monkeys were

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6.08 g/mL (T=3 hours) and 1.24 g/mL (T=8 hours), respectively. The nature of the metabolites was revealed following incubation of samples with specific glucuronidase and arylsulfatase enzymes (i.e., glucuronide and sulfate conjugates) and subsequent analysis by TLC. Prior to enzyme hydrolysis the levels of parent compound were less than 0.8%; however, following enzyme hydrolysis the levels of parent triclosan compound were greater than 80%. Data on the systemic (not dermal) metabolism of dermally-applied triclosan are available from a 90-day bathing study conducted in Rhesus monkeys and its accompanying pilot study [Parkes, 1978a (29), Hazleton Laboratories, 1979b (30)]. In the pilot, single-dose study, 2 Rhesus monkeys (3 days old) were washed with a soap solution containing triclosan (1 mg/mL, volume was not disclosed) [Parkes, 1978a (29)]. Blood samples taken at 1, 3, 5, 8, 12, and 24 hours after washing showed that both glucuronide and sulfate conjugates were present and that no free, unconjugated triclosan was detectable. Blood levels reached plateau levels (0.43 to 0.68 ppm) by 8 to 12 hours after washing and were maintained for 8 to 24 hours. In the 90-day study, blood levels of total triclosan reached a plateau by 15 days and ranged from 0.17 to 0.97 ppm [Parkes, 1978a (29)]. Triclosan was present almost exclusively as glucuronide and sulfate conjugates in the blood; however, the glucuronide conjugate was predominant in samples from Days 1 to 2, after which point the sulfate conjugate predominated, such that sulfate conjugate levels in blood samples at the end of the study were 80 to 90% of the dose. Urinary concentrations ranged from 0.3 to 4.8 ppm and primarily contained the glucuronide conjugate, while faecal levels ranged from less than 0.1 to 10.5 ppm (primarily free triclosan). Overall, the monkey data show that triclosan is absorbed and metabolised to both glucuronide and sulfate conjugates following dermal application and that repeated dermal exposures to triclosan result in the formation of the sulfate conjugate of triclosan as the primary metabolite. 3.3.9.4.2 Dermal Metabolism

An in vitro diffusion skin cell model was used to assess the ability of the skin (rat and human) to metabolise triclosan applied using an ethanol:water (9:1) vehicle [Moss et al., 2000 (21)]. Glucuronidation and sulfation of triclosan were demonstrated to occur in this model (i.e., the conjugates were found in the collecting reservoir following absorption through the skin), with the glucuronide being the primary metabolite at levels up to 1.4%. These findings were supported by in vivo studies with rats in which 0.4 and 1.5% of the parent glucuronide were reported to occur in the urine and skin, respectively, following the dermal application of triclosan [Moss et al., 2000 (21)]. These data show that triclosan is metabolised in the skin. 3.3.9.5 Excretion

The excretion of 14C-labelled and 3H-labelled triclosan was studied in mice, rats, guinea pigs, hamsters, dogs, and monkeys. These studies were the most frequently conducted of all triclosan pharmacokinetic studies. In particular, studies to ascertain the elimination halflife, the routes of excretion, and enterohepatic circulation were conducted for triclosan. 3.3.9.5.1 Elimination Half-Life The terminal plasma half-life of orally administered 14C-labelled triclosan and its metabolites was comparable in rats and mice, but much greater in hamsters (up to 3-fold). In mice, rats, and hamsters the half-life of radiolabelled triclosan is 9 to 12, 10 to 13, and 24 to 32 hours, respectively (Table 23) [van Dijk, 1995 (81); Lin and Smith, 1990 (86); van Dijk, 1996 (82); van Dijk, 1994 (80)]. In hamsters, the much longer half-life of radioactive triclosan is likely attributed to the long residence time of the non-parent M6 and M8/9 metabolites. Different routes of administration [e.g., intraperitoneal (i.p.)] also have been associated with extended or longer half-lives. In rats given tritiated DP300 by i.p. injection, the half-life of radioactive triclosan was 18 hours as compared to 9 to 12 hours [van Dijk, 1996 (82)].

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3.3.9.5.2

Routes of Excretion Following Oral Applications of Triclosan

In mice, rats, hamsters, and monkeys, unlabeled and radiolabelled 14C-triclosan was used to assess the routes of excretion for triclosan following oral doses of triclosan. For studies using radioactive triclosan, the predominant route of excretion in mice and rats was the faeces, whereas in hamsters it was the urine. Based on a survey of the available data, the excretion rates for these species are presented in Table 32. Table 32: Routes of Excretion of Triclosan Following Oral Dosing in Rodent Species
Faeces (%) Mouse Rat Hamster 25-89 81-84 0.1-0.3 Urine (%) 26-44 4-12 60-80 Reference Howes et al., 1989a (95); Howes et al., 1989b (96); van Dijk, 1995 (81) van Dijk, 1996 (82); Lin and Smith, 1990 (86); Hong et al., 1976 (24) Howes and Moule, 1989 (97); van Dijk, 1994 (80)

Routes of excretion in monkeys, have been shown to be urinary in a study using unlabelled triclosan administered to monkeys via oral gavage [Parkes, 1978b (90)], and faecal in a study using dermal applications (washing with soap containing 0.1% triclosan) [Hazleton Laboratories, 1979b (30)]. 3.3.9.5.3 Routes of Excretion Following Dermal Applications of Triclosan

Excretion following dermal applications of triclosan showed that the faecal route predominated in rats, where triclosan was 70 to 90% excreted in the faeces compared to 3 to 4% eliminated in the urine [Ciba-Geigy, 1976b (25)]. Comparable data showing primarily faecal excretion of triclosan regardless of the formulation of the dose were found in other rat studies [Hong et al., 1976 (24); Moss et al., 2000 (21)]. In contrast to rats, rabbits exposed to triclosan in a dermally-applied solution or cream showed moderately greater urinary excretion compared to faecal elimination (47 to 53% vs. 38% of the applied triclosan solution excreted in the faeces; 29 to 48% in urine vs. less than 2% in faeces following the cream application) [Ciba-Geigy, 1976b (25)]. Triclosan levels in the urine and faeces of monkeys bathed daily from birth to 90 days with 15 mL of a soap solution containing triclosan (1 mg/mL) were found to range from 0.3 to 4.8 ppm in the urine and 0.1 to 10.5 ppm in the faeces [Hazleton Laboratories, 1979b (30)]. These data suggest that the faecal route may have dominated in the excretion of triclosan. For the purposes of comparison, it should be noted that the primary route of excretion in humans exposed to triclosan via the dermal route is urinary. In summary, the primary route of excretion following dermal exposure to triclosan differs between species, with the faecal route predominating in the rat, but the urinary route predominating in the rabbit. Neither faecal nor urinary elimination appear to be strongly favoured in the case of primates based on the available data.

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3.3.9.6

Summary of Pharmacokinetic and Toxicokinetic Data in Animals

Triclosan is rapidly absorbed (via the oral, i.p., and i.v.g. routes of administration) in all species. Specific studies conducted in rats indicated that the level of absorption was between 70 to 80% following oral administration in this species. Subsequent to absorption after oral administration, 2 peaks in plasma triclosan levels were detected in mice and rats at 1 and 4 hours, which is indicative of enterohepatic circulation. Evidence from both hamster and rat studies provide evidence of a lack of bioaccumulation/bioretention of triclosan following repeated oral doses. Triclosan is widely distributed in organs and tissues, with well-perfused, and excretory, organs such as liver and kidney, as well as lung, heart, GI tract, and gall bladder showing highest levels following oral, dermal, or i.v. administration in rodents and monkeys. Levels of triclosan in mouse liver were higher than in plasma following repeated dosing with triclosan. Following absorption, the parent triclosan compound was found to be metabolised to both glucuronide and sulfate conjugates. Although different ratios of the individual glucuronide and sulfate conjugates were observed among species, no unique species-specific metabolites have been identified to date. Repeated high-dose administration of triclosan was also shown to change the ratio of these 2 metabolites in hamsters, mice, and monkeys with the sulphate shown to predominate following chronic oral administration. Triclosan was shown to be excreted primarily in the faeces of mice and rats following oral administration, while the predominant excretion route in hamsters and monkeys was via the urine. The faecal excretion route also predominated in rats following dermal applications of triclosan. In addition, evidence of enterohepatic circulation was found for rats and mice, based on autoradiography data, dual peak plasma concentrations, excretion data following i.v. dosing, and bile absorption studies. However, the data available for hamsters do not provide evidence for enterohepatic circulation in this species. Some notable parallels and differences exist among the 3 rodent species, namely mice, rats, and hamsters, that are the most well-characterized species regarding triclosan nonclinical pharmacokinetics. For a target single-dose level of 200 mg/kg body weight, the maximal blood concentrations of both sexes were 213 to 267, 153 to 183 (only males), and 359 to 385 g/g in mice, rats, and hamsters, respectively, with the resulting exposures for this dose of 4,505 to 6,322, 3,237 (average for males) and 4,298 to 6,010 g*hr/mL (Table 23). Thus, in these single-dose studies, rats had the lowest blood levels, whereas mice and hamsters had comparable ranges in exposure levels. In contrast, in rodent chronic carcinogenicity bioassays of at least 18 months duration (i.e., at least 18 months daily exposure in the diet) using different target dose levels, plasma levels in mice were slightly higher or comparable to plasma levels in rats (based on dose-normalized data) and much higher than plasma levels in hamsters (greater than 4- to 5-fold) (Table 29). Evidence from pharmacokinetic studies suggests that the mouse liver is highly exposed to triclosan levels. Repeated dosing in mice led to an increased half-life for triclosan and its sulfate conjugate as measured in the kidney, and elevated liver triclosan levels (~2- to 3-fold) in male mice (with respect to levels measured in the plasma and kidney) [van Dijk, 1995 (98)]. The higher levels of triclosan in mouse liver than in plasma may be correlated with toxicology findings in mouse toxicology studies. Differences between mice, rats, and hamsters in hepatic levels of triclosan [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82)] also may correlate with differences in the incidence of liver-related findings in these species, with the mouse showing greater sensitivity to liver effects, but both the rat and hamster showing either a lower incidence or no liver effects (see Sections 3.3.5 and 3.3.7).

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NOTE Kinetic studies in HUMANS are described in section 3.3.11 3.3.10. Photo-induced toxicity

The data from these studies show that there is no evidence of photosensitisation in the guinea pig, mouse, and pig following exposure to triclosan. As in the case of the sensitisation studies in the guinea pig, these photosensitisation studies also were conducted prior to 1980, preceding the implementation of GLP regulations and OECD guidelines. In a photosensitisation study in the guinea pig, the majority of animals in all dose groups, including negative controls showed an inflammatory response 4 hours post-irradiation with unfiltered light; by 24 hours, none of the animals in the triclosan (1% in solution) or negative control groups showed an erythemic reaction [Thomann and Maurer, 1978 (7)]. It should be noted that a phototoxic response is expected with unfiltered light, as UVB is highly cytotoxic; hence the positive response in the negative control animals. No positive responses in any dose groups were reported following irradiation with filtered light. The authors concluded that there was no indication of photosensitisation activity with triclosan in the guinea pig. There was also no evidence of phototoxicity in the mouse or pig with triclosan (0.1% in methanol, or 0.1 and 1.0% in petrolatum) as reported in another study; however, detailed results were lacking in this study report [Urbach, 1973 (101)]. Based primarily on the guinea pig data, with supporting evidence from the mouse and pig data, there appears to be no potential for photosensitisation with triclosan. Table 33: Findings from Photosensitisation Studies with Triclosan
Species (Strain) Application Details Major Findings Reference, GLP and OECD Status Thomann and Maurer, 1978 (7) Predates GLP and OECD

Guinea pig, Pirbright white

1.0% triclosan in solution, 0.1 mL single application. Irradiation: unfiltered light (UV-A, UV-B), 5 minutes, or filtered light (Pyrexfilters), 15 minutes

Unfiltered light: The majority of animals including controls showed an inflammatory response 4 hours postirradiation in all dose groups. By 24 hours, none of the animals in the triclosan or negative control groups showed an erythemic reaction. Filtered light: No positive responses. No evidence of phototoxicity was observed.

Mouse, Albino Skh:hair-less-1; Pig, Hanford Labco miniature swine

Triclosan (0.1% in methanol, 0.1 and 1.0% in petrolatum) Irradiation sources: UVC; UVB; UVA, and UVB.

Urbach, 1973 (101) Predates GLP and OECD

Summary of Photosensitisation Data In summary, the results of these non-GLP studies indicate that there is no evidence for photosensitisation with triclosan in various formulations and concentrations (up to 1% in petrolatum) in the guinea pig, mouse, and pig. 3.3.11. Human data

Triclosan has been used in consumer products for over 30 years as an antibacterial agent and disinfectant. It is widely used in external-use, over-the-counter personal care products such as dentifrices, deodorants, soaps, creams, and lotions. Triclosan was approved for use at a level of 0.3% in cosmetics products in 1986 by the European Community Cosmetic Directive. The world-wide population of consumers exposed to triclosan includes both adults and children, with not-unexpected exposure in infants occurring via breast milk at low levels [Adolfsson-Erici et al., 2002 (102), Allmyr et al., 2006 (103), Dayan, 2007 (104)].

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Studies on triclosan levels in human blood samples from Sweden [Allmyr et al., 2006 (103), Sandborgh-Englund et al. 2006 (116)] and from Australia [Allmyr et al. 2008 (AR1)] and a recent biomonitoring study with urine samples from the US [Calafat et al. 2008 (AR2) indicate widespread exposure of the general population to triclosan from various sources, including personal care products. The following sections describe the general safety data for triclosan based on consumer use information and on results from clinical studies, the pharmacokinetics of triclosan in both adults and children following single and repeated oral and/or dermal exposures, and the irritation, sensitisation, and photosensitisation data for triclosan as evaluated in clinical tests. Estimates and findings of exposure levels to triclosan are presented in Sections 3.3.11.2.6 (for infants) and 3.3.11.2.7 of this opinion (and in the discussion). 3.3.11.1 Human Safety/Tolerability Data

3.3.11.1.1 Safety Data from Consumer Use Information Consumer use information such as listings of adverse events for cosmetic products containing triclosan is generally unavailable. No serious adverse events have been reported for triclosan-containing toothpaste. The low number of reported non-serious adverse events for triclosan-containing toothpaste (frequency of 0.27 complaints/100,000 units sold) included reactions that were associated with the use of dentifrices in general, as identified during clinical testing (e.g., dry mouth, mouth irritation/burning, sensitive teeth, altered taste or oral sensation, exfoliation). Other non-serious adverse events were those that represented a variety of much more infrequently reported effects (i.e., usually reported by only one or two consumers, resulting in a reporting frequency of 0.09 complaints/100,000 units sold). These rare non-serious events, which may not have been related to the use of the triclosan-containing toothpaste, included stomach ache, belching, alopecia, anxiety, headache, black or coated tongue, dizziness, excess saliva, upper respiratory infection, increased urge to urinate, and shortness of breath. Thus, based on consumer use information for triclosan-containing toothpaste, which represents a wide-spread use that includes the potential for systemic exposure through the oral route, data indicate that triclosan can be used safely and with good tolerability at levels that also are found in personal care products. 3.3.11.1.2 Safety/Tolerability Data from Clinical Studies In addition to the indications of good tolerability and safety from historical and consumer use of personal care products containing triclosan, a number of clinical studies have investigated the safety and tolerability of triclosan. Table 34: Findings from Human Safety Studies for Triclosan
No. Subjects 153 Dosing Regimen Twice daily brushing, for 12 weeks, with 0.2% triclosan in toothpaste (75 subjects) or NaF/silica toothpaste (78 subjects). Major Findings For triglycerides, the triclosan group differed from the placebo group (p<0.01) at 3 weeks, but not at 12 weeks. There was no difference between treatment and control groups for any of the other liver function tests. Reference ColgatePalmolive, 1994 (105)

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No. Subjects 112

Dosing Regimen Normal daily use of a toothpaste containing 0.2% triclosan and 0.5% zinc citrate for 52 weeks, followed by a 13week washout period.

Major Findings The data showed increases in white blood cells and lymphocytes throughout the study, with decreases in mean corpuscular haemoglobin/concentration. Biochemical analyses showed increases in sodium, cholesterol (total, LDL, and HDL), uric acid, and creatinine and decreases in bilirubin at all time points in the study in either males or females or both sexes. These changes were not attributed to triclosan. Blood samplings at baseline and 4, 12, 24, 38, 52, and 65 weeks. Standard haematology and clinical biochemistry results showed some statistically significant changes periodically (compared to baseline values), but no pattern to the changes. Some significant correlations were found between parameters and triclosan blood levels, but these were inconsistent over the course of the study in terms of time or sex of subject. Therefore, these changes were not attributed to triclosan toothpaste use. Oral irritation data including subjective complaint level for the whole trial period were summarised and found to be similar to effects experienced in similar trials involving toothpaste. There were no withdrawals from the trial due to adverse reactions. No treatment-related changes were reported for any parameter measured, including haematology, clinical chemistry, and urinalysis parameters. The data shows that the use of toothpaste containing 0.3% triclosan produces no adverse effects compared to triclosan-free toothpaste formulations.

Reference Safford, 1991 (106)

112 (86 subjects completed study)

Normal daily use (according to subjects normal brushing habits) of a toothpaste containing 0.2% triclosan and 0.5% zinc for 12 weeks, with an extension of the study to 52 weeks (followed by a 13-week observation period)

Barnes, 1991 (107)

3,000

Normal dentifrice use, for 3 years, with silicon dioxide-based toothpaste containing 0.243% fluoride and 0.3% triclosan, or 0.243% NaF, or 0.331% NaF. 0, 1, 5, 9, 12, 15, 18, 21, 24, 27, or 30 mg triclosan in capsule, depending on study phase. Single-dose and repeat-dose depending on study phase (up to 30 days).

Fishman, 1994 (108)

20

There were 6 dropouts (5 triclosan and 1 placebo) due to adverse effects (skin reactions - 3 subjects including 1 placebo subject; increase in liver enzymes - 2 subjects; and, antrum gastritis - 1 subject). These findings were not considered to be attributable to triclosan, as they are not uncommon in pharmacological studies. Based on vital signs, ECG, lung function, neurological examination and most laboratory parameters, triclosan was considered to be well-tolerated. There were slight increases in liver enzyme values including SGPT, SGOT, and gammaGT.

Lucker et al., 1990 (109)

Four human studies have evaluated the safety of triclosan in toothpaste products. These studies tested triclosan-containing toothpaste preparations in study populations over periods ranging from 12 weeks to 3 years at concentrations of 0.2% triclosan [ColgatePalmolive, 1994 (105)], 0.2% triclosan with 0.5% zinc citrate [Safford, 1991 (106); Barnes, 1992 (107)], and 0.3% triclosan with 0.243% fluoride [Fishman, 1994 (108)]. Endpoints measured included blood chemistry and haematological parameters in all 4 studies. In addition, urinalysis parameters were evaluated in 1 study [Fishman, 1994 (108)]. In all studies, there were no changes indicative of overt toxicity. Reported changes in haematological and/or clinical chemistry parameters that did occur could not be attributed to the use of the triclosan-containing toothpaste. In a separate study of pharmacokinetics and tolerability, consumption of escalating daily doses of triclosan in capsule form (0 to 30 mg/capsule) by 20 volunteers was reported to be without overt effects on ophthalmic, neurologic, cardiac, and lung function evaluations [Lucker et al., 1990 (109)]. There were slight increases in liver enzyme values of the treated subjects, but this is in contrast to the four human studies (described above) that reported a lack of findings in liver enzyme parameters. Five of the treated participants dropped out during the course of the study; however, the adverse events reported in these subjects could not be attributed to triclosan.

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One of the safety studies [Safford, 1991 (106)] reported on the concentrations of triclosan in the blood following the use of a toothpaste containing 0.2% triclosan and 0.5% zinc citrate. In this study of 112 participants, the average triclosan concentration in the blood of 15.5 ng/mL was reported to rise to 31.2 ng/mL after a period of 4 weeks on study. This concentration was reported to subsequently level off with further use of the triclosancontaining dentifrice. In summary, the human safety studies reviewed showed no signs of overt toxicity in over 3,000 subjects that used triclosan-containing toothpaste for 12 weeks to 3 years. 3.3.11.2 Human Pharmacokinetics and Metabolism

A total of over 30 pharmacokinetic studies investigating the absorption, metabolism and excretion of triclosan in humans have been reviewed. In these studies, several different routes of administration, including oral exposure to triclosan-containing products (e.g., toothpaste), oral ingestion of capsules, aqueous solutions, and dental slurries (i.e., following brushing with triclosan-containing toothpaste), and percutaneous exposure (in vivo and in vitro) have been investigated. Overall, ingested triclosan is almost completely absorbed, whereas oral cavity and percutaneous exposure to triclosan-containing products (e.g., toothpaste, soap, cream, etc.) results in limited absorption. Following all routes of administration, absorbed triclosan is nearly totally converted to glucuronic and sulphuric acid conjugates (varied relative proportions), with only trace amounts of the parent compound detected in the plasma, and the predominant route of excretion for triclosan is the urine, with the majority of the compound appearing as the glucuronide conjugate. 3.3.11.2.1 Pharmacokinetics The pharmacokinetic parameters assessed for triclosan in humans include the Cmax, the time required to reach Tmax, the AUC values for plasma concentrations versus time, and the elimination half-life (t) of plasma concentrations. These parameters were assessed following several different routes of administration, including oral exposure to triclosancontaining toothpaste (expelled dental slurry), and oral ingestion of triclosan-containing capsules, aqueous solutions, and dental slurries (i.e., ingestion of dental slurry following brushing with triclosan-containing toothpaste). In general, the pharmacokinetic studies reviewed herein analysed samples using HPLC or GC with electron capture detector. The pharmacokinetic data for triclosan, as measured in children and adults following various routes of administration, are summarized in Table 35. Table 35: Summary of Plasma Pharmacokinetics of Triclosan in Children and Adult Subjects
Parameters1 Route Oral (capsules) Oral (aqueous solution) Subject Adults Dose/Duration 1 mg/single dose 15 mg/day for 36 days Adults 10 mg/single dose Cmax (ng/mL) 23.3 330.9 974.1 Tmax (h) 5.00 4.08 1.6 AUC2 (ng*h/mL) 208 4,296 11,237 t (h) 13.4 19.0 19.9 Reference Lucker et al., 1990 (109) Concordia Research Laboratories, 1997a (110) ColgatePalmolive, 1989 (113)

4 mg/day for 21 days

191.2

16.0

2,788

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Parameters1 Route Subject Dose/Duration 4 mg/single dose Cmax (ng/mL) 218 Tmax (h) 1.5 AUC2 (ng*h/mL) 2,600 t (h) 21 Reference SandborghEnglund et al., 2006 (116) ColgatePalmolive, 1997a (111) Concordia Research Laboratories, 1997b (112) Concordia Research Laboratories, 1997a (110) BIBRA International, 1997 (114) ColgatePalmolive, 1989 (113) BIBRA International, 1997 (114) ColgatePalmolive, 1997b (115) ColgatePalmolive, 1997b (115)

Children3

3.0 mg/single dose

495.9

1.8

6,545

16.8

N/A

N/A

N/A

12.7

Oral (dental slurry)

Adults

6 mg/single dose

574.2

3.0

7,235

20.0

18 mg/day for 14 days (dental slurry swallowed after brushing) Oral (toothpaste) Adults 4 mg/day for 21 days 18 mg/day for 14 days (dental slurry expelled after brushing) 3.75 mg/single dose 11.25 mg/day for 12 days (3.75 mg x 3 brushings daily)

878.0

N/A

218,856

N/A

26.7

12.0

329

N/A

145.5

N/A

34,855

N/A

242.9

4.0

2,818

14.6

384.04,5

2.0

2,8446

N/A

N/A, not available 1 Cmax =maximal concentrations in blood and tissues, Tmax =the time required to reach the peak concentration, AUC= the plasma area under the concentration-time curve, T1/2= the elimination half-life of the radioactivity 2 When both AUC(0) and AUC(024h) values were reported, the former was used. 3 Children ranged from 8 to 12 years of age. Due to limited blood collection intervals in the study, limited or no Cmax and Tmax data were available, and no AUC values were calculated for the available data [Concordia Research Laboratories, 1997b (112)]. 4 Sampling was conducted following the first of 3 brushings on Day 12 of the multiple-dose phase of the study. 5 Plasma concentrations ranged from 353 to 384 between hours 1 to 7 of the study. 6 AUC value is dose-normalized (mean AUC(024) corrected for number of brushings on that day (AUC24/3)

As outlined in Table 35, Cmax and AUC values obtained from adult subjects generally increased with increasing dose levels, with Cmax values ranging from 23.3 to 974.1 ng/mL, and AUC values ranging from 208 to 11,237 ng*h/mL for single doses ranging from 1.0 mg/dose to 10 mg/day (corresponding to approximately 0.017 and 0.17 mg/kg body weight, respectively, for a 60 kg adult). For multiple doses ranging from 4 to 18 mg/day (corresponding to 0.067 and 0.30 mg/kg body weight/day for a 60 kg adult), Cmax values ranged from 26.7 to 878 ng/mL, and AUC values ranged from 329 to 21,8856 ng*h/mL. Corresponding Tmax values ranged from 1.5 to 5.0 hours and from 4.08 to 16.0 hours for single and multiple doses, respectively. t values obtained from adult subjects remained fairly constant across all the studies reviewed, ranging from 13.4 to 21.0 hours. There are limited pharmacokinetic data for children, and no direct comparisons to adults were possible, given differences in doses and dosing formulations in all of the studies, with the exception that elimination was determined to be essentially the same for children and adults. Two single oral dose (3.0 mg triclosan from aqueous solution) studies were

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conducted in children [n = 11 children, aged 8 to 12 years in Colgate-Palmolive, 1997a (111) and n = 4 children, aged 9 to 12 years in Concordia Research Laboratories, 1997b (112)]. The mean elimination rate constants (Kel) for each study were calculated to be 0.0453 h-1 (n = 11) (3) and 0.062 h-1 (n = 4) [Concordia Research Laboratories, 1997b (112)]. These values are similar to those calculated in adult subjects exposed to single oral doses of triclosan from aqueous solution (Kel = 0.043 h-1), dental slurry (Kel = 0.043 h-1) and toothpaste use (Kel = 0.0742 h-1) [Concordia Research Laboratories, 1997a (110)]. Thus, the rate of elimination is comparable in children and adults. Pharmacokinetic parameters have also been assessed in the saliva of adult subjects exposed to 4 mg triclosan from toothpaste (not outlined in Table 35). Throughout the 2hour period following tooth brushing, mean saliva triclosan concentrations and Ke and t values revealed first-order kinetics [Gilbert, 1987 (117)]. Intra-subject comparisons were made following single and multiple oral doses of triclosan [Lucker et al., 1990 (109), Colgate-Palmolive, 1997b (115)], and following ingestion of triclosan-containing dental slurry and triclosan aqueous solution [Concordia Research Laboratories, 1997a (110)]. The mean AUC values for the single- and dose-normalized multiple-dose exposures were not different (dose-normalized AUC values for multiple-dose exposures not shown in Table 35), suggesting a linear disposition for triclosan after repeated brushing [Colgate-Palmolive, 1997b (115)]. Dose-normalized AUC and Cmax values were similar following ingestion of triclosan-containing dental slurry and triclosan aqueous solution, suggesting that each route of administration results in similar levels of exposure to triclosan [Concordia Research Laboratories, 1997a (110)]. To investigate differences in exposure levels following different formulae of administration, inter-subject comparisons were made between triclosan-containing toothpaste (dental slurry expelled) use versus ingestion of the dental slurry [BIBRA International, 1997 (114)], and between triclosan-containing toothpaste (dental slurry expelled) use versus ingestion of triclosan aqueous solution [Colgate-Palmolive, 1989 (113)]. As would be expected, based on AUC and Cmax values, ingestion of triclosan aqueous solutions (at levels simulating the maximum absorption of triclosan that would be possible from a triclosan-containing toothpaste) resulted in higher levels of exposure to triclosan compared with the use of triclosan-containing toothpaste (expectorated) [Colgate-Palmolive, 1989 (113)]. Similarly, higher AUC and Cmax values were observed in subjects who ingested the dental slurry compared with those who expelled the slurry following brushing with triclosan-containing toothpaste [BIBRA International, 1997 (114)]. For each study, there were no differences between groups with respect to time to reach steady-state plasma triclosan concentrations [4 days in the 14-day study (BIBRA International, 1997 (114), and approximately 14 days in the 21-day study (Colgate-Palmolive, 1989 (113)]. In summary, AUC and Cmax values appear to increase with increasing doses of triclosan. There does not appear to be a correlation between the duration of triclosan exposure (measured in days) from tooth brushing and the levels of exposure (i.e., AUC values); however, the formula of administration does appear to affect both AUC and Cmax values. While the ingestion of triclosan-containing dental slurry and triclosan aqueous solution each results in similar levels of exposure to triclosan, both routes of exposure result in higher levels when compared with the use of triclosan-containing toothpaste followed by expectoration. These data show that there is limited absorption under normal conditions of toothpaste use (i.e., expectoration and rinsing) compared with oral ingestion of triclosancontaining solutions (see absorption section). Bioaccumulation/Bioretention In the absence of data showing tissue levels of triclosan following single and repeated exposures, evidence of a lack of bioaccumulation or bioretention of triclosan is provided by an examination and comparison of plasma triclosan levels in a number of studies.

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The plasma AUC0-inf level of triclosan was 2,818 ng*h/mL following single use of dentifrice containing 0.3% triclosan (3.75 mg dose, with ingestion of the dentifrice) in a study of 21 adult subjects [Colgate-Palmolive, 1997b (115)] (see Table 35). In the repeated dose phase of the same study, subjects brushed 3 times daily for 12 days with ingestion of the dental slurry (3.75 mg/dose). After 12 days of brushing, the plasma AUC levels following a single dose was calculated to be 2,844 ng*h/mL after normalizing for the number of doses (AUC024h = 8,833 ng*h/mL per 3 doses per 24 hours). There was no significant difference between the single- and repeated-dose AUC values, suggesting a complete elimination of the daily triclosan dose, and no increase in the triclosan level during repeated brushing/ingestion. Additional supporting evidence for a lack of bioaccumulation or bioretention of triclosan is shown in several studies in which there was no further increase in plasma triclosan levels once steady-state blood concentrations had been reached. Triclosan levels in plasma were comparable from Days 7 to 21 in a study in which subjects either ingested 20 mL of an aqueous solution containing 0.01% triclosan or brushed twice daily with 1 g of a dentifrice containing 0.2% triclosan (with expectoration and rinsing) (plasma levels ranged from 15 to 21 ng/mL in the dentifrice group) [Colgate-Palmolive, 1989 (113)]. In a longer-term parallel, double-blind 12-week study with dentifrice containing 0.2% triclosan, blood samples showed comparable mean levels of 16 and 14 ng/mL at 3 and 12 weeks, respectively [Lin, 1989 (135)]. In a 52-week tooth brushing study using dentifrice containing 0.2% triclosan, total triclosan levels in plasma were consistently in the range of 27 to 40 ng/mL from 4 weeks to the end of the 52-week exposure period [Safford, 1991 (106)]. Altogether, the data from these 3 studies show consistency in plasma triclosan levels following the use of dentifrice containing 0.2% triclosan. In a review of the data from Colgate-Palmolive, 1989 (113), Lin, 1989 (135), and Colgate-Palmolive, 1997b (115), this conclusion was extended to dentifrices containing triclosan at levels up to 0.3% [Bagley and Lin, 2000 (142)]. In addition, the data suggest that there is no accumulation of triclosan levels as reflected in comparable plasma levels over the time course of each study, suggesting a lack of evidence of bioaccumulation of triclosan. In the absence of studies examining tissue concentrations over time, relatively invariable plasma concentrations of triclosan provide evidence of a lack of bioaccumulation following dermal application in human studies. Plasma levels of total triclosan ranged between 85 and 101 ng/mL between Days 5 to 20 in males and 41 to 47 ng/mL in females over the same time period in which triclosan exposure occurred through the use of hand wash containing 1% triclosan [Ciba Specialty Chemicals, 2002 (134)]. These data suggest a balance between absorption and elimination and a lack of bioaccumulation following dermal absorption. In summary, plasma AUC data for triclosan following either single or repeated oral dosing indicate a lack of accumulation of triclosan. The blood or plasma AUC is a measurement of exposure to a given dose of drug or substance, encompassing both amount absorbed and amount eliminated. If the AUC of a 24-hour dose interval in a continuous dosing regimen is equal to the AUC of an equivalent single dose, the data would indicate a lack of accumulation (i.e., the complete elimination of the daily triclosan dose, with the absorption equal to the elimination from the body in a given single-dose interval). In addition, the steady and relatively invariable plasma levels of triclosan in long-term dosing (brushing) studies and in dermal application studies further suggest that triclosan does not accumulate in the body. 3.3.11.2.2 Absorption The absorption of triclosan was measured following several different routes of administration, including oral exposure to triclosan-containing products (e.g., toothpaste),

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oral ingestion of capsules, aqueous solutions, and dental slurries (i.e., following brushing with triclosan-containing toothpaste), and percutaneous exposure (in vivo and in vitro). Absorption Following Oral Administration Several studies of oral absorption have been conducted for exposures via capsules, solutions, dental slurries, mouthwash, or toothpaste. Overall, triclosan administered via the oral route in capsules is almost completely absorbed, as indicated by urinary elimination data [Lucker et al., 1990 (109); Stierlin, 1972b (121)]. Following exposure to 15 mg/day of triclosan (corresponding to 0.25 mg/kg body weight/day) for 36 days, 90% of the administered dose was eliminated in the faeces (10%) and urine (80%), as shown in an intra-subject study [Lucker et al., 1990 (109)]. Similarly, more than 98% of the administered dose was recovered in the urine (87%) and faeces (11%) following a single 200.5 mg dose of radiolabelled triclosan in a gelatine capsule (corresponding to 3.3 mg/kg body weight) [Stierlin, 1972b (121)]. Two separate studies examined the effects of the formulation of the administered triclosan on oral absorption [Concordia Research Laboratories, 1997a (110); Colgate-Palmolive, 1989 (113)]. A study comparing oral ingestion of triclosan from dental slurry (following brushing with triclosan-containing toothpaste) with oral ingestion of triclosan from aqueous solution revealed that the onset and rate of absorption of triclosan was faster for the aqueous solution compared with the dental slurry [Concordia Research Laboratories, 1997a (110)]. Results from a study comparing triclosan-containing toothpaste use (expectoration of dental slurry) with ingestion of triclosan aqueous solution confirmed that the amount of triclosan absorbed from normal toothpaste use (including expectoration and rinsing) is extremely low (i.e., 5 to 10% of the dose, which corresponds to 9 to 14% of the amount absorbed and excreted following ingestion of an equivalent amount of triclosan in aqueous solution) due to decreased ingestion [Colgate-Palmolive, 1989 (113)]. The results of these studies show that normal toothpaste use would be expected to result in low levels of total absorption together with a slow onset and rate of absorption compared with oral ingestion of triclosan in an aqueous solution. Oral retention of triclosan following the use of triclosan-containing products (toothpaste and mouth rinse) was examined in 2 studies [Gilbert, 1987 (117); Lin, 2000 (118)]. In one study, saliva samples were collected after the first of two daily brushings with triclosancontaining toothpaste (2 mg triclosan per brushing). Mouth rinse samples were collected following the use of a mouth rinse formulated to recover triclosan after the second brushing. The results indicated that approximately 25% of the triclosan dose is retained in the mouth following tooth brushing, with the remainder being recovered on the toothbrush, expectorated and rinsed out. The use of a non-triclosan mouth rinse following brushing decreases oral retention further, with approximately 14% of the retained triclosan (i.e., of the 25%) being recovered by the mouth rinse [Gilbert, 1987 (117)]. In a separate study, 4.5 mg of triclosan was administered in a mouth rinse twice daily for 21 days. Oral retention of triclosan was measured to be 4 to 13% of the daily dose, and buccal absorption of triclosan was estimated to be 2 to 4% of the daily dose [Lin, 2000 (118)]. Absorption Following Percutaneous Administration The main findings from in vivo and in vitro percutaneous absorption studies for triclosan are summarized in Table 36, with discussion in the paragraphs that follow. Table 36: Findings from Human In Vivo and In Vitro Percutaneous Absorption Studies for Triclosan
Subject In Vitro Studies Method Major Findings Reference

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Subject In vitro

Method Breast or abdominal skin (0.64 cm2) was obtained and exposed to a tritiated triclosan solution for up to 24 hours.

Major Findings Non-GLP. After 24 hours, approximately 6.3% of the applied dose appeared in the receptor fluid, and 46%, 24%, and 22% remained on the skin surface, in the epidermis and dermis, and stratum corneum, respectively. Of the radioactivity in the receptor fluid at 24 hours, 0.8% of the applied dose was present as triclosan, 3.5% as triclosan glucuronide, and 0.2% as triclosan sulfate. Of the radioactivity in the skin at 24 hours, 12% of the dose was recovered as triclosan, 3% as triclosan glucuronide, and 3% as triclosan sulfate. GLP-compliant. Conducted comparable to OECD Guideline No. 428. After 24 h, 95% recovery of the applied dose of triclosan. Period of penetration was between 8 and 24 h following a lag phase of approximately 8 h. The rate of skin permeation reached ~0.008 g/cm2/h. After 24 h, 3.90.4% of the applied dose of triclosan had penetrated into the receptor fluid (0.140.01 g/cm2). As measured at 24 h, triclosan in the surface material was 76% of the applied dose (65% in the 24-h surface wipe and 11% in the first 3 tape strips of skin). Succeeding tape-strips (strips 4-20) contained 6.8% of the applied dose (0.25 g/cm2), and 7.4% of the dose was recovered from the remainder of the sample of skin (0.280.05 g/cm2). Percutaneous absorption was calculated to be 11.3% (0.42 g/cm2) based on receptor fluid plus remainder of skin after removal of tape strips 1-20. GLP-compliant. Conducted comparable to OECD Guideline No. 428. After 24 h, there was 89% recovery of the applied dose of triclosan. Period of penetration was between 2 and 6 h following a lag time of about 2 h. The rate of skin permeation reached ~0.01 g/cm2/h. After 24 h, 2.30.3% of the applied dose had penetrated into the receptor fluid (0.0930.01 g/cm2). As measured at 24 h, triclosan in the surface material was 4.3% of the applied dose (<1% of wiped off surface); 70% had been recovered in the 30-minute rinsate. Succeeding tape-strips (strips 4-20) contained 3.0% of the applied dose (0.25 g/cm2), and 9.71.7% of the dose was recovered from the remainder of the sample of skin (0.390.06 g/cm2). Percutaneous absorption was calculated to be 12.0% (0.483 g/cm2) based on receptor fluid plus remainder of skin after removal of tape strips 1-20. GLP-compliant. Conducted comparable to OECD Guideline No. 428. After 24 h, 84% recovery of the applied dose of triclosan. Period of penetration was between 8 and 24 h following a lag phase of 6-8 h. The rate of skin permeation reached ~0.002 g/cm2/h. After 24 h, 0.850.13% of the applied dose of triclosan had penetrated into the receptor fluid (0.0330.006 g/cm2). As measured at 24 h, triclosan in the surface material was 64% of the applied dose (40% in the 24-h surface wipe and 24% in the first 3 tape strips of skin). Succeeding tape-strips (strips 4-20) contained 13.2% of the applied dose (0.50 g/cm2), and 6.8% of the dose was recovered from the remainder of the sample of skin (0.270.09 g/cm2). Percutaneous absorption was calculated to be 7.65% (0.303 g/cm2) based on receptor fluid plus remainder of skin after removal of tape strips 1-20.

Reference Moss et al., 2000 (21)

In vitro

Female epidermal skin samples from cosmetic surgery, mounted in diffusion cells; single application of 0.2% triclosan in w/o emulsion; leave-on.

Ciba Specialty Chemicals, 1998a (130)

In vitro

Female epidermal skin samples from cosmetic surgery mounted in diffusion cells; single application of dishwashing liquid (0.2% triclosan); rinseoff after 30 minutes.

Ciba Specialty Chemicals, 1998b (131)

In vitro

Female epidermal skin samples from cosmetic surgery mounted in diffusion cells; single application of a deodorant formulation (0.2% triclosan); leaveon.

Ciba Specialty Chemicals, 1998c (132)

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Subject In vitro

Method Female epidermal skin samples from cosmetic surgery, mounted in diffusion cells; single application of a soap solution (0.02% triclosan); rinse off after 10 minutes.

Major Findings GLP-compliant. Conducted comparable to OECD Guideline No. 428. After 24 h, there was 91% recovery of the applied dose of triclosan. Period of penetration was between 2 and 6 h (following a short lag time of <1 h). The rate of skin permeation reached ~0.001 g/cm2/h between 2 and 6 hours. There was marked decrease in penetration between 6 and 8 h, reaching a plateau by 24 hours. After 24 h, 2.30.25% of the applied dose of triclosan had penetrated into the receptor fluid (0.00960.0011 g/cm2). As measured at 24 h, triclosan in the surface material (with stratum corneum) was 9%. Succeeding tape-strips (strips 4-20) contained 4.3% of the applied dose (0.018 g/cm2) and 4.9% (0.0210.0042 g/cm2) of the dose was recovered from the remainder of the sample of skin. Percutaneous absorption was calculated to be 7.2% (0.0306 g/cm2) based on receptor fluid plus remainder of skin after removal of tape strips 1-20. Pre-dates GLP. Autoradiography of skin at 10 minutes after a single application of the various [3H]DP300 soap suspensions showed very low silver grain densities on the stratum corneum and in the epidermis, low or very low densities in the upper dermis, and very low or nil densities in the lower dermis. No grains were seen in the follicles or sebaceous glands. At 48 h after the single application, no silver grains were seen, except in the corneum after application of the fresh soap preparation. Scintillation counting showed no significant differences between the soap vehicles or in the single vs. repeated applications. Blood levels reached a plateau (7.0 to 19.1 ng/mL) immediately (i.e., within 2 hours following first bath) and did not accumulate throughout study. Blood levels increased as duration of study increased for 0.25% (28 to 68 ng/mL) but not 1.0% Irgasan DP 300 (87 to 94 ng/mL). Dose-dependent increase in blood levels was observed. Blood levels return to near baseline (16 ng/mL, baseline = <10 ng/mL) by the end of withdrawal period. Irgasan was present in the plasma in a conjugated form (not specified): no free Irgasan detected. Only a very small percentage of the administered dose was absorbed and was excreted completely; 2 to 7% was recovered in the urine, 0.5 to 2% in the faeces and the remainder in the dressing. The blood concentrations of GP 41 353 were 0.01 g/mL at all time points measured (1, 3, 5, 9 and 24 h after application). Following intravenous administration, the majority of CH 3565 was accounted for in the urine (65.4%13.5% of the injected dose) and faeces (20.6%10.4%). The half -life of CH 3565 was approximately 10 h. Following percutaneous application, an average of 8.9%3.2% of the CH 3565 dose was absorbed percutaneously.

Reference Ciba Specialty Chemicals, 1998d (133)

In vitro

Single versus 6 applications in 3 days of 0.25 mL of an 8% (w/v) conventional soap (freshly prepared or equilibrated over 1 week) or non-soap detergent suspension (freshly prepared) each containing 0.08% (w/v) [3H]DP300 was applied to the lower back (20 cm2) for 2 minutes. 10.0g soap containing 0.75% Irgasan DP 300 used for full body bathing for 75 days. 21-day hand washing study with diluted derma-san containing 0.25% Irgasan DP 300 or scrub containing 1.0% Irgasan DP 300. 7-day withdrawal period. 0.34 and 0.51 mg radiolabelled GP 41 535/kg body weight applied to area of skin measuring 8 x 8 cm and covered for 24 h with occlusive dressing (1 dose/subject). Group of 3 subjects received single intravenous dose of 14 C-irgasan CH3565; group of 6 subjects received single application (52 g) of 14 C-irgasan CH3565 suspended in Ivory soap to 13 cm2 area of forearm for 24 h.

Black et al., 1975 (27)

In Vivo Studies, Percutaneous Absorption Only 3 subjects (2 treatment, 1 control) 125 male and female Ciba-Geigy, 1972a (119) Ciba-Geigy, 1973b (120)

2 females

Stierlin, 1972b (121)

9 males

Maibach, 1969 (123)

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Subject 1 female and 3 male

Method 10 g of surgical scrub product containing 0.75% by weight of Irgasan DP 300 was applied to the surface area of both hands of each subject (2 washings/day for 3 minutes each for a total of 2 weeks).

Major Findings The amount of DP-300 in blood increased immediately after washing, reaching a plateau at the 15 ng/mL level in the blood after the fourth day of hand washing (8 washes). 24 h after discontinuation of the hand washing, 2 subjects had no detectable amounts of Irgasan DP 300 in the blood and by 48 h after discontinuation the third had no detectable amounts of Irgasan DP 300 (1 subject dropped out of the study due to illness). Therefore, small amounts of Irgasan DP 300 are absorbed through the skin and blood levels rapidly drop upon discontinuation of application. Urinary excretion of free triclosan ranged from 0.006 to 0.041% of the administered dose during the 48-h urine collection period. Urinary excretion of free plus glucuronide conjugated triclosan ranged from 2.52 to 6.47% of the administered dose during the 48-h urine collection period. Results indicate that triclosan is slightly absorbed percutaneously. Analysis of the remaining contents of the boats and the washings from the skin revealed 100% recovery of 2% CH3565 from all 3 soap formulations for the first set of subjects. For the second set of subjects, recovery was 96% for the Ivory and Colgate soap bases. The computed average recovery of each formulation was 98%, suggesting that an average of 2% of the CH3565 was absorbed.

Reference Ciba-Geigy, 1972b (124)

6 male

Single dermal application of 5 g of CGP. 433 cream (equivalent to a dose of 150 mg triclosan). Applied to back of each subject (surface area = 900 cm2). 3 different soap formulations (Ivory, Dial base and Colgate base soap), each with and without 2% solution of CH3565 were contained in glass boats. Each subject (2 sets of subjects) had a glass boat taped to each forearm (one with CH3565, the other without). The applications covered a surface area of 15 cm2 for 6 h (single application). Total of 10 mL of Approve skin cleanser (containing 0.75% triclosan) lathered on hands and forearms for a total of 8 minutes (single application).

Caudal et al., 1974 (125)

6 subjects

Schenkel and Furia, 1965 (126)

4 subjects

Absorption of triclosan was minimal and occurred over a period of 8 h, at which time peak plasma concentrations of free plus conjugated triclosan ranged from 15 to 31 ng/mL. Plasma concentrations of free triclosan ranged from <3 to 4 ng/mL. Mean total urinary excretion of free plus conjugated triclosan was 627101 g over a period of 48 hours. Urine concentrations of free triclosan ranged from <3 to 16 ng/mL. In the absence of an occlusive dressing the absorption of triclosan was below the limit of detection (plasma levels <15 ng/mL). The presence of the occlusive dressing enhanced absorption (plasma levels of free plus conjugated triclosan 112 to 192 ng/mL 4 to 8 h after application, declined slowly over 32 to 96 hours). Urinary excretion of free plus conjugated triclosan accounted for 6 to 14% of the dose without occlusive dressing, and 40 to 58% of the dose with occlusive dressing. These values decreased to <2% by 72 to 96 h after application. Absorption was not markedly increased by abrasion by application and removal of cellulose tape.

Thompson et al., 1975a (127)

4 subjects

Application of 1.0 mL of 0.5% GP 41353 Patient Skin Prep (equivalent to 5 mg of triclosan) onto a 100 cm2 area of normal and abraded abdominal skin, the former with and without an occlusive dressing (three 12-hour applications, separated by 4 weeks).

Thompson et al., 1975b (128)

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Subject Single: 6 males 8-day: 6 males (4 Caucasian, 2 African American); 31-day: 11 males (5 Caucasian, 6 African American, including the 2 from the 8day)

Method Single versus 8-day (22 applications) versus 31day (91 applications) scrub study. 10 mL of GP 41353 Surgical Hand Scrub containing 50 mg triclosan applied to hands and forearms per 5-minute application.

Major Findings Single: 10 to 24 hours following single application, maximum plasma concentration of free plus conjugated triclosan reached 62 to 143 ng/mL and returned to baseline over a period of 56 to 96 h. 2.6 to 6.6% of the applied dose was excreted in the urine as free plus conjugated triclosan (elimination half-life 14.4 to 38.4 h). 8-day: Plasma concentration of free plus conjugated triclosan still increasing on Day 8 (maximum concentrations ranging from 490 to 715 ng/mL and elimination half-lives ranging from 1.4 to 2.1 d for Caucasian subjects; maximum concentrations ranging from 1,640 to 1,780 ng/mL and elimination half-lives ranging from 11.3 to 15.6 d for African American subjects). Plasma concentrations of free triclosan ranged from <3 to 13 ng/mL for all subjects. 31-day: maximum plasma concentration of free plus conjugated triclosan (740 to 1,030 ng/mL) was reached at Days 12 to 15 for Caucasians. Plasma concentration of free triclosan was 16 and 6 ng/mL on Days 12 and 19, respectively. African American subjects were categorized as fast- or sloweliminators. Slow eliminators had maximum plasma concentrations of free plus conjugated triclosan ranging from 3,400 to 4,080 ng/mL and baseline values were still not attained by Day 78. The fasteliminators had max concentrations ranging from 554 to 690 ng/mL and baseline values were attained by Day 50. Plasma concentrations of free triclosan for slow- and fast- eliminators, respectively, were <3 and 9 ng/mL on Day 12 and <3 and 10 on Day 19.

Reference Thompson et al., 1976 (129) (single versus 8-day) Thompson, 1975 (122) (8-day versus 31-day)

6 healthy males, 15 leukaemia patients, additional 4 subjects

Healthy males used 1% DP300 soap bar for 11 months. Leukaemia patients bathed daily with 1% DP300 soap bar for 5 weeks. 4 subjects applied an aerosol anti-perspirant containing 0.1% DP300 daily for 4 weeks.

DP300 was not readily absorbed through the skin following repeated topical application of hygienic products. Maximum blood levels of total triclosan were 44, 500, and 18 ng/mL for healthy subjects using soap bar, leukemic subjects using soap bar, and subjects using anti-perspirant, respectively. Blood levels of free triclosan did not exceed 8 ng/mL for healthy subjects using soap bar, or subjects using anti-perspirant. Leukemic subjects using soap bar had blood levels of free triclosan as high as 500 ng/mL. Maximum urine levels of total triclosan were 1,106, >1,001, and 890 ng/mL for healthy subjects using soap bar, leukemic subjects using soap bar, and subjects using anti-perspirant, respectively. Steady-state plasma levels of free and total (free + conjugated) triclosan were measured. Only about 10% of total triclosan was present as free (unconjugated) triclosan. Total plasma triclosan levels were consistently higher in males than females. At steady-state (on Day 20), triclosan levels in plasma were: 95.2 ng/mL in males and 47.4 ng/mL in females; 73.1 ng/mL for both sexes combined. Steady-state plasma levels were estimated to have been reached by Day 7. Plasma levels rapidly decreased after cessation of hand washing. Elimination half-life:1.4 d. There was considerable inter-individual variability in plasma levels. Investigators concluded that use of the 1% hand wash formulation resulted in low levels of systemic exposure (lower than oral route).

Hong et al., 1976 (24)

7 healthy males and 6 healthy females completed study

Volunteers washed their hands with ~3-5 mL of the test material (commercial hand wash containing 1% triclosan) 6 times/day, ~every 2 h during the day, for a lathering time of 15 seconds/washing followed by thorough rinsing, for 20 consecutive days.

Ciba Specialty Chemicals, 2002 (134)

In Vivo Studies, Combined Oral and Percutaneous Absorption

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Subject 20 volunteers (10/group)

Method Group 1: placebo soap, talc, antiperspirant + triclosan-containing toothpaste (0.215%). Group 2: triclosancontaining soap, talc, antiperspirant (concentrations of triclosan not specified) + triclosan-containing toothpaste (0.215%). Exposure for up to 56 d. Group 1: placebo soap and underarm deodorant + triclosancontaining toothpaste (0.28%). Group 2: soap (0.75%, 2X/day), underarm deodorant (0.39% 1X/day) + triclosan-containing toothpaste (0.28%). Daily exposure for 3 months.

Major Findings Total triclosan plasma levels did not generally exceed 100 ng/mL, and levels quickly diminished below detection level upon cessation of treatment (i.e., by Day 64). The amount of unconjugated triclosan levels in all samples was <10 ng/mL. Triclosan was present in plasma mainly as its glucuronide-conjugated metabolite. Wide intersubject variation prevented meaningful comparisons between groups 1 and 2.

Reference BIBRA International, 1988 (137)

68 Caucasian, 54 AfricanAmericans, and 45 Asian subjects, 1865 years

Plasma levels of triclosan at Weeks 3, 6, and 13 showed slight, significantly increased blood levels of total triclosan and of glucuronide-conjugated triclosan in Group 2 (triclosan-containing toothpaste plus triclosan-containing hygiene products) compared to Group 1 (triclosan-containing toothpaste alone) [e.g., 23.79 vs. 18.99 ng/mL total triclosan at Week 13]. Note: sulfate-conjugated triclosan levels were difficult to interpret due to inefficiencies in the analysis. These data show percutaneous absorption of triclosan from personal hygiene products.

Beiswanger and Tuohy, 1990 (138)

Absorption Following In Vitro Percutaneous Administration H-labelled triclosan has been used to examine percutaneous absorption in a number of in vitro studies. Percutaneous penetration of 30.3% of the total applied dose of triclosan in an ethanol/water solution was measured 24 hours after application of the dose to human skin samples in a diffusion cell system [Moss et al., 2000 (21)]. Results from studies using skin samples in diffusion cells showed limited percutaneous absorption following application of triclosan to the skin surface in any of the formulations used. The amount of triclosan tested in most of these studies (0.2%) is within the range of anticipated concentrations in consumer products (from 0.15 to 0.3%). The low concentration in the soap solution study (0.02%) was intended to simulate actual-use conditions (i.e., a soap solution of 0.2% triclosan would be diluted with water when applied to the skin). Appropriate rinse steps were included in the rinse-off product studies. As recommended by SCCP guidelines (SCCP, 2006), calculations of dermal absorption included the amounts of triclosan recovered in the dermis and epidermis layers (i.e., the remaining sample of skin after removing the stratum corneum, represented by tape strips 1 to 20 in these studies) and the amount recovered in the receptor fluid. Table 37 presents a summary of the values from the in vitro studies conducted in human skin samples. The results from the autoradiography study [Black et al., 1975 (27)] indicated no significant differences in the low levels of percutaneous absorption between different soap vehicles or between single versus multiple applications [Black et al., 1975 (27)]. Overall, the dermal absorption of triclosan was shown in these in vitro studies to be lower in human skin than in rat (7.2 to 30.3% versus 41.2% in studies in rats, including the data from the study using ethanol/water formulation)]. Table 37: Summary of Dermal Absorption Values in Human Skin Samples from In Vitro Studies for Triclosan
Test Formulation % Triclosan in test material 0.2% 0.2% Dermal Absorption (SCCP)1 % w/o Emulsion Dishwashing 11.3 12.0 g/cm2 0.420 0.483 Ciba Specialty Chemicals, 1998a (130) Ciba Specialty Chemicals, Reference
3

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Test Formulation

% Triclosan in test material

Dermal Absorption (SCCP)1 % g/cm2

Reference

formulation Deodorant formulation Soap solution Ethanol/water formulation 0.2% 0.02% -7.7 7.2 30.3% 0.303 0.0306 --

1998b (131) Ciba Specialty Chemicals, 1998c (132) Ciba Specialty Chemicals, 1998d (133) Moss et al., 2000 (21)

1 Dermal absorption = amount measured in the dermis, epidermis (without stratum corneum) and the receptor fluid (SCCP, 2006). Thus, for data from the in vitro studies, dermal absorption = remainder of skin + receptor fluid

Absorption Following In Vivo Percutaneous Administration The in vivo percutaneous absorption of triclosan following single applications of triclosancontaining products (e.g., cream and soap) has been consistently reported as only a small proportion of the applied dose (i.e., generally 9%) [Stierlin, 1972b (121); Maibach, 1969 (123); Caudal et al., 1974 (125); Schenkel and Furia, 1965 (126); Thompson et al., 1975a (127)]. Additional studies were conducted to determine blood and urine levels of triclosan (as a measure of absorption) following single and multiple percutaneous applications of triclosan in different vehicles including soaps, creams, other skin cleansers and surgical scrubs. Together, these data support findings indicating that percutaneous triclosan absorption is minimal [Caudal et al., 1974 (125); Thompson et al., 1975a (127); Hong et al., 1976 (24)]. In general, blood levels of triclosan increased immediately after percutaneous application [Ciba-Geigy, 1972a (119); Ciba-Geigy, 1972b (124)], were enhanced by the presence of an occlusive dressing [Thompson et al., 1975b (128)], and were proportional to the dose applied [Ciba-Geigy, 1973b (120)]. Percutaneous absorption of triclosan from the use of triclosan-containing soap, underarm product and talc was also shown to be detectable in subjects already using triclosan-containing toothpaste [BIBRA International, 1988 (137); Beiswanger and Tuohy, 1990 (138)]. 3.3.11.2.3 Metabolism The metabolism of triclosan was investigated for several different routes of administration, including oral exposure to triclosan-containing products (e.g., toothpaste), oral ingestion of capsules and aqueous solutions, and percutaneous exposure (in vivo and in vitro). Table 38 identifies the studies and their designs based on the route of administration of triclosan. Table 38: List of Metabolism Studies of Triclosan
Route of Administration Oral (capsules) Oral (toothpaste) Study Design Single versus multiple dose (intra-subject comparisons) Treatment versus placebo (inter-subject comparisons) Treatment versus placebo (inter-subject comparisons); effect of mouth rinse Toothpaste versus toothpaste plus percutaneous (inter-subject comparisons) Reference Lucker et al., 1990 (109) Lin, 1989 (135) Colgate-Palmolive, 1988 (136) BIBRA International, 1988 (137); Beiswanger and Tuohy, 1990 (138) Lin, 2000

Oral (mouth rinse)

Treatment versus placebo (inter-subject

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Route of Administration comparisons) Percutaneous (in vivo)

Study Design (118) Treatment versus placebo (intra-subject comparisons) (soap bar use) Low versus high dose (hand washing study)

Reference Wagner and Leshar, 1977 (139) Ciba-Geigy, 1973b (120) Moss et al., 2000 (21)

Percutaneous (in vitro)

Human versus rat

Biotransformation of triclosan In general, the studies outlined in Table 38 indicate that, due to a pronounced first-pass effect, only trace amounts of the parent compound (i.e., unconjugated or free triclosan) are detected in the plasma. As a result of the first-pass effect, there is near total conversion of triclosan to glucuronide and sulphate conjugates [Lin, 1989 (135) and 2000 (118)]. Studies indicate that the relative amounts of glucuronide- and sulphate-conjugated triclosan present in the plasma vary depending on the plasma steady state concentrations of total triclosan. Up to study day 14, the main metabolite in plasma was the glucuronide conjugate (97% of total triclosan) in patients receiving 15 mg/day with only trace amounts of sulphate [Lucker et al., 1990 (109)]. Similarly, the glucuronide conjugate predominated in plasma samples from subjects brushing daily with triclosan-containing toothpaste (0.6%) [ColgatePalmolive, 1988 (136)]. However, as steady state concentrations were reached (Cmax = 330 ng/mL) during a 36-day exposure to the capsules, the absolute amount of glucuronide conjugates remained relatively constant, whereas sulphate conjugates increased to approximately 53% [Lucker et al., 1990 (109)]. Two studies comparing oral exposure to triclosan-containing toothpaste alone and in combination with percutaneously applied triclosan-containing products (e.g., soap, talc, anti-perspirant/underarm deodorant) revealed that, in each study and for both groups, circulating metabolites were composed primarily of glucuronide for the duration of the study (8 and 13 weeks) [BIBRA International, 1988 (137); Beiswanger and Tuohy, 1990 (138)]. Plasma concentrations of total triclosan generally did not exceed 100 ng/mL in the 8-week study and ranged from approximately 20 to 30 ng/mL in the 13-week study. In the 13-week study [Beiswanger and Tuohy, 1990 (138)], the group receiving oral plus percutaneous exposure had increased blood levels of total triclosan and glucuronide-conjugated triclosan (approximately 24 to 30 and 26 to 29 ng/mL, respectively) compared with the group receiving oral exposure only (approximately 19 to 22 and 19 to 21 ng/mL, respectively) throughout the study (Weeks 3, 6, and 13). In summary, the relative ratio of glucuronide to sulphate conjugates alters with repeat dosing (i.e., as plasma steady-state triclosan levels are reached) versus single dosing. Generally, lower plasma concentrations of total triclosan are associated with glucuronide conjugates as the predominant metabolite. With increasing plasma levels of total triclosan, there is an increase in circulating sulphate conjugates, which can reach levels greater than those attained by glucuronide conjugates, given high enough plasma levels of total triclosan. Metabolism Following Percutaneous Administration Glucuronidation and sulphation were demonstrated to occur in an in vitro diffusion skin model used to assess the ability of skin to metabolise triclosan [Moss et al., 2000 (21)]. Of the radioactivity in the receptor fluid (i.e., following absorption through the skin) at 24 hours, 3.5% of the applied dose was present as triclosan glucuronide and 0.2% was present as triclosan sulfate.

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A study of percutaneous absorption from 21 days of hand washing with triclosan-containing soap or scrub showed that triclosan was nearly completely converted to a conjugated form detectable in plasma (no free triclosan was detected) [Ciba-Geigy, 1973b (120)]. In vivo percutaneous absorption studies have measured plasma concentrations of free plus conjugated triclosan as well as free triclosan following single and multiple applications of triclosan-containing products (e.g., soap, anti-perspirant, skin cleanser, hand scrub) [Thompson et al., 1975a (127); Thompson, 1975c (122); Thompson et al., 1976 (129); Hong et al., 1976 (24)] (see Table 36). In these studies, although the specific conjugates were not differentiated (i.e., glucuronic acid and sulphuric acid conjugates), results showed that the majority of circulating triclosan was in the conjugated form. In a small study, 6 subjects used triclosan-containing soap for 45 days, with plasma steady-state concentrations ranging from 100 to 2,580 ng/mL. Plasma from subjects with lower steadystate levels contained primarily glucuronide conjugates, whereas plasma from subjects with higher steady-state levels contained primarily sulphate conjugates [Wagner and Leshar, 1977 (139)]. 3.3.11.2.4 Excretion The routes of excretion of triclosan were measured following single and multiple oral doses (triclosan capsules or mouthwash solution), single and multiple percutaneous applications (soap), and intravenous administration. Elimination half-life values for triclosan were outlined in the pharmacokinetic section (see Table 35) and are discussed in more detail below. Elimination half-life The elimination half-life values for orally-administered triclosan are comparable from study to study, irrespective of the formulations used, the duration of exposure, and the age of the subjects. For adults receiving single and multiple administrations of triclosan capsules, the elimination half-life values are 13.42 and 18.97 hours, respectively [Lucker et al., 1990 (109)]. In adults, single doses from aqueous solutions and dental slurries have elimination half-life values approximating 11 to 20 hours [Concordia Research Laboratories, 1997a (110); Sandborgh-Englund et al., 2006 (116)], and single use of triclosan-containing toothpaste has a value of 14.6 hours [Colgate-Palmolive, 1997b (115)]. In children, single doses of aqueous solution result in elimination half-lives up to 16.8 hours [ColgatePalmolive, 1997a (111); Concordia Research Laboratories, 1997b (112)]. One study reported an elimination half-life value of approximately 10 hours based on excretion data following intravenous administration of triclosan [Maibach, 1969 (123)]. This value suggests a slightly shorter elimination half-life for intravenously injected triclosan compared with values obtained from oral studies, which may be reflecting the fact that absorption is by-passed following intravenous administration. The elimination half-life value for dermally-applied triclosan used at 1% in a hand wash formulation was 1.4 days, based on combined data from men and women [Ciba Specialty Chemicals, 2002 (134)]. In vivo percutaneous absorption studies for triclosan conducted in the early 1970s had suggested that differences exist in the rate of elimination between Caucasians and African Americans [Thompson, 1975c (122); Thompson et al., 1976 (129)]. In an 8-day study conducted with triclosan-containing hand scrub, the elimination half-lives ranged from 33.6 to 50.4 hours for Caucasians and from 271.2 to 374.4 hours for African Americans [Thompson, 1975c (122)]. In a similar 31-day study, this difference in rate of elimination was observed between some but not all the African American and Caucasian subjects [Thompson et al., 1976 (129)]. Despite these findings, a subsequent study was designed specifically to evaluate any race differences (Caucasians vs. African Americans vs. Asians) in the metabolism of triclosan [Beiswanger and Tuohy, 1990 (138)]. This study was conducted with triclosan-containing toothpaste, soap and deodorant, and revealed no metabolic differences between these populations.

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Elimination half-life The elimination half-life values for orally-administered triclosan are comparable from study to study, irrespective of the formulations used, the duration of exposure, and the age of the subjects. For adults receiving single and multiple administrations of triclosan capsules, the elimination half-life values are 13.42 and 18.97 hours, respectively [Lucker et al., 1990 (109)]. In adults, single doses from aqueous solutions and dental slurries have elimination half-life values approximating 11 to 20 hours [Concordia Research Laboratories, 1997a (110); Sandborgh-Englund et al., 2006 (116)], and single use of triclosan-containing toothpaste has a value of 14.6 hours [Colgate-Palmolive, 1997b (115)]. In children, single doses of aqueous solution result in elimination half-lives up to 16.8 hours [ColgatePalmolive, 1997a (111); Concordia Research Laboratories, 1997b (112)]. One study reported an elimination half-life value of approximately 10 hours based on excretion data following intravenous administration of triclosan [Maibach, 1969 (123)]. This value suggests a slightly shorter elimination half-life for intravenously injected triclosan compared with values obtained from oral studies, which may be reflecting the fact that absorption is by-passed following intravenous administration. The elimination half-life value for dermally-applied triclosan used at 1% in a hand wash formulation was 1.4 days, based on combined data from men and women [Ciba Specialty Chemicals, 2002 (134)]. In vivo percutaneous absorption studies for triclosan conducted in the early 1970s had suggested that differences exist in the rate of elimination between Caucasians and African Americans [Thompson, 1975c (122); Thompson et al., 1976 (129)]. In an 8-day study conducted with triclosan-containing hand scrub, the elimination half-lives ranged from 33.6 to 50.4 hours for Caucasians and from 271.2 to 374.4 hours for African Americans [Thompson, 1975c (122)]. In a similar 31-day study, this difference in rate of elimination was observed between some but not all the African American and Caucasian subjects [Thompson et al., 1976 (129)]. Despite these findings, a subsequent study was designed specifically to evaluate any race differences (Caucasians vs. African Americans vs. Asians) in the metabolism of triclosan [Beiswanger and Tuohy, 1990 (138)]. This study was conducted with triclosan-containing toothpaste, soap and deodorant, and revealed no metabolic differences between these populations. Excretion Following Oral Administration Following both single and multiple oral doses of triclosan in a gelatine capsule, the predominant route of excretion is the urine (57 to 87% of the administered dose), with much smaller amounts appearing in the faeces (10 to 33% of the administered dose) [Lucker et al., 1990 (109); Stierlin, 1972b (121); Ciba-Geigy, 1976c (140)]. In a study using single doses of aqueous solutions containing triclosan, the major fraction was excreted within 24 hours of exposure, with between 24 and 83% (median 54%) of the oral dose excreted within the first 4 days after dosing [Sandborgh-Englund et al., 2006 (116)]. In the same study, the median urinary excretion half-life was 11 hours and relative renal clearance was 57% of the total dose. Following single oral administration of radioactive triclosan (in capsule form), nearly 100% of the radioactivity detected in the urine was reported to be the glucuronide conjugate [Stierlin, 1972b (121); Ciba-Geigy, 1976c (140)], whereas in the faeces, 30 to 40% was recovered as the free unchanged triclosan compound [Stierlin, 1972b (121)]. Excretion Following Percutaneous Administration The predominant route of excretion following percutaneous application of triclosan is the urine (2 to 14% of the applied dose) [Stierlin, 1972b (121); Caudal et al., 1974 (125); Thompson et al., 1975b (128)], with much smaller amounts appearing in the faeces (0.5 to 2% of the applied dose) [Stierlin, 1972b (121)]. Excretion data revealed that, following single and multiple applications (2 to 3 times/day for 45 days), all the triclosan appearing in

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the urine was present as the glucuronide conjugate [Wagner and Leshar, 1977 (139); Caudal et al., 1974 (125)], whereas the majority of the faecal triclosan was present as the free unchanged compound [Wagner and Leshar, 1977 (139)]. Excretion Following Intravenous Administration Excretion data obtained from an intravenous study were consistent with those obtained from the oral studies. Following injection of radiolabelled triclosan, the majority of the dose (approximately 65%) was excreted in the urine, while approximately 21% was excreted in the faeces [Maibach, 1969 (123)], suggesting biliary excretion into the gut. 3.3.11.2.5 Plasma Protein Binding An in vitro study was conducted with serum derived from a healthy donors blood to estimate the binding of 14C-labelled GP 41353 to serum proteins (specifically albumin) using equilibrium dialysis. At concentrations ranging from 0.36 to 7.9 g/mL, 99.8% of the drug was bound to serum proteins. There was no indication of saturation of binding sites, and the ratio of the unbound fraction to the bound fraction remained constant with increasing concentrations. At concentrations ranging from 0.095 to 9.1 g/mL, 98.9% of the drug was bound to albumin, with no indication of saturation of binding sites. It was therefore concluded that, in plasma, the majority of the drug binds to albumin. Equilibrium dialysis showed that dialysis time is 60 minutes and that GP 41353 is bound to protein more firmly than it adheres to the membrane [Wagner, 1973 (141)]. 3.3.11.2.6 Exposure of Infants by breast milk Distribution into Breast Milk Infant exposure to triclosan was estimated based on levels of triclosan found in 5 random samples of breast milk in a small study [Adolfsson-Erici et al., 2002 (102)]. In this study, the concentrations of triclosan in breast milk were reported to range from less than 20 to 300 g/kg lipid weight. Based on breast milk fat intake rates of 0.0268 kg breast milk fat/day and using the most conservative (highest) concentration for intake estimates, the exposure of infants to triclosan via breast milk was calculated to be approximately 8.04 g/day (0.00804 mg/day). Assuming a small infant of 2 kg weight, the exposure to triclosan from breast milk was calculated to be 4.02 g/kg bw/day. Another study determined triclosan levels in plasma and milk from 36 Swedish nursing mothers with and without known exposure from personal care products [Allmyr et al., 2006]. In this study triclosan was found in breast milk at concentrations ranging from < 0.018 to 0.95 ng/g (levels comparable on a fresh weight basis for milk to those determined by Adolfsson-Erici et al.). Allmyr et al. (Ref 103) calculated that the daily intake of triclosan would be <11570 ng/day for an infant weighing 4 kg with an estimated milk intake of 150 ml/kg/day. As breast milk levels are lower than those in plasma of nursing mothers (ratio M/P <1), the infant is exposed to a considerably smaller dose of triclosan via the breast milk compared to the dose in the mother. A recent risk assessment estimated the maximum daily consumption of triclosan by infants through breast milk, based on the most conservative values for breast milk concentration [Dayan, 2007 (104)]. The study based its calculations on the average triclosan concentration in the 5 breast milk samples with the highest triclosan levels, out of 62 samples, as well as a value for infant milk intake covering 97.5% of the population with the highest intake on a volume/kg body weight basis, i.e., 1-month-old infants. The maximum daily infant consumption of triclosan through breast milk was calculated to be 7.4 g/kg bw/day.

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3.3.11.2.7. Triclosan levels in urine and plasma Information on exposure to triclosan can be derived from biomonitoring and similar studies. Urinary triclosan concentrations were assessed in a representative sample of the U.S. general population from the 2003-2004 National Health and Nutrition Examination Survey (NHANES) by Calafat et al. (2008) [AR2]. They analyzed 2,517 urine samples by means of sensitive analytical methods and detected triclosan in about three-quarters of urine samples: The geometric mean and 95th percentile concentrations were 13.0 g/L (12.7 g/g creatinine) and 459.0 g/L (363.8 g/g creatinine), respectively. Concentrations differed by age and socioeconomic status but not by race/ethnicity and sex. Specifically, the concentrations of triclosan appeared to be highest during the third decade of life and among people with the highest household incomes. Dose estimates for triclosan based on the NHANES measurement data have not been reported by Calafat et al. since the conversion of measured spot urine concentrations to daily doses is not trivial because of several uncertainties related to variable dilution caused by wide variations in fluid intake and excretion and lack of information on route of exposure (i.e. oral, dermal, inhalation) and duration. Three methods proposed to estimate the dose from measured spot urine concentrations in the absence of total urine volume data have been recently used by the US EPA in computations for an aggregate risk assessment on triclosan (US EPA 2008; AR9). SCCP considers these dose estimates for consumers in the US as useful additional information in their evaluation on the safety of triclosan. There are three smaller studies on plasma levels in consumers, two in Swedish and one in Australian subjects: Sandborgh-Englund et al. [ref. 116] found triclosan in plasma at 0.1 8.1 ng/ml of 10 subjects, of which 5 were exposed and 5 not exposed to triclosan via personal care products. In that study there was no apparent difference between the two groups. Allmyr et al. 2006 (103) detected a broader range of plasma concentrations (0.01038 ng/ml) in 36 Swedish nursing mothers, with higher concentrations in both plasma and milk of individuals with known exposure to triclosan via personal care products. Recently, Allmyr et al. 2008 [AR1] reported on their analysis of human blood samples collected in Australia between 2002 and 2005, and pooled according to age, gender and region. The dataset suggests that the exposure to triclosan among different groups of the Australian population is relatively homogeneous [Allmyr et al. 2008]. In comparison to their previous measurements in human plasma from Sweden, triclosan concentrations were a factor of 2 higher in Australian serum than in Swedish plasma. 3.3.11.2.8 Summary of Human Pharmacokinetic Data

Triclosan is very well absorbed following oral ingestion (up to 98% of the dose). However, under normal conditions of toothpaste use (i.e., expectoration and rinsing) or following percutaneous application of several different personal care products, there is only limited absorption (approximately 5 to 10% of the dose via either of these routes of administration). Based on plasma levels and percentage of dose absorbed, it is clear that low exposures to triclosan occur following either toothpaste use or soap/hand wash use and that, with repeated exposures using either route, low steady-state levels of triclosan are reached after approximately 7 to 10 days. Regardless of the formulation administered, only trace amounts of the parent compound are detected in the plasma following exposure to triclosan-containing products. Due to a pronounced first-pass effect, there is near total conversion of absorbed triclosan to glucuronic and sulphuric acid conjugates. The relative proportions of these metabolites vary depending on the plasma steady state concentration of total triclosan, with higher concentrations resulting in a shift from predominantly glucuronide- to predominantly sulphate-conjugates. Following ingestion, percutaneous application, or intravenous administration of triclosan, the predominant route of excretion is the urine, in which

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triclosan is present as the glucuronide conjugate. In contrast, triclosan excreted in the faeces is present as the free unchanged compound. Pharmacokinetic data, in particular, AUC values after single or repeated oral exposures (e.g., after toothpaste use), as well as plasma levels after dermal (soap application) exposures, indicate a lack of evidence of bioaccumulation of triclosan. 3.3.11.3 Human Irritation and Sensitisation

Several irritation, sensitisation, and photosensitisation studies have been conducted with triclosan in humans. Data regarding the irritation and/or sensitisation potential of triclosan was also available from studies that reported results of routine patch tests with a series of compounds in patients presenting with contact dermatitis. 3.3.11.3.1 Irritation/Corrosivity The irritation potential of triclosan was determined in tests on human skin and human mucous membranes. The main findings of these studies are presented in Table 39. Table 39: Findings from Irritation Studies with Triclosan in Humans
No. of subjects N=106 females. Application Details Repeated Insult Patch Test. Soap formulation applied to the back for 48 hours, twice a week for 5 weeks. Concentration of triclosan not reported (test material described as soap bar and P-300 Anti-Bac. Deo. Soap (NDA #16-486)) Hand washing Study. Volunteers with no known allergic reactions to hand washing agents. A commercially available 2% triclosan detergent was used (commercially available). Subjects washed with 5 mL for 1 min, rinsed, and applied a further 5 mL for 2 min of washing before rinsing and drying thoroughly. This procedure was performed 5 times at 20 min intervals. Major Findings There was no evidence of primary irritation following any of the 48-h patch tests (a total of 1155 patch tests). Reference ColgatePalmolive, 1972 (147)

N=20 volunteers

Five of 20 subjects (25%) complained of irritation consisting of itching and soreness. In all cases, the irritation reaction was delayed by 6 to 8 h after the use of the triclosan-containing detergent. In 4/5 cases, the reaction lasted 24 to 36 h. In the worst case, the complaint was of a burning sensation and slight swelling that persisted for 2 days. It is important to note that this study did not use a control group treated with a detergent formulation without triclosan and it was inconclusive whether the irritation reaction was due to triclosan or to some other component of the detergent formulation. No skin reactions were observed with triclosan alone or distilled water. SLS resulted in erythema of the skin of all subjects. SLS and triclosan together did not result in erythema. Pre-treatment with triclosan did not reduce erythema resulting from exposure to SLS. One triclosan-containing toothpaste (triclosan/copolymer/SLS/ propylene glycol) produced mild to severe irritant reactions in 16 of 19 subjects, while the other (triclosan/zinc citrate/SLS/ polyethylene glycol) did not elicit any reaction.

Bendig, 1990 (162)

N=10 subjects

Finn Chamber Patch test. Tests were conducted on the forearms of subjects. Single application of patches saturated with either 1.0% SLS, 0.3% triclosan, 1.0% SLS and 0.3% triclosan, or distilled water. Occluded patch test, doubleblind study. 1 cm of each of 4 toothpastes was applied to the forearm of each subject for 24 hours. Concentration of ingredients in toothpastes was not reported.

Barkvoll and Rolla, 1994 (143)

N=19 subjects

Skaare et al., 1997a (144)

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No. of subjects N=14 or 15 subjects

Application Details Five mouth rinses were tested in groups of subjects: A, 1.5% sodium lauryl SLS B, 1.5% SLS/0.5% zinc citrate; C, 1.5% SLS/polyethylene glycol (1:8); D, 1.5% SLS/0.15% triclosan/0.3% zinc citrate; E, 1.5% SLS/0.15% triclosan Irritation tests in oral mucosa (daily exposures). Experiment I (5 min/d for 5 d): Solidox Fluor (F) (1.5% SLS) or Solidox G (G) (1.5% SLS/0.3% triclosan/0.75% zinc citrate). Experiment II (2 min twice daily for 4 d): A, 1.5% SLS; B, 3% SLS/0.3% triclosan/ 0.75% zinc citrate; C, detergent-free toothpaste (negative control).

Major Findings Mouth rinses containing triclosan (D, E) resulted in decreased frequency and severity of erythemic reactions, compared to mouth rinses without triclosan (A, B, C). Mouth rinse D elicited only 2 erythema reactions and E elicited 5 reactions. These 5 subjects also had mild desquamation. None of the subjects using Solidox G (triclosan-containing toothpaste) showed oral mucosal desquamation, versus 7/10 subjects with positive results using toothpaste without triclosan. Comparison of results overall showed that triclosan eliminated the effects of 1.5% SLS (Experiment I) and reduced the severity of the effects of 3% SLS (Experiment II).

Reference Skaare et al., 1997b (145)

Experiment I: N=10 subjects Experiment II: N=28 subjects

Skaare et al., 1996 (146)

Abbreviations: SLS = sodium lauryl sulfate

The ability of triclosan to cause irritation to human skin or mucous membranes was evaluated in human volunteers. Triclosan (0.3%) was shown not to induce skin irritation in single patch tests in 10 subjects [Barkvoll and Rolla, 1994 (143)]. There was also no evidence of primary skin irritation in repeated patch tests with a bar soap formulation containing triclosan (concentration of triclosan not specified) in 106 female subjects [Colgate-Palmolive, 1972 (147)]. In this study, a total of 1,155 patch tests were conducted, including a challenge application 14 days after the 10th patch test in each subject. Although irritation effects were observed in a hand washing study using a commercial detergent containing a high concentration (2%) of triclosan, the irritation effects could not clearly be attributed to triclosan [Bendig, 1990 (162)]. Thus, taken altogether, the data indicated that triclosan showed low skin irritation potential in clinical irritation tests and, possibly, skin irritation in a commercial detergent formulation containing a high concentration of triclosan (2%). The effect of triclosan on skin and oral mucosa irritation produced by SLS was investigated in four studies. In the same patch test study in which triclosan was shown not to produce skin irritation, it was also shown to eliminate SLS-induced irritation [Barkvoll and Rolla, 1994 (143)]. In another study, the effects of triclosan in toothpaste formulations on SLSinduced irritation was tested in skin, with inconclusive, but suggestive, results with respect to a protective effect of triclosan [Skaare et al., 1997a (144)]. Studies of oral mucosal response to various mouth rinse or toothpaste formulations containing SLS (1.5 or 3%) with/without triclosan (0.15% or 0.3%) showed that formulations containing triclosan reduced or eliminated the severity and frequency of SLS-induced oral mucosal erythema and desquamation compared to formulations without triclosan [Skaare et al., 1997b (145); Skaare et al., 1996 (146)]. Taken altogether, the data from these studies indicate that triclosan has a protective effect against SLS-induced skin or oral mucosa irritation. While the effects of triclosan alone have not been evaluated in stand-alone tests in oral mucosa because triclosan was typically tested in combination with SLS, the lack of irritant effects of triclosan-containing test formulations in oral mucosa studies, together with the protective effects of triclosan against SLS-induced irritation, indicate that triclosan is not a mucosal irritant. In summary, the skin and oral mucosa irritation studies evaluating the effects of triclosan alone, or in combination with SLS, indicate that triclosan is not a skin or oral mucosal irritant.

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3.3.11.3.2.

Sensitisation

Table 40: Findings from human induction studies


No. of Subjects 106 females Application Details Repeated Insult Patch Test. Induction: Soap formulation applied to the back for 48 hours, twice a week for 5 weeks (occluded). Challenge: application 14 d after 10th application. Concentration of triclosan not reported in P-300 AntiBac. Deo. Soap. Modified Draize test (patch test). Induction: triclosan in petrolatum was applied for 48-72 h per application, 10 times over 3.5 weeks. Challenge: After 14 d, using a patch for 72 h. Modified Maximisation Test. Preparation: 5% sodium lauryl sulphate under occlusion for 24 h. Induction: Application of 20% triclosan in petrolatum at the same site on Days 1, 3, 5, 7, and 9. Challenge was 14 d later, using 1%, 2%, or 5% triclosan. Repeat Insult Patch Test (100 volunteers) and the Prophetic Patch Test (50 volunteers). Applications (0.5 mL) of triclosan in solution or as a slurry were for 24 h (no further details were provided). Major Findings There was no evidence of sensitisation potential in any of the 106 volunteers. Reference Colgate-Palmolive, 1972 (147)

144 males

There was no evidence of sensitisation potential in any of the tests conducted with induction/challenge combinations of 5%/5% (none of 61 subjects tested), 20%/1% (none of 83 subjects tested).

Marzulli and Maibach, 1973 (148)

20 males / females

Induction phase: 17/20 subjects showed signs of skin irritation (erythema, slight oedema, moderately painful). However, 0/20 test volunteers and all control volunteers showed no positive patch test reactions up to 7 days post-challenge.

Lachapelle and Tennstedt, 1979 (18)

150, sex of subjects sex not specified

There was no evidence of skin sensitisation in any of the 150 volunteers (note that the concentration of triclosan tested was not provided).

DeSalva et al., 1989 (1)

In total, triclosan was tested in 420 healthy subjects using variations and modifications to the Patch, Draize, and Maximisation Test methods at induction concentrations of up to 20% and challenge concentrations of up to 5%. There were no positive reactions in any of the test subjects, including after repeated patch testing [e.g., Colgate-Palmolive, 1972 (14); DeSalva et al., 1989 (1)]. No positive challenge results were observed, leading investigators to conclude that triclosan has a very low sensitisation potential. Taken altogether, the results from these studies indicate that triclosan has very low sensitisation potential in healthy subjects. Comment SCCP considers human induction studies as unethical Triclosan has also been tested in patients with contact dermatitis or suspected cosmetic allergy. The results of routine patch testing with triclosan as one of a series of preservative or antimicrobial ingredients tested in these patients are shown in Table 41. The data show that triclosan has a low potential to cause positive skin reactions in this sensitive population. Table 41: Findings from Patch Testing with Triclosan in Patients with, or Suspected of Having, Contact Dermatitis

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No. of Patients Tested 5,202 (cosmetic intolerance)

Major Findings Patch test. Seven (7) of 5,202 patients (0.1%) had a positive reaction to triclosan. In particular, 1 of 156 patients with a known pure cosmetic allergy showed a positive reaction (0.6%). Note: concentration of triclosan was not reported. Patch test. No positive reactions to triclosan (2% in petrolatum) were observed (0%). Patch test. Twenty-nine (29) of 11,406 patients (0.3%) had a positive reaction to triclosan (2% in petrolatum). Note that 59 of 11,406 (0.5%) patients had a questionable/irritative reaction. Investigators considered the sensitisation rate to be low or very low. Patch test. Two (2) of 179 patients (1.1%) had a positive reaction to triclosan (2% in petrolatum) All 3 patients in these case studies tested positive in patch tests (2% triclosan in petrolatum) at 48, 72, and 96 h.

Reference Broeckx et al., 1987 (149)

627 (suspected contact dermatitis) 11,406 (consecutive patients)

De Groot et al., 1986 (150) Schnuch et al., 1998 (151)

179 (suspected cosmetic allergy) 3 (patients known to have used cream containing 3% triclosan) 2,295 (suspected allergic contact dermatitis) 2,002 (consecutive patients)

De Groot et al., 1985 (152) Veronesi et al., 1986 (153)

Patch test. Triclosan was described as having a low sensitisation rate, based on the observed rate of 0.8% positive reactions to triclosan (2% in petrolatum). Patch test. In total, 0 of 432, 0 of 470, and 2 of 1,100 patients tested positive to 0.5% triclosan in Vaseline, 1.0% triclosan in ethanol, and 2.0% triclosan in Vaseline, respectively (i.e., 0%, 0%, and 0.18% positive reactions, respectively). Patch test. Three (3) of 103 patients (2.9%) tested positive. Of the 3 positive reactions, 2 patients were known to have used cream containing 3% triclosan (the 3rd patients history of use of triclosan was unknown). Note: triclosan concentration tested was not reported. Patch test (chamber method). One (1) of 1,796 patients (0.06%) tested positive to 1% triclosan.

Perrenoud et al., 1994 (154) Wahlberg, 1976 (155)

103 (suspected contact dermatitis)

Steinkjer and Braathen, 1988 (156)

1,796 (eczema patients with suspected contact allergy) 1,234 (consecutive patients with eczema) 713 (suspected cosmetic dermatitis) 745 (suspected sunrelated skin disease)

Hannuksela et al., 1976 (156) Mitchell et al., 1982 (157) Adams and Maibach, 1985 (158) Wennersten et al., 1984 (160)

Patch test. There were reported to be 1 to 2% positive reactions in 1,234 patients tested with 2% triclosan in petrolatum. Patch test. One (1) of 713 patients (0.14%) with suspected cosmetic dermatitis tested positive to triclosan. Note: triclosan concentration tested was not reported. Photopatch test trial. One (1) of 745 patients (0.13%) tested positive for a contact allergic reaction to 2% triclosan in petrolatum; 2 of 745 (0.27%) tested positive for a photoallergic reaction.

Case reports of contact dermatitis due to triclosan use have been relatively rare (Campbell and Zirwas 2006, AR3), although a few cases have been reported following the use of a cream formulation containing a high concentration of triclosan (3%) together with 0.02% flumethasone pivalate. Three such cases were reported by Veronesi et al. [1986 (153)], and 2 by Steinkjer and Braathen [1988 (159)]. The reason for a third case of reported allergic contact reaction to triclosan was not discovered [Steinkjer and Braathen, 1988 (159)]. All 6 of these patients were found to have positive results in patch tests using 2% triclosan in petrolatum (see Table 41). Investigators concluded that low concentrations of triclosan in cosmetic products do not cause contact dermatitis; however, sensitisation may occur following the use of products containing higher concentrations [Veronesi et al., 1986 (153)].

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In general, the clinical test results have shown that triclosan has a very low sensitisation potential. This is apparent in the interpretation of results from extensive testing in patients with known or suspected allergic contact dermatitis. In total, over 14,000 consecutive patients have been tested for reaction to triclosan (typically tested at a concentration of 2% in petrolatum), with the range of positive results being 0.1 to 0.3% of the tested population. Additionally, triclosan testing in patients with known or suspected cosmetic allergy or intolerance has shown positive reaction rates ranging from 0.06 to 0.8% of a total of 11,887 tests conducted in this population. 3.3.11.3.3 Photo-Induced Toxicity The potential of triclosan to induce photosensitisation reactions was determined in a number of studies. The major findings of these studies are presented in Table 42. Table 42: Findings from Phototoxicity and Photosensitisation Studies with Triclosan in Human Subjects
Application Details N=5 males. 100 L (triclosan, 0.1% in methanol, 0.1 or 1.0% in petrolatum) applied to a 30 cm2 area on the back. Exposed to light sources 1 hour after application; readings at 24 and 48 h. N=104 females. Subjects had undergone repeated insult patch testing (soap formulation with concentration of triclosan not reported; 10 applications and a challenge after 14 d all negative). Photochallenge: 24 h after challenge and again 7 days after 1st photochallenge. Major Findings There was no evidence of phototoxicity. Reference Urbach, 1973 (101)

There was no evidence of photosensitising potential following the photochallenge application. After the second UV light photochallenge, one subject had a minimal reaction, considered to be the result of scratching, which was completely negative by the 48-h reading. There was no evidence of phototoxicity or photoallergenicity.

ColgatePalmolive, 1972 (147)

Phototoxicity (n=10): triclosan (2.5% in petrolatum) applied to skin for 1 hour, followed by irradiation. Readings at 4-6 and 24 h. Photoallergy (n=25): triclosan (10% in petrolatum) applied to the same site for five 48-hour intervals under occlusion. Site was irradiated after each application. A new site was photopatch tested 2 weeks after the last 48-h test. Modified Draize test (patch test). Induction: triclosan in petrolatum was applied for 48-72 h per application, 10 times over 3.5 weeks. Elicitation: After 14 d, using a patch for 72 h. Three (3) minimal erythema doses (MED) of Kromayer light were used during the induction phase, and 10 MED filtered through window glass during the elicitation phase. Photopatch test trial. 745 (patients with suspected sun-related skin disease (photodermatitis)) were tested for reaction to triclosan (2% in petrolatum) as part of a standard series of tests. Patch and photopatch test. Triclosan (2% in petrolatum) was tested; details not provided. 103 patients with suspected contact dermatitis.

Kligman, 1969 (161)

There was no evidence of photosensitisation in induction/challenge combinations using 1%/1%, 5%/1%, 20%/1%, and 20%/5% concentrations of triclosan (i.e., 0/51, 0/60, 0/52, and 0/24 positive responses in the tests, respectively, for a total of 0/187 subjects). 2 of 745 (0.27%) tested positive for a photoallergic reaction.

Marzulli and Maibach, 1973 (148)

Wennersten et al., 1984 (160) Steinkjer and Braathen, 1988 (159)

No positive photoallergic reactions.

Six studies investigated the photosensitising potential of triclosan. In one of the larger studies using a soap formulation containing triclosan (concentration not reported), only one positive reaction was observed following a second photochallenge with the soap formulation; however, this reaction was considered to be the result of scratching [ColgatePalmolive, 1972 (147)]. There was no evidence of photosensitising potential of triclosan in two of the other studies that tested over 100 subjects per study, and only two of 745

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patients tested positive for a photoallergic reaction in the largest study (0.27% of patients) [Wennersten et al., 1984 (160)]. There was also no evidence of phototoxicity or photoallergenic potential in two smaller studies (n#25) with triclosan at concentrations of up to 10% in petrolatum or 0.1% in methanol. In summary, data from the photosensitisation and patch testing studies performed with triclosan indicate that it is unlikely to produce phototoxicity or photosensitisation in human skin at levels used in personal care products. Triclosan was tested at concentrations of up to 10% in petrolatum in the photosensitisation studies.

3.3.12.

Special investigations

Special investigations have been conducted to study potential neurotoxic and nephrotoxic effects of triclosan in rats. In the 14-day neurotoxicity study, clinical signs, organ weights, and brain and nerve histopathology were examined. In the nephrotoxicity study, kidney tissue function from triclosan-treated rats was assessed in vitro. In addition to these studies, the effects of triclosan in rodent liver have been evaluated in rats, mice, and hamsters. Studies have been conducted to determine the effect of triclosan on liver morphology (e.g., size, hepatocyte necrosis, hepatocyte proliferation, changes in hepatocellular organelles) and on biochemical parameters (e.g., protein content, cytochrome P450 content and activity, and fatty acid oxidation activity). 3.3.12.1 Effects of Triclosan in the Brain

A 2-week neurotoxicity study was conducted with triclosan in the rat [Ciba-Geigy, 1973a (163)], the main findings of which are provided in Table 43. No histopathological changes were observed in the brain or sciatic nerve of treated or control animals. There were no differences in brain weights between treated and control animals. Clinical signs included decreased movement and muscular tone, polydipsia, and polyuria at dose levels of 300 mg/kg body weight/day and higher. The results of this study indicate a NOEL of 100 mg/kg body weight/day for triclosan in the rat. There was no evidence of neuropathology at any dose level, as examined in the brain and sciatic nerve tissues. The investigators concluded that triclosan produces no specific neurotoxic effects in the rat; however, the reasons for observations of clinical signs consistent with possible neurotoxicity (e.g., hypoactivity, decreased muscular tone) are unclear. Table 43: Findings from a Two-Week Oral Neurotoxicity Study with Triclosan in the Rat
Species (Strain) Rat, albino SIV 50 Dosing Regimen (mg/kg bw/day) 0, 100, 300, 1,000, or 2,000 mg/kg bw/day via oral administration (specific route not reported) for 14 days Major Findings Reference, GLP and OECD Status Ciba-Geigy, 1973a (163) Predates GLP and OECD

17 deaths in high-dose group (5 of these were sacrificed due to moribund condition). Decreased body weights in highdose group. Dose-dependent inhibition of movement, decreased muscular tone, polydipsia and polyuria at dose levels of 300 mg/kg bw/day and higher. Clinical signs were most severe in the high-dose group and also included spastic respiration for several animals. No difference in brain weights between treated and controls (no other organ weights were measured). No histopathological changes in the brain or sciatic nerve of treated or control animals were observed (no other tissues evaluated). NOEL: 100 mg/kg bw/day

3.3.12.2

Effects of Triclosan in Kidney

The nephrotoxic effects of triclosan have been investigated in a non-GLP study (published report) [Chow et al., 1977 (164)], described in Table 44 by means of in vitro and in vivo

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methods. Data from renal cortical slice incubation assays that measured p-aminohippurate (PAH) and N-methylnicotinamide accumulation as a measure of nephrotoxicity indicate that triclosan may have potential to cause kidney damage, although blood urea nitrogen (BUN) measurements in the same animals did not show indications of overt nephrotoxicity. The reason for the discrepancy between the in vitro results (where triclosan was added to the incubation medium) and in vivo results (where triclosan was administered to rats) is unclear, but may be related to the amounts of triclosan that reached the kidney, potential formation of a metabolite that selectively alters PAH accumulation, or the relative sensitivities of the assays. Table 44: Findings from a Nephrotoxicity Study for Triclosan
Species (Strain) Rat (Wistar) Dosing Regimen 625, 1,250, or 1,875 mg/kg bw 24 h prior to kidney cortical slice assay Duration of Treatment Single dose Major Findings Reference, GLP and OECD Status Chow et al., 1977 (164) Predates GLP and OECD

In vitro assay: There was decreased accumulation of both PAH and NMN. In vivo study: No change in blood urea nitrogen concentration at 24 h. Time-course study of renal cortical function in high-dose rats showed initial decreases in PAH accumulation that recovered to near-control levels by 72 h. N-Methylnicotinamide (NMN) accumulation was not significantly different from control up to 72 h. The significant decreases in PAH accumulation were dose-dependent.

In addition to these early toxicity data, a later (1994) GLP study conducted in hamsters showed that doses of 350 or 900 mg/kg body weight/day in the diet induced renal tubular epithelium proliferation that was evident at 7 and 13 weeks in the 13-week study [See Table 45, Persohn, 1994 (167)]. The authors concluded the increased labelling index (LI) in kidney tubular epithelium was a compensatory response to cell damage in the kidney. However, no histopathology results were available for confirmation of the existence of cell damage. In summary, findings in rat and hamster kidney studies of decreased kidney function and of increased cell proliferation that may be secondary to cellular damage suggest that triclosan may induce kidney damage at relatively high-dose levels. 3.3.12.3 Effects of Triclosan in Rodent Liver

The effects of triclosan on liver morphology, hepatocyte proliferation, and biochemical parameters, including the activities of cytochrome P450 enzymes, have been investigated in a number of studies in mice, rats, and hamsters. The major findings of these studies are presented in Tables 45 and 46. 3.12.3.1 Cell Proliferation in Rodent Liver The effect of triclosan on hepatocyte replicative DNA synthesis has been examined in studies conducted in the CD-1 mouse, Sprague-Dawley rat, and Syrian hamster at doses ranging from 25 to 900 mg/kg body weight/day [Eldridge, 1993 (165); Persohn and Molitor, 1993 (166); Persohn, 1994 (167)]. In both the mouse and hamster cell replication studies, the effect of triclosan on replicative DNA synthesis was monitored by using the proliferating cell nuclear antigen (PCNA) technique, the results of which are summarized in Table 45.

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Table 45: Findings from Cell Proliferation Studies for Triclosan


Species (Strain) Mouse (CD-1) Dosing Regimen Oral (diet) doses of 0, 25, 75, 200, 350, or 900 mg/kg bw/d Duration of Treatment 90 days (45 day interim data also available) Major Findings Reference, GLP and OECD Status Eldridge, 1993 (165) GLP and OECD not specified, but original animal study was GLPcompliant and consistent with OECD

In general, labelling index (PCNA-stained Sphase cells) increased with dose and time, and was slightly greater in males vs. females. Cell proliferation was significantly increased at 90 days by 4.8-, 3.5-, 11-, and 15-fold in M at doses of 75, 200, 350, and 900 mg/kg/d, respectively, and by 1.5-, 3.3-, 6.1-, and 7.1fold in F. Control group labelling indices were 0.0350.016 and 0.0420.036 in males and females, respectively. Cell proliferation was not increased at 25 mg/kg bw/d. Liver morphology changes showed dose-related hepatocyte hypertrophy starting at 25 mg/kg, together with necrosis of individual hepatocytes. Hypertrophy was reported as the most consistent and prominent change at 45 d, along with large areas of necrosis in the 350 and 900 mg/kg groups. Necrosis was accompanied by proliferating cells (bile duct epithelial cells, fibroblasts, Kupffer cells). Findings at 90 days were comparable to those at 45 d, except that necrosis was occasionally more severe, with panlobular necrosis in the most severe cases. Mean scores for liver necrosis at 90 days were 0, 0, 0.2, 0.6, 1.2, and 1.8 for males in the 0, 25, 75, 200, 350, and 900 mg/kg groups and 0, 0, 0, 1.2, 0.6, and 1.6 for females, respectively (scale of 04). Lipofuscin staining, lipid vacuolization, biliary hyperplasia were minimal. No deaths occurred, and no clinical signs of toxicity were observed. Feed consumption was initially decreased in high-dose animals, but was subsequently increased vs. controls. Body weight gains were initially decreased, but subsequently normal in the high-dose group. Absolute and relative liver weights were increased at 6,000 ppm compared to controls. Decreased numbers of hepatocyte nuclei per microscopic field together with increased liver weights indicated hepatic hypertrophy in highdose animals. Triclosan did not increase cell proliferation at doses of up to 6,000 ppm in the diet, for treatment durations of up to 42 days. Slight but significant decreases in cell proliferation at the high dose were observed after at least 7 days of treatment. There were no effects in the lower dose groups. No histopathology results were available. No increases in hepatic labelling indices (LI) vs. control were observed at the high dose of 900 mg/kg bw/d at 13 weeks. Kidney tubular epithelial cell nuclear mean LI were examined at 200, 350, and 900 mg/kg bw/d doses at 13 weeks. Kidney LI was significantly increased in males at 350 and 900, but not 200 mg/kg bw/d (3.7 and 6X greater than control, respectively). Kidney LI was significantly increased in high dose females at 13 weeks (8.8X greater than control). No histopathology results were available.

Rat (SpragueDawley)

Oral (diet) doses of 0, 300, 1,500, or 6,000 ppm (actual doses of 0, ~25, ~125, and ~500 mg/kg bw/d)

2, 4, 7, 14, or 42 days (1 group received 6,000 ppm for 14 days followed by 28 days of recovery)

Persohn and Molitor, 1993 (166) GLP: not specified OECD: comparable

Hamster (Syrian)

Oral (diet) doses of 0, 75, 200, 350, 750, or 900 mg/kg bw/d

13 weeks (7-week data also available)

Persohn, 1994 (167) GLP-compliant. OECD not specified, but original animal study was consistent with OECD

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The cell proliferation data demonstrate that triclosan treatment produces a dose-related increase in cell replication in male and female mice. Morphological examination of liver sections from this study revealed various histological changes including hepatocyte hypertrophy and necrosis in both sexes of the higher dose groups. In contrast, DNA synthesis data in male rats, and in male and female hamsters showed that triclosan did not increase hepatic replicative DNA synthesis in either sex after 7 or 13 weeks of treatment [Persohn and Molitor, 1993 (166); Persohn, 1994 (167)] (Table 45). Indeed, a significant decrease in hepatocyte proliferation relative to control was observed in male rats treated with ~500 mg/kg body weight/day for at least 7 days, and in male hamsters treated with 900 mg/kg body weight/day for 7 weeks. Altogether, the data show that cell proliferation following administration of triclosan is observed in mice, but not in rats or hamsters. 3.3.12.3.2 Liver Morphology and Biochemical Studies for Triclosan Several investigations into changes in liver morphology and selected biochemical parameters induced by triclosan have been conducted. Of the six studies, the findings of which are presented in Table 46, three [Molitor et al., 1992 (168); Molitor and Persohn, 1993 (169); and Thomas, 1994 (170)] can be considered to be pivotal in the scope of the endpoints examined within the investigation, even though 2 of the 3 did not contain statements of GLP compliance. Table 46: Findings from Liver Morphology and Biochemical Studies for Triclosan
Species (Strain) Mouse (CD-1) Dosing Regimen Oral (diet) doses of 0, 18, 54, 258, or 951 mg/kg bw/d (males) or 0, 20, 271, or 1,105 mg/kg bw/d (females) Duration of Treatment 14 days + recovery (28 days) Major Findings Reference, GLP and OECD Status Molitor et al., 1992 (168) GLP: not specified OECD: not applicable

Increases in protein and cytochrome P450 (P450) content, and enzyme activities were dose-dependent and reversible. Highlights of the results include (high-dose data reported as % of control): increased lauric acid hydroxylation (up to 833%), ethoxyresorufin Odeethylase activity (up to 502%), and testosterone hydroxylation (up to 619%); peroxisomal fatty acid beta-oxidation (~340% in M and F) and pentoxyresorufin O-depentylase (up to 2,390 and 1,580% in males and females, respectively). Immunoblot analyses showed increases in CYP3A1/2 proteins (842 and 5,851% in M and F, respectively) and in CYP4A proteins (~800% in M and F). Electron microscopy results show dose-dependent and reversible effects of an increase in smooth endoplasmic reticulum (ER) membranes, reduction and disorganization of rough ER membranes, and increase in lipid vacuolization in hepatocytes. Dose-dependent increases in numbers of peroxisomes (moderate to striking increases in numbers) at doses of 54 mg/kg bw/d.

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Species (Strain) Rat (SpragueDawley)

Dosing Regimen Oral (diet) doses of 0, 23, 108, or 518 mg/kg bw/d

Duration of Treatment 14 days + recovery (28 days); 42 days

Major Findings

Reference, GLP and OECD Status Molitor and Persohn, 1993 (169) GLP: not specified OECD: not applicable

Increased, but reversible, absolute and relative liver weights in high-dose group. Increases in enzyme activities and cytochrome P450 content were generally dose-dependent and reversible. Highlights of the results include (high-dose data reported as % control): lauric acid hydroxylation (up to 281%); pentoxyresorufin O-depentylase activity (up to 1,143%). Peroxisomal fatty acid beta-oxidation (FAO) was unaffected. Immunoblot analyses showed dose-dependent, large, and reversible increases in CYP2B1/2 enzymes (at least >100X control at 6,000 ppm); only slight or small increases in CYP3A1/2 and CYP4A proteins (209% and170% of control, respectively). Electron microscopy findings show reversible proliferation of smooth endoplasmic reticulum, increased cytoplasmic lipid vacuoles, dilated mitochondrial cristae, transient formation of matrical plates in peroxisomal matrices in high-dose rats. Increased, but reversible, absolute and relative liver weights in high-dose females. Increases in enzyme activities and cytochrome P450 content were generally dose-dependent and reversible. Highlights of the results showed increases in: cytochrome P450 content; ethoxyresorufin O-deethylase and pentoxyresorufin Odepentylase activities; 7-hydroxytestosterone production in females; 15 -HT, 16-HT, and androstenedione production, lauric acid hydroxylation. Peroxisomal fatty acid betaoxidation was unchanged. Immunoblot analyses showed no increases in levels of CYP1A- and CYP3A-related proteins, but slight to mild increases in CYP4A-related proteins (levels in males were 140% greater than controls, but increases in females were slight, with no overall change in total CYP4A levels). Electron microscopy findings show no changes to hepatocytes, including the number and size of peroxisomes. Triclosan induced aminopyrine N-demethylase (APND) activity slightly, but significantly, in mice at a dose of 100 mg/kg bw/d. Slight (<100% increase vs. control), but significant increases in biphenyl 4-hydroxylase, APND, and phenacetine O-deethylase activities at 100 mg/kg bw/d in rats. Triclosan induced moderate increases (100-130%) in biphenyl 2hydroxylase, ethoxycoumarin O-deethylase, and p-nitrophenerole O-deethylase (p-NPOD) activities at 100 mg/kg bw/d in rats. p-NPOD activity was also increased slightly at 50 mg/kg bw/d in rats.

Hamster (Syrian)

Oral (diet) doses of 0, 50, 310, or 800 mg/kg bw/d in males, or 0, 46, 314, or 960 mg/kg bw/d in females

14 days + recovery (28 days)

Thomas, 1994 (170) GLP: compliant OECD: not applicable

Mouse (ddY); Rat (Wistar)

Daily i.p. doses of 50 or 100 mg/kg bw/d

3 days

Kanetoshi et al., 1992 (4) GLP: not reported OECD: no comparable guidelines

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Species (Strain) Rat (Wistar)

Dosing Regimen In vitro concentrations of 0.1, 1, 10, or 100 M

Duration of Treatment Male rats were pretreated with cytochrome P450 inducers prior to preparation of microsomes

Major Findings

Reference, GLP and OECD Status Hanioka et al., 1996 (171) GLP: not specified OECD: not applicable

Ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) activities were inhibited by 93 and 86%, respectively at a concentration of 10 M triclosan. Inhibition of phenacetine Odeethylase and 4-nitrophenol hydroxylase activities was 5-60% vs. control levels at 10 M and up to 80% vs. controls at 100 M. Testosterone 6-hydroxylase and lauric acid hydroxylase activities were inhibited by up to 50% at 100 M. Ki values for EROD and PROD activities were 0.24 and 1.48 M, respectively. The effect on EROD activity was consistent with competitive inhibition, whereas the effect on PROD indicated noncompetitive inhibition. Increases in biochemical parameters were generally dose-related, reaching significance at the high dose in rats. Increases included: microsomal protein, cytochromes P450 and b5, and NADPH-cytochrome C reductase. Benzyloxyresorufin O-debenzylase (BROD) and pentoxyresorufin O-depentylase (PROD) activities were induced up to 22- and 20-fold, respectively. Levels of other P450 enzymes were either 2.4- to 4.9-fold increased at the high dose, or were unchanged. Immunoblotting data show levels of CYP 2B proteins (associated with PROD activity) were increased from 10.8 up to 34-fold.

Rat (Wistar)

0, 58, 116, or 232 mg/kg bw/d

5 days

Hanioka et al., 1997 (172) GLP: not specified OECD: not applicable

A series of 3 critical studies were conducted in mice, rats (males only), and hamsters to examine the effects of triclosan on selected biochemical and morphological liver parameters following dietary administration of triclosan for 14 days [Molitor et al., 1992 (168); Molitor and Persohn, 1993 (169); Thomas, 1994 (170)]. Biochemical investigations included measurements of levels of protein and cytochrome P450 (P450) content and P450 enzyme activities. Morphology was examined using electron microscopy (EM). As can be seen from Table 46, and summarized in Table 47, there exist differences in responses between species. There was little difference between sexes, although females appeared slightly less sensitive to the effects of triclosan than males. With regard to morphology changes, the most notable was a dose-dependent, moderate to striking increase in the numbers of peroxisomes in mouse liver electron micrographs [descriptors taken from the original report by Molitor et al., 1992 (168)]. In contrast, there were no increases in numbers of peroxisomes in rats and hamsters. Liver weight and microsomal P450 content were strongly increased in mice, mildly increased in higher dose rats and relatively unaffected in hamsters. Similarly, induction of various enzyme activities was most pronounced and occurred at lower doses in mice when compared to other species. Cyanide-insensitive fatty acid -oxidation activity (palmitoyl-CoA oxidation), a diagnostic indicator of peroxisome proliferation, showed a dose-dependent increase in mice, but not in rats or hamsters. Cytosolic glutathione S-transferase activity was mildly increased in mice, less so in rats, and slightly depressed in hamsters. Lauric acid 12-hydroxylation activity, as catalysed by isoenzymes of the cytochrome P450 CYP4A gene subfamily, was strongly increased in mice but less so in rats and hamsters. Lauric acid 11-hydroxylation activity, as catalysed by isoenzymes of the cytochrome P450 CYP1A, CYP2B, and CYP2C families, was again strongly increased in mice, but less so in hamsters. No effects were found in rats. Ethoxyresorufin O-deethylase (EROD) activity showed a mild dose-dependent increase in mice, with lesser increases in hamsters, and was decreased in rats. A strong induction of 7-pentoxyresorufin O-depentylase (PROD), a CYP2B marker, was found in mice and rats; hamsters showed significantly less potent induction of this activity.

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The pattern of regio- and stereo-selective testosterone hydroxylation was used as a tool to assess treatment-related effects on the activities of several isoenzymes of the microsomal P450 enzyme family. Only in the mouse was total testosterone hydroxylation significantly induced. At the highest dose level of 951 mg/kg body weight/day, a 6-fold dose-dependent increase was observed. The most prominent changes in the testosterone hydroxylation profile were increased production of the 2-, 6-, 15-, and 16-hydroxy metabolites. Production of the 2- and 6-hydroxy metabolites is associated with the expression of cytochrome CYP3A, while 16-hydroxylation is catalysed by the CYP2B cytochrome family. Rats showed a different pattern of testosterone hydroxylation after triclosan administration. Production of the 16-hydroxy metabolite was most prominent and, except for a mild induction of 15-hydroxylation which is associated with cytochromes CYP2C12 and CYP2C13, no other hydroxy metabolite was produced in significant amounts. Hamsters were much less sensitive to P450 enzyme induction than mice. The only significant change observed in the hydroxylation profile was a small increase in 17-oxidation to form androstenedione. Immunoblot analyses using monoclonal antibodies generated against, and specific for, inducible isoenzymes of rat liver cytochrome P450 were performed to further clarify the nature of triclosan-induced changes in liver enzymes. Mice demonstrated a slight dosedependent decrease in the content of the isoenzymes of the CYP1A gene subfamily. Rats had an increased content of this isoenzyme family and hamsters a marginal increase. Triclosan strongly induced isoenzymes of the CYP3A subfamily in mice (8.4x control levels), produced a small increase (2X) at the highest dose in rats, and produced a decrease of this enzyme in female hamsters, with no effect in male hamsters. CYP4A induction was also extremely strong (7.8x at the highest dose) in mice and much less so in the rat and hamster (1.6x and 2.4x at the highest dose, respectively). The effect of triclosan on P450 enzyme activities was also investigated in 3 published reports of an in vivo study in mice and in vitro and in vivo studies in rats [Kanetoshi et al., 1992 (5); Hanioka et al., 1996 (171); Hanioka et al., 1997 (172)]. Both of the rat studies support the findings in the critical 1993 rat investigation [Molitor and Persohn, 1993 (169)]. In the in vitro study, the effects of triclosan on P450 enzyme activities were investigated using liver microsomes from rats pretreated with one of several P450 inducers (3methylcholanthrene, phenobarbital, pyridine, dexamethasone, or clofibrate) [Hanioka et al., 1996 (171)]. The data from this study, which assessed the effect of triclosan on the activity levels of P450 enzymes previously increased by P450 inducers, suggest that triclosan competitively inhibits enzymes of the cytochrome P450 1A family, as indicated by inhibition of EROD and phenacetin O-deethylase activities, and non-competitively inhibits cytochrome P450 2B enzymes, as evidenced by its effect on PROD activity. Interaction of triclosan with cytochrome P450 2B1/2 enzymes was also suggested by the pattern of induction of various P450 enzyme activities in the published in vivo rat study which investigated the effects of 5 days of dosing with triclosan on P450 enzyme activities [Hanioka et al., 1997 (172)]. Data from the in vivo study show slight to no effects of triclosan administered for 3 days in mice on a variety of P450 activities, but did show increases in P450 activities in rats [Kanetoshi et al., 1992 (5)]. The reason for the inconsistency with the pivotal 1992 mouse biochemical investigation is unclear, but may be related to the length of time of dosing or the difference in the route of administration (intraperitoneal in this published report vs. dietary administration in the pivotal report). Table 47 summarizes the effects of triclosan on selected biochemical parameters.

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Table 47:

Summary of Effects on Selected Biochemical Parameters Following 14-Day Dietary Administration of Triclosan

End-Point Minimum Effect Level (mg/kg bw/day) 518 518 518 20 20 20 ND 50 ND 518 23 108 20 ND 50 270 none 800 310 310 310 250 ND 518 50 ND 310 800 20 270 800 50 270 none (sl.) none Effect Minimum Effect Level (mg/kg bw/day) Effect Minimum Effect Level (mg/kg bw/day) Effect Minimum Effect Level (mg/kg bw/day) Effect

Male Rat 1

Male Mouse 2

Female Mouse 2

Male Hamster 3

Female Hamster 3 Minimum Effect Level (mg/kg bw/day) 960 960 314 960

Effect

Liver weight (absolute)

Protein content, microsomal

none

Cytochrome P450 content

Glutathione S-transferase

Fatty acid -oxidation

none

Lauric acid 11-hydroxylation

none

960 960 314 314

Lauric acid 12-hydroxylation

Ethoxyresorufin O-deethylase

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20 250 518
* *

Pentoxyresortufin O-depentylase

* * *

Testosterone hydroxylation (total) 1-hydroxylase 2-hydroxylase 6-hydroxylase 7-hydroxylase 15-hydroxylase 16-hydroxylase 2-hydroxylase 6-hydroxylase 15-hydroxylase 16-hydroxylase Androstenedione 108
*

none none none none none none none ND ND none ND 50 20 20 50 950 20

ND ND ND ND ND ND ND ND ND ND ND ND

none ND ND ND none ND none none none none

350

none ND ND ND ND none none none none

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Table 47:

Summary of Effects on Selected Biochemical Parameters Following 14-Day Dietary Administration of Triclosan

End-Point Minimum Effect Level (mg/kg bw/day) 50 20 20 800 ND 20 20 270 Effect Minimum Effect Level (mg/kg bw/day) Effect Minimum Effect Level (mg/kg bw/day) Effect Minimum Effect Level (mg/kg bw/day) Effect

Male Rat 1

Male Mouse 2

Female Mouse 2

Male Hamster 3

Female Hamster 3 Minimum Effect Level (mg/kg bw/day)

Effect

Cyt. P/-450 protein levels CYP1A CYP2B CYP3A CYP4A 108 23 518 518 ND none ND none

(sl.)

none ND none

314

Rat data from Molitor and Persohn, 1993 (169). Mouse data from Molitor et al., 1992 (168) 3 Hamster data from Thomas, 1994 (170). * Not statistically significant but dose-response effect observed. ND, not done sl., slight

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3.3.12.3.3 Summary of Liver Morphology and Biochemistry Data for Triclosan The data showing strong increases in peroxisomal fatty acid beta-oxidation, 11- and 12hydroxylation of lauric acid, and levels of CYP4A proteins (diagnostic for peroxisome proliferation), and increases in numbers and size of peroxisomes provide biochemical and EM evidence that triclosan has peroxisome proliferator-type activity in mouse liver at doses 50 mg/kg body weight in males. In contrast, key events associated with PPAR agonists, such as lauric acid 12-hydroxylation and an increase in total cytochrome P450, were significantly increased in the high dose group in rats; however, the magnitude of the increase was less than that seen in mice. The hamster data provide only limited evidence of any peroxisome proliferator activities for triclosan, with increased CYP4A proteins and lauric acid hydroxylation activity occurring only at high doses [Thomas, 1994 (170)]. However, the lack of induction of peroxisomal fatty acid oxidation and of morphological evidence suggests that triclosan is not a peroxisome proliferator in hamster liver. Mice and rats, but not hamsters, produced a dose-related increase in PROD, which was significant at all doses in the mouse [Molitor et al., 1992 (168)], but only significant in the highest dose group in rats [Molitor and Persohn, 1993 (169)]. Activation of PROD is typically associated with induction of CYP2B which is induced by phenobarbital. However, as is discussed more fully in Section 3.13.3, several other known PPAR agonists also significantly increase induction of PROD. In examining the evidence for peroxisome proliferator-like effects of triclosan in mice, it is interesting to note that the effects of the known peroxisome proliferator clofibrate in rats treated for 3 days at the dose of 400 mg/kg body weight/day were shown to include induction of PROD (3.7X), 4-nitrophenol hydroxylase (2.9X), and lauric acid hydroxylase (12.5X) activities compared to untreated rats [Hanioka et al., 1996 (171)]. Overall, both biochemical and morphological evidence from special investigations of the effects of triclosan in rodent liver serve to differentiate the response to triclosan in mice as that of a peroxisome proliferator. 3.3.12.4. Effects of Triclosan on Rat Thyroid

Effects of triclosan on thyroid hormone levels in rats have been investigated in two recent studies, one with 4-day oral exposure (10, 30, 100, 300, 1000 mg/kg bw/d) in weanling female Long-Evans rats (Crofton et al., 2007, AR4), the other with oral administration (3, 30, 100, 200, 300 mg/kg bw/d) from postnatal day (PND) 23 to 53 in male Wistar rats (Zorilla et al., 2009, AR10). Short term oral exposure in female rats resulted in dose dependent decreases in serum thyroxine levels: serum T4 was decreased 28, 34 and 53% following treatment with 100, 300 and 1000 mg/kg bw/day triclosan, respectively. No significant changes were seen at 10 and 30 mg/kg bw/day triclosan in female weanling rats. The authors of this study (Crofton et al. 2007) suggest that decreases in T4 may result from increases in the sulfation or glucuronidation via PXR-linked genes. This view is consistent with triclosan-induced upregulation of liver enzymes documented in other studies that have been described above (section 3.3.12.3. and Tab. 3.3.12.3.2-1 and Tab. 3.3.12.3.2-2). The purpose of the second study was to determine effects of triclosan on pubertal development and thyroid hormone levels in the male rat. After 31 days of exposure, triclosan significantly decreased serum thyroxine (T4) in a dose-dependent manner at 30 mg/kg bw/day and higher (Zorilla et al., 2009). The active thyroid hormone triiodothyronine (T3) was decreased significantly only at 200 mg/kg bw/day, and thyroid stimulating hormone (TSH) was not statistically different from controls at any dose. Liver weights were increased at 100 mg/kg bw/day triclosan and above suggesting that induction of hepatic enzymes have contributed to the altered T4 and T3 levels. The authors did not consider the levels of change in glucuronidation (UDPGT) activity at 30 mg/kg as sufficient to explain the observed decrease in T4 levels; however, sulfation activity was not assessed. Triclosan did not alter the age at onset of puberty (assessed by preputial separation) or the

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development of androgen-dependent tissues, even though there was a 60% decrease in androgen serum levels in the 200 mg/kg dose group. In conclusion, alterations in thyroid hormone levels induced by triclosan in juvenile male rats did not lead to any apparent functional consequences. Thus, the lowest observed effect level for a decrease in T4 (30 mg/kg bw/day) is regarded as biochemical effect marker, but neither this nor the no observed effect level (3 mg/kg bw/day) are used for a risk assessment for triclosan since they have not been linked to an adverse effect. Moreover, it is important to acknowledge major differences in the thyroid hormone physiology and regulation between rats and humans (SCCNPF 2004, AR7). Since the rat is a very sensitive model for chemical induced changes in the thyroid hormone axis, limitations exist with respect to extrapolation of rat data to human physiology/pathophysiology. 3.3.13. 3.3.13.1 Safety evaluation (including calculation of the MoS) Consumer Exposure Assessment

For cosmetics, consumer exposure, measured as systemic exposure dose (SED) is typically based on dermal absorption data. In the case of triclosan, because of exposure through toothpaste use and mouthwash, oral exposure data are also relevant. Calculations were made for individual products and for product groups in which triclosan is used according to the industry submission, i.e. most prevalently in toothpaste, deodorant, hand and body soaps (referred to as common-use products) and for products in which triclosan is used less frequently, such as facial cosmetic products, body lotion, and mouthwash (referred to as marginal-use products). For the purpose of SED calculations for oral for mu lat ion s (toothpaste, mouthwash) it was assumed that triclosan is 100% bioavailable (see Table 48). In the dossier that was submitted, calculations were based on current-use triclosan concentrations in different product types as given by the applicant, which, for some product categories, are below the maximally allowed content of 0.3% triclosan. However, the SCCP was requested to evaluate the safety of triclosan at the currently authorised level. Therefore, SEDs from both the current-use and the maximally allowed concentrations of triclosan are given in the calculations below. Table 48: SED Calculation for Oral Products
Product Assumed bioavailability (%)1 100 100 100 Amount applied (mg)2 2,750 mg per day 10,000 mg per use 10,000 mg per use Retention3 Frequency of application (times/d)4 NA 3 3 Triclosan content (%) 0.3 0.26 0.37 BW (kg) SED (mg/kg bw/d)5 0.0234 0.1000 0.1500

Toothpaste Mouthwash Mouthwash

0.17 0.1 0.1

60 60 60

Abbreviations: BW, body weight; d, day; NA, not applicable; SED, systemic exposure dose 1 For the purposes of these calculations (i.e., for oral products), it was assumed that the bioavailability of triclosan was 100%. 2 Amount of application value was taken from Table 2, Section 6-2 in SCCP, 2006. 3 Retention value was taken from Table 2, Section 6-2 in SCCP, 2006. 4 Frequency of use per day value for toothpaste was taken from Table 3, Section 6-2 in SCCP, 2006, which takes into account frequency of application. Frequency of use per day value for mouthwash was taken from Table 2, Section 6-2 of the SCCP, 2006 guidance.

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6 7

Formula: SED = (Bioavailability in % x amount of product applied (mg) x Frequency x Retention factor x amount of triclosan in product) / BW. Current use concentration as given by the applicant maximally authorised concentration

For d er ma l f or mu lat ions , SED calculations were based on percutaneous absorption data from in vitro human studies (Table 37). SED calculations for individual personal-care products containing triclosan were carried out based on dermal absorption values (g/cm2) from in vitro percutaneous absorption studies conducted with deodorant and w/o formulations containing 0.2% triclosan and dilute soap solution formulation containing 0.02% triclosan. In each calculation for dermal products, an extrapolated value for flux (g/cm2 absorption) was used, based on the assumption that skin penetration is by passive diffusion, such that flux would be proportional to the concentration of triclosan applied to the skin. For hand soap and body soap, the conversion of the g/cm2 dermal absorption value to a current-use value for amount of triclosan in the product type assumed a 10-fold dilution of 0.3% triclosan. For both soaps, a retention factor was not used due to the inclusion of a rinse-off step in the relevant in vitro percutaneous absorption study. The results of these calculations are provided in Tables 49 and 50, below, for leave-on and rinse-off products, respectively. Table 49: SED Calculation for Leave-On Products
Product 24-h dermal absorption based on 0.2% triclosan (g/cm2) 1 0.303 0.420 0.420 0.420 0.420 0.420 0.420 Triclosan content (%) Calculated 24-h dermal absorption based on triclosan content (g/cm2) 2 0.455 0.315 0.630 0.420 0.630 0.315 0.630 SA (cm2)
3

F (per d)

Conversion (mg/g)

BW (kg)

SED (mg/kg bw/d)5

Deodorant stick Body Lotion Body Lotion Face powder Face powder Blemish Concealer Blemish Concealer

0.3 0.156 0.37 0.26 0.37 0.156 0.37

200 15670 15670 565 565 57 57

1 1 1 1 1 1 1

1x10-3 1x10-3 1x10-3 1x10-3 1x10-3 1x10-3 1x10-3

1 1 1 1 1 1 1

60 60 60 60 60 60 60

0.0015 0.0823 0.1646 0.0040 0.0060 0.0003 0.0006

Abbreviations: BW, body weight; d, day; F, frequency of application; h, hour; R, retention; SA, surface area of application; SED, systemic exposure dose 1 Dermal absorption values based on in vitro data using 0.2% in deodorant formulation (for deodorant stick) and 0.2% water/oil emulsion (for body lotion, face powder, and stick concealer). 2 Calculation: (Absorption from 0.2% triclosan applied in the relevant in vitro study) x (triclosan content for the product/0.2%) = Absorption from 0.3% triclosan. This assumes that skin penetration is by passive diffusion, such that flux would be proportional to the concentration of triclosan applied to the skin. 3 Area of application values were taken from Table 1, Section 6-2 in SCCP, 2006. The skin area for blemish concealer was assumed to be 1/10th of the face. 4 Frequency of application values for use per day were taken from Table 2, Section 6-2 in SCCP, 2006. Frequency of application values for face powder and stick concealer were assumed to be once per day. 5 Formula: SED = (24-h dermal absorption in g/cm2 based on use levels of triclosan in product x surface area of application in cm2 x frequency of application per day x retention x conversion factor) / BW. 6 Current use concentration as given by the applicant 7 Maximally authorised concentration

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Table 50: SED Calculation for Rinse-Off Products


Product 24-h dermal absorption based on 0.02% triclosan (g/cm2) 1 0.0306 0.0306 Calculated 24-h dermal absorption based on 0.03% triclosan (g/cm2) 2 0.046 0.046 SA (cm2) 3 F (times/d) 4 Conversion (mg/g) BW (kg) SED (mg/kg bw/d)5

Hand soap Shower gel/body soap

860 17,500

10 2

1x10-3 1x10
-3

60 60

0.0066 0.0268

Abbreviations: BW, body weight; d, day; F, frequency of application; h, hour; SA, surface area of application; SED, systemic exposure dose 1 Dermal absorption value based on in vitro data using 0.02% soap solution. 2 Calculation: (Absorption from 0.02% triclosan applied in the relevant in vitro study) x (0.03%/0.02%) = Absorption from 0.03% triclosan solution. This assumes that 1) a 10X dilute solution of 0.3% triclosan is applied and 2) skin penetration is by passive diffusion, such that flux would be proportional to the concentration of triclosan applied to the skin. 3 Area of application values were taken from Table 1, Section 6-2 in SCCP, 2006. 4 Frequency value for shower gel/body soap use per day was taken from Table 2, Section 6-2 in SCCP, 2006. Frequency for hand washing was not provided in SCCP, 2006, so was assumed to be 10 times per day for this calculation. 5 Formula: SED = (24-h dermal absorption in g/cm2 based on 0.03% triclosan x surface area of application in cm2 x frequency of application per day x conversion factor) / BW.

The combined SED for common-use triclosan-containing personal care products and marginal-use triclosan-containing personal care products was also calculated. The SED calculations are presented in the Table below.

Summary of Triclosan SED Values from the Use of Personal Care Products Type of Product(s) Toothpaste Hand Soap Body Soap/shower gel Deodorant (Stick) Mouthwash Mouthwash Face powder Face powder Body lotion Body lotion Stick-type concealer Stick-type concealer Common-Use Products (toothpaste, hand soap, body soap/shower gel, deodorant stick) Marginal-Use Products (mouthwash, body lotion, face powder, stick concealer) Marginal-Use Products (mouthwash, body lotion, face powder, stick concealer) All Products (toothpaste, hand soap, body Triclosan content (%) 0.3 0.3 0.3 0.3 0.2 0.3 0.2 0.3 0.15 0.3 0.15 0.3 0.3 0.15-0.2 0.3 0.15-0.3 SED 0.0234 0.0066 0.0268 0.0015 0.1000 0.1500 0.0040 0.0060 0.0823 0.1646 0.0003 0.0006 0.0583 0.1866 0.3212 0.2449

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Summary of Triclosan SED Values from the Use of Personal Care Products Type of Product(s) soap/shower gel, deodorant stick, mouthwash, body lotion, face powder, stick concealer) All Products (toothpaste, hand soap, body soap/shower gel, deodorant stick, mouthwash, body lotion, face powder, stick concealer) Abbreviations: SED, systemic exposure dose 0.3 0.3795 Triclosan content (%) SED

Internal exposure and absorption of triclosan under simulated use conditions by humans can be approximated also from in vivo studies with volunteers that applied triclosan-containing personal care products for a prolonged time period (see table 36). Precise information on the use-pattern and the level of triclosan content of the formulations was available in the study by Beiswanger and Tuohy (1990), in which 182 subjects used a toothpaste (0.28 % triclosan), a bar soap (0.75% triclosan) and a deodorant (0.39% triclosan) for 13 weeks. The results of this study indicated that all subjects reached a stable plateau plasma level after 3 weeks of use of the toothpaste, deodorant, and soap. The results showed plasma levels of: 19-23 ppb (exposure to toothpaste only), and 29-31 ppb (exposure to toothpaste, deodorant, and soap). 3.3.13.2 Safety Assessment

The human plasma levels can be compared to the plasma level (of 28,160 ng/ml) reported in studies with rats that received triclosan doses at the NOAEL of 12 mg/kg bw/day (see Tab. 27) to derive plasma level based MoS.

Types of Products Used

SED (mg/kg bw/d)

MoS Based on Rat NOAEL of 12 mg/kg bw/d

MoS Based on Plasma Levels

Toothpaste Toothpaste, deodorant stick, and hand soap Common-Use Products 0.3% triclosan (toothpaste, hand soap, body soap/shower gel, deodorant stick) All Products 0.15 0.3% triclosan (toothpaste, hand soap, body soap/shower gel, deodorant stick, mouthwash, body lotion, face powder, blemish concealer) All Products 0.3% triclosan (toothpaste, hand soap, body soap/shower gel, deodorant stick, mouthwash, body lotion, face powder, blemish concealer)

0.0234 0.0315 0.0583

513 381 206

1408 939 Not done (no human plasma data available) Not done (no human plasma data available)

0.2449

49

0.3795

32

Not done (no human plasma data available)

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From the calculations above it can be deduced that the use of triclosan in all products is not safe because of the magnitude of the aggregate exposure. This conclusion is reached regardless whether current use levels as given by the applicant or the maximally authorised use level is considered. However, the use in the common-use products (toothpaste, hand soap, body soap/shower gel and deodorant stick) is considered safe. The exposure to triclosan from face powder and blemish concealer (up to 0.3% triclosan) is low and not considered to be of concern in addition to exposure from common-use products. However, the use of triclosan in body lotions and mouthwashes results in high exposures and is not recommended. Safety of Triclosan in Children and Neonates The very low levels of exposure as measured in breast milk indicate that maternal use of triclosan immediately post-partum is unlikely to be a safety concern for neonates (see 3.3.11.2.6). Infant exposure to triclosan from breast milk has been shown to be significantly lower than exposure to the mother, based on a comparison of triclosan concentrations in breast milk and plasma [Allmyr et al., 2006 (103)]. No measured exposure data for babies and young children following use of consumer products containing triclosan was identified in the literature, except spot urine measurements in the age group 6-11 years from the NHANES study [Calafat et al. 2008; AR3]. Based on the conversion of spot urine concentrations to estimated dose, this subpopulation had a lower aggregate exposure to triclosan than children of 11-19 years and adults [US EPA 2008 (AR9)]. The rapid increase in maturation of glucuronidation ability within the first year and the maturity of the sulfation pathway, indicate that capabilities of children to metabolise triclosan through glucuronidation or sulfation are likely comparable to those of adults. Also, glomerular filtration rates normalised to body weight approach adult values by around 6 months of age and renal tubular function matures to near-adult values by around 1 year of age (Alcorn and McNamara, 2002). Accordingly, studies have shown that elimination is comparable in adults and children (see Section 3.11.2.1). 3.3.14. Discussion

Physico-chemical properties Trichlosan is a phenol and a weak acid (pKa 8.1). This and its partition coefficient (logPo/w 4.8) facilitate transfer of the protonated (non-ionized) form of triclosan across lipid membranes. General toxicity Triclosan is not acutely toxic via the oral route of administration, with high oral intubation LD50 values in the range of 3,750 to 5,000 mg/kg body weight in mice and rats, and an oral capsule LD50 value of greater than 5,000 mg/kg body weight in dogs. SCCP considers the NOAEL as 12 mg/kg bw/d due to haematoxicity and decreased absolute and relative spleen weights (Mid Dose Females) in the long term toxicity study in rats. Irritation / sensitisation The irritation/corrosivity data from either irritation studies in the hamster, guinea pig, and rabbit, or skin toxicity studies conducted in the mouse, rat, monkey, and dog suggest that triclosan may cause slight reversible skin irritation at concentrations of 0.5 to 5% under experimental conditions. Triclosan at concentrations of 1 to 10% produced only slight, reversible irritation in the rabbit eye. Data from human use evaluating the skin and oral mucosa irritation effects of triclosan alone, or in combination with SLS, indicate that triclosan 0.3% is not a skin or oral mucosal irritant. In the guinea pig no sensitisation with triclosan in various formulations and concentrations (up to 10% in petrolatum) was found. However, clinical experience has shown that triclosan does have a low sensitisation potential in humans. In over 14,000 patients patch tested with triclosan (typically tested at a concentration of 2% in petrolatum), the range of positive

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results was 0.1 to 0.3% of the tested groups. When tested in patients with known or suspected cosmetic allergy or intolerance positive reaction rates ranged from 0.06 to 0.8% of a total of 11,887 tests conducted. Possible photocontact allergy has been rarely reported. Dermal and oral absorption Data from percutaneous absorption studies indicate that triclosan is well absorbed through the skin in all species tested with the extent of absorption being dependent on the formulation in which it was delivered. In the rat, percutaneous absorption was approximately 23 to 28% of the applied dose of triclosan in ethanol, ethanol/ water, soap suspension, or a cream formulation. Triclosan is highly absorbed following oral administration, with no species-related differences, and in humans this is up to 98% of the dose. However, under normal conditions of toothpaste use (i.e., expectoration and rinsing) or following percutaneous application of several different personal care products, absorption is more limited (approximately 5 to 10% of the dose via either of these routes of administration). See also brief summary of in vivo data under Kinetics Mutagenicity / genotoxicity The genotoxic potency of triclosan has been investigated in a number of tests which can be broadly sub-divided in non-regular and normal (regulatory accepted) tests. Most of the tests are rather old and performed before the introduction of OECD guidelines. Consequently, the latter tests are not performed under currently accepted protocols. Since, next to non-standardised protocols, the tests have limited value but may occasionally give supportive evidence. Only two of the (non-regular) tests indicate a putative genotoxic potential of triclosan: Irgasan DP 300 (triclosan) induced mutations in an in vitro gene mutation assay in yeast. This positive result is not confirmed in an appropriate gene mutation test in mammalian cells. The same compound also induced mutations in a mouse spot test. However,, in a similar experiment with lower and thus less toxic concentrations this result could not be confirmed. Triclosan was investigated in (regular) genotoxicity tests covering the 3 endpoints: gene mutations, structural and numerical chromosome aberration. Triclosan exposure did not result in gene mutations in bacteria or mammalian cells nor did it induce UDS in vitro in primary hepatocytes. Triclosan induced chromosome aberrations in V79 cells, but was tested negative in assays with CHO cells. The positive result could not be confirmed in an in vivo micronucleus test in bone marrow cells of mice. Consequently, triclosan can be considered to have no relevant genotoxic potential in vivo. Carcinogenicity Three rodent lifetime bioassays have been conducted to evaluate the carcinogenic potential of triclosan. Triclosan produced hepatic effects and hepatic tumours in mice, but little evidence of toxicity and no tumours in rats. Hamsters showed increased liver toxicity relative to the rat, but no tumours. According to the EU classification system, triclosan is not considered classifiable as a carcinogen. It should be noted that triclosan is a peroxisome proliferator in mice liver. Reproductive/developmental Toxicity Triclosan was not teratogenic nor a reproductive toxicant in a full complement of reproductive and developmental toxicity studies conducted in mice, rats, and rabbits conducted at doses of up to 350 mg/kg body weight/day. NOAEL (NOEL) values from the definitive GLP studies were summarized in Table 21. It is important to note the determination of the foetal NOAEL value for each study was based on foetal variation effects that were most likely secondary to general maternal toxicity, and not direct effects of triclosan per se. It is also worth noting that the low NOAEL value for foetal effects in the mouse study (25 mg/kg body weight/day) is likely attributable to the sensitivity of the maternal mice to the liver effects of triclosan, also observed in the repeated dose and carcinogenicity studies in mice.

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Kinetics Numerous human and animal studies are available on the toxicokinetics of triclosan following oral and dermal exposure to single and repeated doses. The studies cover all important aspects, i.e. absorption, distribution, metabolism and excretion. Upon oral administration absorption of triclosan from the gastrointestinal tract is rapid and extensive in both humans and animals. But, limited buccal absorption was seen in humans following normal toothpaste use (up to 14% of the amount that would be absorbed upon ingestion of an equivalent dose). Upon dermal application in humans, absorption was at least 3% to 7%, and at least 14% in one volunteer. Triclosan is rapidly distributed in the organism following oral or dermal exposure. The main metabolic pathways in humans and animals involve glucuronidation and sulfation by phase2 enzymes. The half-life of elimination for orally administered triclosan ranged from 13 to 29 h in humans compared to 10 to 15 h in rats, 8-12 in mice and 25 to 32 h in hamsters. The major route of excretion in humans, hamsters, rabbits and primates is via urine, with excretion via faeces being of secondary importance in these species. The reverse situation is observed in rats, mice and dogs where biliary excretion is more important than renal excretion. The human oral and dermal data provide no evidence for a bioaccumulation potential. Likewise, the kinetic data in rats and hamsters provide no evidence for a bioaccumulation in these species, whilst in mice retention of triclosan (and/or metabolites) appears to occur in liver. In conclusion, kinetics of triclosan are qualitatively similar, but the observed quantitative differences between humans and several animals make human data the first choice for the safety evaluation of triclosan-containing consumer products. Other aspects Recently, the US EPA (2008) utilized population-based biological monitoring data for triclosan (available from the NHANES study) to assess the co-occurrence of uses to develop an aggregate exposure assessment. Because of some uncertainties in converting spot urine concentrations to estimated dose, three conversion methods were used. Calculated exposure was then compared to the selected oral NOAEL of 30 mg/kg/day (from the chronic toxicity study in baboons). Based on the results at the mean and 99th percentile, the aggregate risks to triclosan from a ll (personal care and other consumer products) uses did not trigger a risk of concern. The mean MOEs ranged from 4,700 to 19,000. The MOEs at the 99th percentile ranged from 260 to 1,500 [US EPA 2008, AR9]. Exposure estimates based on biological monitoring data from the US are considered by SCCP as useful additional information in their overall evaluation on the safety of triclosan. The difference in SCCP and US-EPA evaluations of triclosan may be explained as follows: - USA-EPA chose a NOAEL of 30 mg/kg/d whereas SCCP selected a NOAEL of 12 mg/kg/d (based on haemotoxicity) as the critical effect level against which human exposure to triclosan is compared (for subsequent MOE or MOS calculations). The SCCP approach is in line with the evaluation of triclosan by EFSA for its use in food contact materials. - US-EPA has estimated triclosan exposure in the US population on the basis of biomonitoring data from spot urine samples. Although this approach probably reflects exposure from current use concentrations in various products on the US market, it cannot be applied directly to the evaluation regarding the safe use of triclosan in cosmetic products by SCCP, since: - The current use concentrations in the USA may have been lower than the max imal triclosan concentration limit of 0.3% as preservative in cosmetic products in the EU, the safety of which SCCP was asked to evaluate according to this mandate (Question 1) - In estimating human exposure, the SCCP followed its Notes of Guidance to calculate systemic exposure doses (SED) from triclosan-containing products (at 0.3%) applied

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orally and dermally. This may be viewed as a worst-case scenario. The alternative approach, i.e. MOS calculations that are based on plasma levels (measured under simulated use-conditions) were only available for certain products, not for all triclosancontaining products. Representative biomonitoring data are not available for the European population. It is important to note that the two evaluations followed different objectives: While US-EPA in principle looked at real exposure that occurred in the population to derive a conclusion about a possible concern, the SCCP is asked to evaluate the safety of a hypothetical maximum exposure according to the authorised concentrations and applications in the cosmetic legislation.

4. CONCLUSION Taking into account the provided toxicological data, the SCCP considers that the continued use of triclosan as a preservative at the current concentration limit of maximum 0.3% in all cosmetic products is not safe for the consumer because of the magnitude of the aggregate exposure. However, its use at a maximum concentration of 0.3% in toothpastes, hand soaps, body soaps/shower gels and deodorant sticks ("common-use products" as defined by the applicant) is considered safe. Any additional use of triclosan in face powders and blemish concealers at this concentration is also considered safe but the use of Triclosan in other leave-on products (e.g. body lotions) and in mouthwashes is not considered safe for the consumer due to the resulting high exposures. Importantly, before a final conclusion on the safety of triclosan in cosmetic products can be reached, the potential development of resistance to triclosan and cross-resistance by certain micro-organisms must be assessed. This aspect is not covered in this document and will be discussed in a separate opinion. Inhalation exposure to triclosan from spray products (e.g. deodorants) was not assessed.

5. MINORITY OPINION Not applicable

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116. Sandborgh-Englund, G.; Adolfsson-Erici, M.; Oldham, G.; Ekstrand, J. Pharmacokinetics of triclosan following oral ingestion in humans.. J Toxicol Environ Health A Oct. 69(20): 1861-73. 2006 117. Gilbert, RJ. The oral clearance of zinc and triclosan after delivery from a dentifrice. J. Pharm. Pharmacol 39: 480-483. 1987 118. Lin, Y.-J. Buccal absorption of triclosan following topical mouthrinse application. Am J Dent 13: 215-217. 2000 119. Ciba-Geigy. Hexachlorophene- Irgasan DP 300 Blood Level Pilot Study (Full Body Bathing). Notebook # 239; pages 38 etc. CIBA-GEIGY Corporation. May 31, 1972 120. Ciba Geigy. Irgasan DP-300 and Hexachlorophene content of plasma samples: 21 days handwashing study. Report No: 73-019 and 73-034. Ciba Geigy Corporation, Greensboro, NC, USA. October 5, 1973 121. Stierlin, H. GP 41 353: Scouting studies to ascertain the cutaneous resorption of GP 41 353 in humans after topical application in a crme excipient. Ciba Geigy Ltd., Basel, Switzerland. November 23, 1972 122. Thompson, T.A. TriclosanResults to date of multiple scrub studies with GP 41353 Surgical Scrub. Ciba-Geigy Medical Research. December 11, 1975 123. Maibach, H.I. Percutaneous Penetration of Irgasan CH 3565 in a soap solution. Department of Dermatology, University of California Medical Center, San Francisco, CA. May 9, 1969 124. Ciba-Geigy Corporation. Irgasan DP-300 skin absorption pilot study. CS 239, pages 316. Ciba-Geigy Corporation. February 2, 1972 125. Caudal, F.; Grimault, D.; Sioufi, A. Urinary excretion (free and glucuronide) in man after topical application of CGP. 433 cream. C.R.B. R 4/1974. Ciba-Geigy Biopharmaceutical Research Center. April 30, 1974 126. Schenkel, A.; Furia, T. The absorption of CH3565 through intact human skin from soap solutions. 65-16-W221. Giegy Industrial Chemicals. December 30, 1965 127. Thompson, T.A.; Goodblatt, R.S.; Borman, C.H. Triclosan (Report 1): A method for determining triclosan in human plasma and urine, and results of a pilot handwashing study. Drug Metabolism Report 1975-17. Ciba-Geigy. September 1975 128. Thompson, T.A.; Borman, C.H.; Goodblatt, R.S. Triclosan (report 2): Concentration in human plasma and urinary excretion after dermal application of GP 41353 patient skin prep. Drug Metabolism Report 1975-18. September 1975 129. Thompson, T.A.; Goodblatt, R.S.; Borman, C.H. Triclosan (report 3): Concentration in human plasma and urinary excretion after use of GP 41353 surgical scrub. Drug Metabolism Report 1976-3. Ciba-Geigy. February 1976 130. Ciba Specialty Chemicals Inc. In vitro human skin penetration and distribution of 14Clabelled triclosan from a w/o emulsion. CSC/4e1/98. An-eX Analytical Services Ltd. October 8, 1998 131. Ciba Specialty Chemicals Inc. In vitro human skin penetration and distribution of 14Clabelled triclosan from a dishwashing liquid. CSC/4e2/98. An-eX Analytical Services Ltd. October 8, 1998 132. Ciba Specialty Chemicals Inc. In vitro human skin penetration and distribution of 14Clabelled triclosan from a deodorant formulation. CSC/4e3/98. An-eX Analytical Services Ltd. October 8, 1998 133. Ciba Specialty Chemicals Inc. In vitro human skin penetration and distribution of 14Clabelled triclosan from a soap solution. CSC/4e4/98. An-eX Analytical Services Ltd. October 8, 1998 134. Ciba Specialty Chemicals Corporation (CSCC) and Dial Corporation (Dial). A pilot study for the in vivo evaluation of the percutaneous absorption of triclosan. CIBA-03-010131. Arizona Clinical Research Center, Tucson, Arizona, USA. January 17, 2002 135. Lin, Y.-J. Triclosan Canadian Study. Hazleton Blood Report HLA Study No. 6002-773. January 24, 1989 136. Colgate-Palmolive Company. Triclosan (DP-300) dentifrice plaque clinical study (Pharmacokinetic section). Study No. 87-03. Colgate-Palmolive Technology Center. November 15, 1988

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137. BIBRA International, Woodmansterne Rd, Carshalton, Surrey, UK, SM5 4DS; The free and conjugated triclosan concentrations, clinical chemical analysis and haematology of blood samples from a human volunteer study. Beechams Project SR 396 (project no. 3.0720, report no. 720/3/88). BIBRA International, Woodmansterne Rd, Carshalton, Surrey, UK, SM5 4DS. April 1988 138. Beiswanger, B.B. and Tuohy, M.A. Analysis of blood plasma samples for free triclosan, triclosan-glucuronide, triclosan sulfate and total triclosan from subjects using a triclosan dentifrice or a dentifrice, bar soap and deodorant. OHRI Study #89-A-111. Indiana University School of Dentistry. October 17, 1990 139. Wagner, W.E.; Le Shar, A. Triclosan (Irgasan DP-3000) soap bar plateau plasma levels in man (report no.5). The Research Department, Pharmaceuticals Division, CIBA-CEIGY Corporation. April 1977 140. Ciba-Geigy. Pharmacokinetic and metabolic studies in man following oral administration of a 14C-labelled preparation (report no. B 6/1976). Ciba-Geigy Ltd., CH-Basel, 27.1.76 141. Wagner, J. Preparation GP 41 353: Binding to human serum proteins in vitro. Report No. B34/73. Ciba-Geigy Ltd., Basel, Switzerland. October 18, 1973 142. Bagley, D.M.; Lin, Y.-J. Clinical evidence for the lack of triclosan accumulation from daily use in dentifrices. Am J Dent 13:148-152, 2000 143. Barkvoll, P. and Rolla, G. Triclosan protects the skin against dermatitis caused by sodium lauryl sulphate exposure. J Clin Periodontol 21(10): 717-719. 1994 144. Skaare, A., Kjrheim, V., Barkvoll, P., Rlla, G. Skin reactions and irritation potential of four commercial toothpastes. Acta Odontol Scand 55:033-136, 1997 145. Skaare, A., Rlla, G., Barkvoll, P. The influence of triclosan, zinc or propylene glycol on oral mucosa exposed to sodium lauryl sulphate. Eur J Oral Sci 105:527-533. 1997 146. Skaare, A., Eide, G., Herlofson, B., Barkvoll, P. The effect of toothpaste containing triclosan on oral mucosal desquamation. A model study. J Clin Periodontol 23:11001103. 1996 147. Colgate Palmolive Co. P-300 Soap Bar (Primary Irritation or Sensitisation and Repeated Insult Patch Test in Humans) (Ref. number 5). Colgate Palmolive Company. 11.10.72 148. Marzulli, F.N., Maibach, H.I. Antimicrobials: Experimental contact sensitisation in man. J Soc Cosmet Chem 24:399-421. 1973 149. Broeckx, W., Blondeel, A., Dooms-Goossens, A., Achten, G. Cosmetic intolerance. Contact Dermatitis 16:189-194. 1987 150. De Groot, A.C., Weyland, J.W., Bos, J.D., Jagtman, B.A. Contact allergy to preservatives (I). Contact Dermatitis 14(2):120-122. 1986 151. Schnuch, A., Geier, J., Uter, W., Frosch, P.J. Patch testing with preservatives, antimicrobials and industrial biocides. Results from a multicentre study. British Journal of Dermatology 138:467-476. 1998 152. De Groot, A.C., Liem, D.H., Nater, J.P., van Ketel, W.G. Patch tests with fragrance materials and preservatives. Contact Dermatitis 12:87-92, 1985 153. Veronesi, S., De Padova, S.M.P., Vanni, D. and Melino, M. Contact dermatitis to triclosan. Contact Allergy 15:257, 1986 154. Perrenoud, D., Bircher, A., Hunziker, T., Suter, H., Bruckner-Tuderman, L., Stager, J, Thurlimann, W., Schmid, P., Suard, A., and Hunziker, N. Frequency of sensitisation to 13 common preservatives in Switzerland. Swiss Contact Dermatitis Research Group. Contact Dermatitis 30:276-279, 1994 155. Wahlberg, J.E. Routine patch testing with Irgasan DP 300. Contact Dermatitis 2(5):292, 1976 156. Hannuksela, M., Kousa, M., and Piril, V. Allergy to ingredients of vehicles. Contact Dermatitis 2:105-110, 1976 157. Mitchell, J.C., Adams, R.M., Glendenning, W.E., Fisher, A., Kanof, N., Larsen, W., Maibach, H.I., Rudner, E.J., Schnorr, W., Storrs, F., and Taylor, J.S. Results of standard patch tests with substances abandoned. North American Contact Dermatitis Research Group. Contact Dermatitis 8:336-337, 1982

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158. Adams, R.M.; Maibach, H.I. A five-year study of cosmetic reactions. J Am Acad Dermatol 13:1062-1069, 1985 159. Steinkjer, B. and Braathen, L.R. Contact dermatitis from triclosan (Irgasan DP 300). Contact Dermatitis 18(4)243-244, 1988 160. Wennersten, G., Thune, P, Brodthagen, H., Jansen, C., and Rystedt, I. The Scandinavian multicenter photopatch study. Preliminary results. Contact Dermatitis 10:305-309, 1984 161. Kligman, A.M. Report to Geigy Chemical Corporation on Phototoxicity and Photoallergy Study on L-2. 1969 162. Bendig, J.W.A. Surgical hand disinfection: comparison of 4% chlorhexidine detergent solution and 2% triclosan detergent solution. J. Hosp. Infect. 15:143-148, 1990 163. Ciba-Geigy Ltd. GP 41 353: Report on the Two Weeks Neurotoxicity Study Oral Administration to Albino Rats. PH 2.632. Biological Research Laboratories of the Pharmaceuticals Division of CIBA-GEIGY Ltd. Basel, Switzerland. October 2, 1973 164. Chow, A.Y.K., Hirsch, G.H., Buttar, H.S. Nephrotoxic and hepatotoxic effects of triclosan and chlorhexidine in rats. Toxicology & Applied Pharmacology 42:1-10, 1977 165. Eldridge, S. Cell proliferation in rodent liver. Final Report. Pathology Associates, Inc., 4915D Prospectus Drive, Durham, NC. January 13, 1993 166. Persohn, E. and Molitor, E. The effect of FAT 80023/Q (Irgasan DP 300) on replicative DNA synthesis in hepatocytes following dietary administration to male rats. Report No.: CB 92/28-2. Toxicology Services/Cell Biology, Ciba-Geigy Ltd, CH-Basel, 17.9.93 167. Persohn, E. FAT 80023/R. Assessment of replicative DNA synthesis in the course of a 13-week oral toxicity study in the hamster (RCC Project 356490). CB 93/47. Toxicology Services/Cell Biology, Ciba Limited, Basel, Switzerland. 19.9.1994 168. Molitor, E., Persohn, E., Thomas, H. The effect of FAT 80023/Q (Irgasan DP 300) on selected biochemical and morphological liver parameters following subchronic dietary administration to male and female mice. (Laboratory Report No. CB 91/18). CibaGeigy Limited, Toxicology Services/Cell Biology, Basel, Switzerland. May 22, 1992 169. Molitor, E., Persohn, E. The effects of FAT 80023/Q (Irgasan DP 300) on selected biochemical and morphological liver parameters following dietary administration to male rats. Laboratory Report No. CB 92/28. Ciba-Geigy Limited, Toxicology Services/Cell Biology, Basel, Switzerland. August 2, 1993 170. Thomas, H. CB 93/40. Toxicology Services/Cell Biology, CH-4002 Basel, Switzerland. September 16, 1994 (Final Report) 171. Hanioka, N., Omae, E., Nishimura, T., Jinno, H., Onodera, S., Yoda, R., Ando, M. Interaction of 2,4,4-trichloro-2-hydroxydiphenyl ether with microsomal cytochrome P450-dependent monooxygenases in rat liver. Chemosphere 33(2):265-276, 1996 172. Hanioka, N., Jinno, H., Nishimura, T., Ando, M. Effect of 2,4,4-trichloro-2hydroxydiphenyl ether on cytochrome P450 enzymes in the rat liver. Chemosphere 34(4):719-730, 1997 Additional References given in the dossier Alcorn J. and McNamara PJ. Ontogeny of hepatic and renal systemic clearance pathways in infants: Part I. Clinical Pharmacokinetics 41(12), 959-998 (2002). Ashby J., Brady A., Elcombe C.R., Elliott B.M., Ishmael J., Odum J., Tugwood J.D., Kettle S. and Purchase I.F.H. Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. Human Experimental Toxicology, 13, S1S117 (1994). Auboeuf, D., Rieusset, J., Fajas, L., Vallier, P., Frering, V., Riou, JP., Staels, B., Auwerx, J., Laville, M. and Vidal, H. Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of obese and NIDDM patients. Diabetes 46, 1319-1327 (1997). Babich H. and Babich J.P. Sodium lauryl sulfate and triclosan: in vitro cytotoxicity studies with gingival cells. Toxicology Letters 91, 189-196 (1997).

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Bartek M.J., LaBudde J.A. and Maibach H.I. Skin permeability in vivo: Comparison in rat, rabbit, pig and man. The Journal of Investigative Dermatology 58, 114-123 (1972). Bentley P., Calder I., Elcombe C., Grasso P., Stringer D. and Weigand J.-J. Hepatic peroxisome proliferation in rodents and its significance for humans. Food and Chemical Toxicology 31, 857-907 (1993). Bhargava H.N. and Leonard P.A. Triclosan: Applications and safety. American Journal of Infection Control 24, 209-218 (1996). Billoni, N., Buan, B., Gautier, B., Collin, C., Gaillard, O., Mahe, YF. and Bernard, BA. Expression of peroxisome proliferator activated receptors (PPARs) in human hair follicles and PPAR involvement in hair growth. Acta Dermato Venereol 80, 329-334 (2000). Cariello, NF., Romach, EH., Colton, HM., Ni, H., Yoon, L., Falls, JG., Casey, W., Creech, D., Anderson, SP., Benavides, GR., Hoivik, DJ., Brown R. and Miller RT. Gene expression profiling of the PPAR agonist ciprofibrate in the Cynomolgus monkey liver. Toxicological Sciences 88(1), 250264 (2005). Cattley R.C., DeLuca J., Elcombe C., Fenner-Crisp P., Lake B.G., Marsman D.S., Pastoor T.A., Popp J.A., Robinson D.E., Schwetz B., Tugwood J. and Wahli W. Do peroxisome proliferating compound pose a hepatocarcinogenic hazard to humans? Regulatory Toxicology and Pharmacology: RTP 27, 47-60 (1998). Cattley, RC. Peroxisome proliferators and receptor-mediated hepatic carcinogenesis. Toxicologic Pathology 32(2), 6-11(2004). Chevalier, S. and Roberts, RA. Perturbation of rodent hepatocyte growth control by nongenotoxic hepatocarcinogens: mechanisms and lack of relevance for human health (review). Oncology Reports 5(6), 1319-1327 (1998). Cohen, SM. Human carcinogenic risk evaluation: an alternative approach to the two-year rodent bioassay. Toxicological Sciences 80, 225-229 (2004). Cohen, SM., Meek, ME., Klaunig, KE., Patton, DE. and Fenner-Crisp, PA. Invited Review: The human relevance of information on carcinogenic modes of action: Overview. Critical Reviews in Toxicology 33(6), 581-589 (2003). Elcombe, CR., Odum, J., Foster, JR., Stone, S., Hasmall, S., Soames, AR., Kimber, I. and Ashby, J. Prediction of rodent nongenotoxic carcinogenesis: Evaluation of biochemical and tissue changes in rodents following exposure to nine nongenotoxic NTP carcinogens. Environmental Health Perspectives 110(4), 363-375 (2002). Evans, D., Aberle, J., Wendt, D., Wold, A., Beisiegel, U. and Mann, WA. A polymorphism, L162V, in the peroxisome proliferator-activated receptor (PPAR) gene is associated with lower body mass index in patients with non-insulin-dependent diabetes mellitus. Journal of Molecular Medicine 79, 198-204(2001). Flavell, DM., Pineda Torra, I., Jamshidi, Y., Evans, D., Diamond, JR., Elkeles, RS., Bujac, SR., Miller, G., Talmud, PJ., Staels, B. and Humphries, SE. Variation in the PPAR gene is associated with altered function in vitro and plasma lipid concentrations in Type II diabetic subjects. Diabetologia 43, 673-680 (2000). Gow PJ., Ghabrial H., Smallwood RA., Morgan DJ. and Ching MS. Neonatal hepatic drug elimination. Pharmacology and Toxicology 88(1), 3-15 (2001). Hanefeld, M., Kemmer, C. and Kadner, E. Relationship between morphological changes and lipid-lowering action of p-chlorphenoxyisobutyric acid (CPIB) on hepatic mitochondria and peroxisomes in man. Atherosclerosis 46(2), 239246 (1983). Hinton, RH., Mitchell, FE., Mann, A., Chescoe, D., Price, SC., Nunn, A., Grasso, P. and Bridges, JW.Effects of phthalic acid esters on the liver and thyroid. Environmental Health Perspectives 70, 195210 (1986). Hoivik, DJ., Qualls, CW., Mirabile, RC., Cariello, NF., Kimbrough, CL., Colton, HM., Anderson, SP., Santostefano, MJ., Morgan, RJO., Dahl, RR., Brown, AR., Zhao Z., Mudd Jr., PN., Oliver, WB., Brown, HR. and Miller, RT. Fibrates induce hepatic peroxisome and mitochondrial proliferation without overt evidence of cellular proliferation and oxidative stress in cynomolgus monkeys. Carcinogenesis, 25(9), 1757-69 (2004).

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Holsapple, MP., Pitot, HC., Cohen, SH., Boobis, AR., Klaunig, JE., Pastoor, TA., Dellarco, VL. and Dragan, YP.Forum: Mode of action in relevance of rodent liver tumors to human cancer risk. Toxicological Sciences 89(1), 51-56 (2006). IARC. IARC Technical Report No. 24. Peroxisome Proliferation and its Role in Carcinogenesis. WHO International Agency for Research on Cancer. IARC Press, Lyon, France (1995). ICH. Dose selection for carcinogenicity studies of pharmaceuticals. Harmonised Tripartite Guideline. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). S1C (R1). Parent Guideline (27.10.1994), and Addendum on Limit Dose (17 July 1994) incorporated November 1995. ICH. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Harmonised Tripartite Guideline. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). S2B. July 1997. ICH. Reproductive Toxicology: Detection of toxicity to reproduction for medicinal products & toxicity to male fertility. Harmonised Tripartite Guideline. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). S5(R2). November 2005. International Programme on Chemical Safety. IPCS framework for analyzing the relevance of a cancer mode of action for humans. Draft. IPCS Workshop 1-29 (2005). Klaunig, JE., Babich, MA., Baetcke, KP., Cook, JC., Corton, JC., David, RM., DeLuca, JG., Lai, DY., McKee, RH., Peters, JM., Roberts, RA. and Fenner-Crisp, PA. PPAR agonistinduced rodent tumors: modes of action and human relevance. Critical Reviews in Toxicology 33(6)655-780 (2003). Lai, DY. Rodent Carcinogenicity of peroxisome proliferators and issues on human relevance. Journal of Environmental Science and Health C22(1), 27-55 (2004). Lake, B.G. Mechanisms of hepatocarcinogenicity of peroxisome-proliferating drugs and chemicals. Annual Review of Pharmacology and Toxicology 35, 483-507 (1995). Lake, BG., Rijcken, WR., Gray, TJ., Foster, JR. and Gangolli, SD. Comparative studies of the hepatic effects of di- and mono-n-octyl phthalates, di-(2-ethylhexyl) phthalate and clofibrate in the rat. Acta Pharmacologica et Toxicologica 54(3), 167-176 (1984). Ledwith, BJ., Johnson, TE., Wagner, LK., Pauley, CJ., Manam, S, Galloway, SM. and Nichols, WW. Growth regulation by peroxisome proliferators: Opposing activities in early and late G1. Cancer Research 56, 32573264 (1996). Lee, SS., Pineau, T., Drago, J., Lee, EJ., Owens, JW., Kroetz, DL., Fernandez-Salguero, PM., Westphal, H. and Gonzalez, FJ. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Molecular Cell Biology 15(6), 30123022 (1995). Makowska, JM., Anders, C., Goldfarb, PS., Bonner, F. and Gibson GG. Characterization of the hepatic responses to the short-term administration of ciprofibrate in several rat strains. Co-induction of microsomal cytochrome P-450 IVA1 and peroxisome proliferation. Biochemical Pharmacology 40(5), 1083-1093 (1990). Mandard, S., Muller, M. and Kersten, S. Review: Peroxisome proliferators-activated receptor target genes. Cellular and Molecular Life Sciences 61, 393-416 (2004). McGuill M.W. and Rowan A.N. Biological Effects of Blood Loss: Implications for sampling volumes and techniques. ILAR News 31, 5-20 (1989). Meek, ME., Bucher, JR., Cohen, SM., Dellarco, VL., Hill, RN., Lehman-McKeeman, LD., Longfellow, DG., Pastoor, TA., Seed, J. Patton, DE. A framework of human relevance analysis of information on carcinogenic modes of action. Critical Reviews in Toxicology 33(6), 591-653 (2003). Morimura, K., Cheung, C., Ward, JM., Reddy, JR. and Gonzalez, FJ. Differential susceptibility of mice humanized for peroxisome proliferator-activated receptor to Wy-14,643induced liver tumorigenesis. Carcinogenesis 27, 1074-1080 (2005). Mortensen, J.T., Brinck, P. and Lichtenberg, J. The minipig in dermal toxicology. A literature review. Scandinavian Journal of Laboratory Animal Science 25, 77-84 (1998).

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Oliver, JD. and Roberts, RA. Receptor-mediated hepatocarcinogenesis: role of hepatocyte proliferation and apoptosis. Pharmacology and Toxicology 91(1), 1-7 (2002). Omiecinski, CJ., Remmel, RP. and Hosagrahara, VP. Forum: Concise review of the cytochrome P450s and their roles in toxicology. Toxicological Sciences 48, 151-156 (1999). Palmer, CNA., Hsu, MH., Griffin, KJ., Raucy, JL. and Johnson, EF. Peroxisome proliferator activated receptor- expression in human liver. Molecular Pharmacology 53, 14-22 (1998). Palut, D., Kostka, G., Wiadrowska, B. and Bankowski, R. Effect of diclofop on the activity of some drug-metabolizing enzymes in the liver of male Wistar rats. Roczniki Panstwowego Zakladu Higieny 53(1), 1-9 (2002). (Abstract in English). Priborsky J. and Muhlbachova E. Evaluation of in-vitro percutaneous absorption across human skin and in animal models. The Journal of Pharmacy and Pharmacology 4, 468472 (1990). Pugh, G. Jr., Isenberg, JS., Kamendulis, LM., Ackley, DC., Clare, LJ., Brown, R., Lington, AW., Smith, JH. and Klaunig, JE. Effects of di-isononyl phthalate, di-2-ethylhexyl phthalate, and clofibrate in cynomolgus monkeys. Toxicological Sciences 56(1), 181188 (2000). Richert L., Price S., Chesne C., Maita K. and Carmichael N. Comparison of the induction of hepatic peroxisome proliferation by the herbicide oxadiazon in vivo in rats, mice, and dogs and in vitro in rat and human hepatocytes. Toxicology and Applied Pharmacology 141, 35-43 (1996). SCCP. The SCCPs Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation. 6th Revision. Health & Consumer Protection, Directorate-General. European Commission. December 19, 2006. Shaban, Z., El-Shazly, SI., Ishizuka, M., Kimura, K., Kazusaka, A. and Fujita, S. PPAR dependant modulation of hepatic CYP1A by clofibric acid in rats. Archives of Toxicology 78(9), 496-507 (2004a). Shaban, Z., El-Shazly, SI., Abdelhady, S., Fattouh, I., Muzandu, K., Ishizuka, M., Kimura, K., Kazusaka, A. and Fujita, S. Down regulation of hepatic PPAR function by AhR ligand. Journal of Veterinary Medical Science 66(11), 1377-1386 (2004b). Shaban, Z., Soliman, M., El-Shazly, SI., El-Bohi, K., Abdelazeez, A., Kehelo, K., Kim, HS., Muzandu, K., Ishizuka, M., Kazusaka, A., and Fujita, S. AhR and PPAR: antagonistic effects on CYP2B and CYP3A, and additive inhibitory effects on CYP2C11. Xenobiotica 35(1), 51-68 (2005). Stier, H., Fahimi, HD., Van Veldhoven, PP., Mannaerts, GP., Vlkl, A. and Baumgart, E. Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): Possible involvement of the peroxisome proliferator-activated receptor (PPAR). Differentiation 64, 55-66 (1998). Tucker, MJ. and Orton, TC. Comparative toxicology of hypolipidaemic fibrates. Taylor and Francis, Britol, PA (1995). Cited In: IARC, 1995. U.S. EPA. Guidelines for carcinogen risk assessment. Risk assessment Forum. Washington, DC: U.S. Environmental Protection Agency, EPA/630/P-03/001B, March 2005. U.S. EPA. Proposed OPPTS Science Policy: PPAR-Mediated Hepatocarcinogenesis in Rodents and Relevance to Human Health Risk Assessment. U.S. Environmental Protection Agency (US EPA), Washington, Office of Prevention, Pesticides, & Toxic Substances, 5.11.2003 U.S. FDA. Integration of study results to assess concerns about human reproductive and developmental toxicities. Draft Reviewer Guidance. U.S. Food and Drug Administration Center for Drug Evaluation and Research (CDER), Rockville, Maryland. October 2001. Vohl, MC., Lepage, P., Gaudet, D., Brewer, CG., Btard, C., Perron, P., Houde, G., Cellier, C., Faith, JM., Desprs, JP., Morgan, K. and Hudson, TJ. Molecular scanning of the human PPAR gene: association of the L162V mutation with hyperapobetalipoproteinemia. Journal of Lipid Research 41, 945-952 (2000). Ward, JM., Peters, JM., Perella, CM. and Gonzalez, FJ. Receptor and non-receptor mediated organ-specific toxicity of di(2-ethylhexyl) phthalate (DEHP) in peroxisome proliferatoractivated receptor -null mice. Toxicological Pathology 26, 240-246 (1998).

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Wester R.C. and Noonan P.K. Relevance of animal models for percutaneous absorption. International Journal of Pharmaceutics 7, 99-110 (1980). Zanger, RC., Woodcroft, KJ., Kocarek, TA. and Novak, RF. Xenobiotic-enhanced expression of cytochrome P450 2E1 and 2B1/2B2 in primary cultured rat hepatocytes. Drug Metabolism and Disposition 23(7), 681-687 (1995). Zanger, RC., Woodcroft, KJ. and Novak, RF. Differential effects of ciprofibrate on renal and hepatic cytochrome P450 2E1 expression. Toxicology and Applied Pharmacology 141, 110-116 (1996) Additional references added by SCCP Allmyr M, Harden F, Toms LM, Mueller JF, McLachlan MS, Adolfsson-Erici M, Sandborgh-Englund G (2008). The influence of age and gender on triclosan concentrations in Australian human blood serum. Sci Total Environ. 393(1):162-7. AR2. Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL (2008). Urinary concentrations of triclosan in the U.S. population: 2003-2004. Environ Health Perspect. 116(3):303-7. AR3. Campbell L, Zirwas MJ. (2006). Triclosan. Dermatitis 17(4):204-7. AR4. Crofton KM, Paul KB, DeVito MJ, Hedge JM (2007). Short-term in vivo exposure to the water contaminant triclosan: evidence for disruption of thyroxine. Environmental Toxicology and Pharmacology 24: 194-197. AR5. Danish Environmantal Protection Agency (2007). Survey and risk assessment of chemical substances in deodorants. Survey of Chemical Substances in Consumer Products, No. 86 http://www2.mst.dk/Udgiv/publications/2007/978-87-7052-6258/pdf/978-87-7052-626-5.pdf AR6. EFSA (2004). Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food. The EFSA Journal 37, 1-7 AR7. SCCNFP (2004). Opinion concerning Iodopropynyl carbamate (P91). Adopted by the SCCNFP on 1.7.2004 http://ec.europa.eu/health/ph_risk/committees/sccp/documents/out288_en.pdf AR8. SCF (2000). Opinion of the Scientific Committee on Food on the 10th additional list of monomers and additives for food contact materials (adopted by the SCF on 22/6/2000) http://europa.eu.int/comm/food/fs/sc/scf/out62_en.pdf AR9. United States Environmental Protection Agency (2008). 5-Chloro-2-(2,4dichlorophenoxy)phenol (Triclosan): Risk Assessment for the Reregistration Eligibility Decision (RED) Document. Case No 2340. (Docket EPA-HQ-OPP-2007-0513; PC Code: 054901. DP Barcode: 373535) available under http://www.regulations.gov AR10. Zorilla LM, Gibson EK, Jeffay SC, Crofton KM, Setzer WR, Cooper RL, Stoker TE (2009). The effects of triclosan on puberty and thyroid hormones in male Wistar rats. Toxicological Sciences 107: 56-64. AR1.

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EUROPEAN COMMISSION HEALTH AND CONSUMERS DIRECTORATE-GENERAL Public Health and Risk Assessment Risk assessment Brussels, SANCO.C7 EXPLANATORY NOTE FOR THE MODIFICATIONS OF THE SCCS OPINION ON TRICLOSAN (ANTIMICROBIAL RESISTANCE) FOLLOWING THE PUBLIC CONSULTATION ON THE DRAFT FINAL OPINION

This note sets out the rationale for the modifications made to the opinion of the European Commission Scientific Committee on Consumer Safety (SCCS) on triclosan (antimicrobial resistance) following a public consultation conducted between 29 March 2010 and 26 May 2010. Introduction In May 2009, the European Commission requested the Scientific Committee on Consumer Safety to assess the effects of triclosan on the emergence of bacterial resistance. A SCCS Working Group comprising of a member of the SCCS, a member of the Scientific Committee on Emerging and Newly Identified Risks (SCENIHR), a member of the Scientific Committee on Health and Environmental Risks (SCHER) and an expert from academia with experience on the subject was formed. The WG produced a draft opinion which was discussed and adopted by the SCCS plenary on 23 March 2010 as a preliminary opinion suitable for public consultation. In line with its procedures for stakeholder dialogue, implemented in the Rules of Procedures of the new Scientific Committees set up by Commission Decision 2008/721/EC of 5 September 2008, the European Commission Health and Consumers Directorate General (DG SANCO) conducted a public consultation on the preliminary opinion of SCCS between 29 March and 26 May 2010. Results/participation By the deadline, DG SANCO received a total of 10 contributions. All of them were reviewed by the Working Group during its meeting on 8 June 2010 and appropriate modifications were introduced into the opinion which was then discussed and adopted as the final opinion by the SCCS at its plenary of 21 June 2010. Modifications to the opinion The opinion has been modified to take into account all submitted comments which were assessed by the Working Group to be pertinent and relevant for the subject matter and which were within

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the competences of the Scientific Committees and respected the clear separation between risk assessment and risk management that underpins the Scientific Advisory structure of the European Commission. Comments on policy, risk management, legal clarification, ethics, the precautionary principle, were not considered as, although pertinent to the subject matter, they are outside the competences of the Scientific Committees. Detailed explanations of the way the comments received were treated by the SCCS are provided below. The numbering of paragraphs and lines correspond to the sections of the final opinion adopted by the SCCS on the 21 June 2010 and published together with this document. Changes to the opinion 1. On page 16, section 5.2, a sentence reporting on the reference Bojar et al. (2009) was added as a complement of information. 2. On page 26, section 6.3, a sentence reporting on the reference Bayston et al. (2007) was added. This reference reinforces the evidence base presented in the section on by-pass metabolic blockage. 3. On page 36, with respect to the negative results from the in situ studies, the text was amended as follows: "While these results are at first sight reassuring, the differences of methodologies used to measure resistance and to analyse the data make it premature at this stage to conclude that triclosan exposure never leads to developing microbial resistance. In addition, these useful in situ studies do not provide information on expression of genes involved in resistance, maintenance of resistance and virulence genes and transfer of resistance determinants. Thus this opinion strongly recommends to perform additional in situ studies looking at these aspects and bacterial phenotypes where known concentrations of triclosan have been found in the environment." 4. On page 30, section 6.6.2, text on the reference Pycke et al was added: "Pycke et al. (2010) observed that triclosan exposure of the environmental -proteobacterium Rhodospirillum rubrum led to an increase in triclosan MIC. The extent of this increase as well as the generation of different antibiotic susceptibility profiles was triclosan-concentration dependent, indicating the expression of distinct resistance mechanisms." Triclosan resistance is rarely found in clinical strains because it is rarely looked for. 5. On page 27, section 6.3, the following text was added: "It is however interesting to note that Tabak et al. (2009) observed a synergistic action of sequential treatment of triclosan (500 g/ml) followed by ciprofloxacin (500 g/ml) against biofilm of S. enterica serovar Typhimurium. There is little information in the literature about the potentiation of activity between a biocide and an antibiotic and such a study is important and provides interesting application/effect of triclosan." 6. The reference of Cottell et al is already mentioned in the text, page 30. 7. On pages 37-38, the text of the opinion (section 12) has been modified to clarify the use of in situ data in relation to in vitro investigations. 8. On page 29, section 6.6.2, text referring to Pycke et al (2010) on the characterization of triclosan-resistant mutants was added 9. On page 26, section 6.3, text referring to Yu et al (2010) on the signature gene expression

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profile of triclosan-resistant Escherichia coli was added. 10. On page 25, section 6.3, text referring to Zhu et al (2010) on triclosan resistance of Pseudomonas aeruginosa was added. 11. On page 27, section 6.3, text referring to Tabak et al (2009) on the synergistic activity of triclosan and ciprofloxacin on biofilms of Salmonella Typhimurium was added. 12. The conclusion of the opinion has been clarified to reflect comments received suggesting additional clarity. Comments for which no changes could be made In addition to the comments received which resulted in the above changes, the following comments were received and were evaluated by the SCCS but no changes were introduced in the opinion. The main reasons for this are : 1) comments were outside the scope of the terms of reference for this opinion; 2) comments were outside the competences of the Scientific Committees (and SCCS in this case) as they concerned policy, risk management, on field use of DU ammunition and on site sampling and surveillance; 3) in the scientific judgement of the SCCS, the submitted scientific evidence and argumentation were not of sufficient quality and strength to support changes and modifications in the opinion and its conclusions. For reasons of clarity, a brief rationale underpinning its evaluation of each comment is provided for each comment. 1. Regarding the comment on stability and solubility of triclosan, the normal storage conditions are not defined in the submission. Quantitative data on the solubility of triclosan in DMSO are not available. The SCCS considers that triclosan is soluble in DMSO. This opinion is not dealing with the ecotoxicity of triclosan. 2. Regarding the comment on evidence of the potential of triclosan to induce or transmit antibacterial resistance stating that since 2006 "there do not appear to be any compelling reasons or scientific data to support different conclusions regarding the potential for triclosan to induce or transmit antibacterial resistance. In fact, there are several studies that provide support for a lack of antibacterial resistance in situ (Cole et al. 2003; Jones, 2000; Ledder et al. 2006; McBain et al. 2004; Sullivan et al. 2002)", the WG agrees. This is actually in the text of the opinion in section 5.5.2. 3. The WG agrees on the lack of standardized methods for MIC determination. These points are covered in the document. There is no recognized bacterial model for the study of biocide resistance (SCENIHR, 2009). 4. Regarding the comment on the in situ clinical and environmental studies that "the 2010 SCCS draft document should give more consideration to the in situ clinical and environmental studies of triclosan and its impact on antimicrobial resistance as recommended by Russell (2004)", the SCCS considers that the information provided by Russell (2004) is limited and largely agrees with the data and conclusion of the opinion. 5. The SCCS considers that number of in situ studies conducted is limited. In addition, the methodologies used in these studies differ, notably in the measurement of resistance and although useful, the data provided by these studies showing a lack of correlation between triclosan usage and selection of triclosan resistance are limited. In particular, they do not incorporate any genetic

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aspect of resistance. Therefore, the SCCS concluded that it is still not possible to quantify the genetic risk associated with triclosan usage (and any other biocide) and recommended in its opinion that additional studies be performed. 6. The in situ studies focused on measuring triclosan resistance and antibiotic resistance following triclosan exposure. They did not look into the expression of genes involved in resistance, maintenance of resistance and virulence genes and transfer of resistance determinants (SCENIHR 2010). The six in situ studies reported in this opinion showed no increase in bacterial resistance following exposure to triclosan. However, in these studies, not all hazards (e.g. genetic aspects) have been measured. 7. The points regarding dental plaque and gingival health and the lack of resistance found in a number of environmental isolates are already covered by the opinion. The SCCS considers that: The status of gingival health is not an indication of the lack of bacterial resistance. While the Walker study and the other studies cited were state-of-the art at the time they were performed, they did not have the modern tools (e.g. proteomic or genomic analysis) available today to investigate the complete bacterial population and the bacterial response to biocides.

8. Regarding the comment according to which triclosan-induced antibacterial resistance has not been convincingly demonstrated in the group of in situ studies discussed, the SCCS is of the following view. Only few in situ studies have been conducted. Through the use of different methodologies and analysis of data, these studies did not find a correlation between triclosan exposure and emerging resistance. This contrasts with studies performed in vitro and emphasizes the need for translational research. The development of bacterial resistance through well-defined mechanisms, notably following triclosan exposure, has been very well-described. In situ studies have only focused on MIC and cross-resistance to antibiotics and demonstrated a lack of both following triclosan exposure. These studies have however not looked at the phenotypic expression of these mechanisms, nor at the maintenance of the gene pool and transfer of resistance determinants. With this in mind, the information obtained in situ is limited, and it is premature at this point to conclude that triclosan is not of any concern. It must be emphasized that, although this opinion focuses on triclosan, the conclusion and observation drawn in this document are also valid for other biocides. 9. Regarding the comment that the statement that environmental concentrations in selected geographical regions are high enough that triggering of bacterial resistance could also occur in the environment is speculative and not supported by the studies conducted by Ledder et al. (2006) and McBain et al. (2004), the SCCS is of the following view. The McBain et al. (2004) study is an in vitro study. The Ledder et al. (2006) is an ex situ study and actually showed that bacterial microcosm exposure to triclosan did not result in widespread high level resistance, except for enteric bacteria, especially E. coli. The McBain et al. (2003) ex situ investigation actually highlighted a change in bacterial microcosm composition although the overall microcosm resistance to triclosan as measured by an increase in MIC did not change. Again, this is not unique to triclosan. Where a selective pressure exerted by a biocide is present, then alteration of a microcosm is to be expected. 10. The SCCS considers that reports investigating triclosan low level resistance in clinical Acinetobacter strains (Chen et al. 2009) should be taken seriously and further research should be

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conducted on the mechanisms and conditions leading to increase of resistance in the environment is already mentioned in the text (reference to Chen et al, page 25). 11. The SCCS considers the comment that environmental conditions favourable for induction of triclosan resistance is an interesting point and that it should be considered in future research projects as previously mentioned in the SCENIHR (2010) opinion on biocides research. 12. The SCCS agrees with the comment on limiting the use of triclosan without proven benefit for human health but also accepts however, that where evidence exists that triclosan use in beneficial in e.g. preventing disease in humans, it should be encouraged. Hence prudent use is mentioned in the conclusion of the opinion: When used appropriately, biocides, including triclosan, have an important role to play in disinfection, antisepsis and preservation. 13. Concerning the comment on the difference between the previous SCCP opinion and this one by the SCCS , the main difference is the scientific information available on proteomic and genetic aspects of triclosan resistance. 14. This opinion is based on the weight and quality of the available scientific evidence regardless of its source. On that basis, the SCCS notes that the lack of resistance reported in some in situ studies did not take into account the recent developments in genetic and proteomic methodologies. 15. Concerning the comments on the value of the comparative study by Lambert (2004) with clinical isolates of methicillin-resistant and methicillin-sensitive Staphylococcus aureus and Pseudomonas aeruginosa which showed no indications for a connection between triclosan resistance and antibiotic resistance under real-world conditions, the views of the SCCS are the following: this was a retrospective study looking at the MIC profile of various clinical isolates to a range of biocides. There were indeed no differences in susceptibility of the clinical isolates to various biocides over time. This study confirmed that antibiotic resistant bacterial isolates might not necessarily be less susceptible to a range of biocides. However, in the view of the SCCS, this study did not inform on the continuous use of biocides on antibiotic resistance in the clinical settings. This view was first formulated by Russell in 2002. 16. Concerning comments on the continued use of triclosan, due to the limited number of in situ studies of resistance induced by triclosan to date, the SCCS can only recommend the prudent use of triclosan, for example in applications where a health benefit can be demonstrated. However, the SCCS considers that conclusions from in vitro studies cannot be ignored, notably, the role of triclosan (and other biocides) in triggering resistance and in the dissemination (or lack of) resistance determinants. Hence, the SCCS appreciates that research investment from industry will be maintained to contribute to a better understanding of the potential risks associated with triclosan applications. Research in triggering mechanisms of resistance, maintenance of the gene pool and the transfer of resistance and virulence determinants, and improving the translational application of laboratory results to situations in situ are needed. In that spirit, the SCCS appreciates the comments received that product manufactures are taking approaches which limits the use of triclosan to a limited number of products with a demonstrated health benefit. 17.Comemnts on ecotoxicity were not considered as they are out of the scope of this opinion. 18. With respect to comments on the essential differences between the concepts of resistance to antibiotics and resistance to disinfectants (Cerf et al 2010), the SCCS will point to the 2009 SCENIHR opinion on the antibiotic resistance effect of biocides.

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19. Concerning the comments on the significance of the Chen et al (2009), and Stickler and Jones (2008) studies the views of the SCCS are the following: These are not in situ studies. Chen et al. 2009 observed that the majority of the A. Baumannii was susceptible to triclosan with only 3% showing reduced susceptibility (max. 16 mg/L). They further observed that all triclosan resistant isolates were also resistant to important chemotherapeutic antibiotics while the susceptible isolates showed a resistance percentage between 40% and 55%. Stickler and Jones (2008) has been cited several times in the opinion. 20. Concerning comments on the link between triclosan exposure and resistance to important antibiotics in vitro this has been made clear in the opinion. The role of biocides, including triclosan, in emerging resistance to clinically effective antibiotics and their impact in clinical settings needs to be clarified. In the views of the SCCS, this is certainly of a lesser concern than the improper use of antibiotics in clinical settings triggering resistance as mentioned in the SCENIHR 2009 opinion. 21. Concerning the comments on the use concentrations of triclosan, the information available to the SCCS for the elaboration of its opinion are from 2007. The SCCS has no information on the triclosan use patterns before 2007 and therefore is not in a position to establish a link between the environmental concentrations of triclosan and its use in cosmetic products only. 22. Concerning the comment on soil bacteria, the opinion (section 10.2) makes clear that the exposure to some biocides favours the dissemination and maintenance of genetic mobile elements. However, there is no such information available concerning triclosan. On that basis, the SCCS in it opinion (section 10.3), formulates a number of questions that need to be answered to be able to perform a risk assessment on this issue. 23. The comment that the environmental concentrations of triclosan depend upon the efficiency of WWTPs is already stated in the opinion (section 5.5, page 18) so no further revision was deemed necessary. 24. Concerning the comment on the relevance of the Beier et al. (2008) study on triclosan resistance, the SCCS is of the view that the study investigated the susceptibility profile of vancomycin-resistant Enterococcus faecium isolates from community wastewater. One third of the isolates showed MICs of up to 8 g/ml. This confirms that there was no correlation between antibiotic susceptibility profile and biocide susceptibility. In the view of the SCCS the study showed a specific mechanism of high resistance to vancomycin and is not correlated to any known mechanism of biocide resistance. 25. Concerning the comment on the possibility to predict changes in antibiotic resistance of bacteria following exposure to triclosan, the SCCS agrees with the view of the SCENIHR (2010) that, on the basis of the available evidence in the scientific literature, it is not possible at present to predict changes in the antibiotic resistance profiles of bacteria following exposure to triclosan or to any other of the biocides currently used in various applications.

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SCCP/1251/09

Scientific Committee on Consumer Safety SCCS

Opinion on triclosan

Antimicrobial Resistance

The SCCS approved this opinion at its 7th plenary of 22 June 2010 after public consultation

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SCCP/1251/09 Opinion on triclosan antimicrobial resistance

About the Scientific Committees Three independent non-food Scientific Committees provide the Commission with the scientific advice it needs when preparing policy and proposals relating to consumer safety, public health and the environment. The Committees also draw the Commission's attention to the new or emerging problems which may pose an actual or potential threat. They are: the Scientific Committee on Consumer Safety (SCCS), the Scientific Committee on Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) and are made up of external experts. In addition, the Commission relies upon the work of the European Food Safety Authority (EFSA), the European Medicines Evaluation Agency (EMEA), the European Centre for Disease prevention and Control (ECDC) and the European Chemicals Agency (ECHA). SCCS The Committee shall provide opinions on questions concerning all types of health and safety risks (notably chemical, biological, mechanical and other physical risks) of non-food consumer products (for example: cosmetic products and their ingredients, toys, textiles, clothing, personal care and household products such as detergents, etc.) and services (for example: tattooing, artificial sun tanning, etc.). Scientific Committee members Jrgen Angerer, Ulrike Bernauer, Claire Chambers, Qasim Chaudhry, Gisela Degen, Gerhard Eisenbrand, Thomas Platzek, Suresh Chandra Rastogi, Vera Rogiers, Christophe Rousselle, Tore Sanner, Kai Savolainen, Jacqueline Van Engelen, Maria Pilar Vinardell, Rosemary Waring, Ian R. White Contact European Commission Health & Consumers Directorate C: Public Health and Risk Assessment Unit C7 - Risk Assessment Office: B232 B-1049 Brussels Sanco-Sc6-Secretariat@ec.europa.eu

European Union, 2010 ISSN 1831-4767 Doi:10.2772/11162 ISBN 978-92-79-12484-6 ND-AQ-09-001-EN-N

The opinions of the Scientific Committees present the views of the independent scientists who are members of the committees. They do not necessarily reflect the views of the European Commission. The opinions are published by the European Commission in their original language only. http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm

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ACKNOWLEDGMENTS

Dr. J. Davison, Retired, Member of SCHER, FR Dr. J.-Y. Maillard, Cardiff University, UK Dr. J.-M. Pags, University of Marseille, Member of SCENIHR, FR Dr K. Pfaff, BfR, DE Dr. S.C. Rastogi, Retired, Member of SCCS, DK (Chairman)

(Rapporteur)

Keywords:

SCCS, scientific opinion, preservative, triclosan, P32, antimicrobial resistance directive 76/768/ECC, CAS 3380-34-5, EC 222-182-2

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on triclosan (antimicrobial resistance), 22 June 2010

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SCCP/1251/09 Opinion on triclosan antimicrobial resistance

Table of Content

ACKNOWLEDGMENTS...........................................................................3 ABSTRACT..........................................................................................6 EXECUTIVE SUMMARY..........................................................................6 1. 2. 3.


3.1. 3.2. 3.3. 3.4.

BACKGROUND..............................................................................8 TERMS OF REFERENCE ..................................................................8 INTRODUCTION ...........................................................................9


Scope................................................................................................................................................................. 9 Physico-chemical properties......................................................................................................................... 10 Triclosan in biocidal formulations............................................................................................................... 12 Mode of action ............................................................................................................................................... 13

4. 5.
5.1. 5.2. 5.3. 5.4. 5.4.1. 5.4.2. 5.4.3. 5.4.4. 5.4.5. 5.5. 5.5.1. 5.5.2. 5.6.

DEFINITIONS............................................................................. 13 PRODUCTION, USE AND FATE OF TRICLOSAN................................. 15


Triclosan in cosmetics ................................................................................................................................... 15 Triclosan in healthcare and medical devices............................................................................................... 16 Triclosan in household and other consumer products ............................................................................... 16 Triclosan in food and feed ............................................................................................................................ 17 Triclosan in food production.................................................................................................................... 17 Triclosan as disinfectant in food and feed production ........................................................................... 17 Triclosan as food preservative ................................................................................................................. 17 Triclosan in animal husbandry ................................................................................................................ 18 Triclosan as feed preservative.................................................................................................................. 18 Triclosan in the environment ....................................................................................................................... 18 Fate of triclosan in the environment........................................................................................................ 18 Effect of triclosan on micro-flora and toxicity of metabolites ............................................................... 21 Triclosan in the human body........................................................................................................................ 22

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6.
6.1. 6.2. 6.3. 6.4. 6.5. 6.6. 6.6.1. 6.6.2. 6.7.

MECHANISMS OF RESISTANCE TO TRICLOSAN ............................... 22


General considerations on biocide resistance in bacteria .......................................................................... 22 General considerations on the study of triclosan........................................................................................ 24 Mechanisms of bacterial resistance to triclosan ......................................................................................... 24 Mutation rates and transfer of resistance ................................................................................................... 27 Induction of resistance.................................................................................................................................. 27 Bacterial cross-resistance to triclosan and antibiotics ............................................................................... 28 General considerations ............................................................................................................................. 28 Triclosan and cross-resistance ................................................................................................................. 28 Triclosan resistance in bacteria in situ ........................................................................................................ 30

7. 8. 9.
9.1. 9.2.

TRICLOSAN BIOAVAILABILITY AND FORMULATION EFFECTS............. 31 MEASUREMENT OF RESISTANCE AND CROSS-RESISTANCE .............. 32 DATA GAPS ON SCIENTIFIC KNOWLEDGE ...................................... 33
Scientific gaps:............................................................................................................................................... 33 Technical gaps:.............................................................................................................................................. 33

10.
10.1. 10.2. 10.3. 10.4.

RISK ASSESSMENT................................................................... 33
Limitation in activity................................................................................................................................. 34 Genetic and bacterial point of view ......................................................................................................... 34 Environment point of view ....................................................................................................................... 34 Biofilm formation in specific environmental conditions ........................................................................ 35

11. 12. 13. 14. 15.

CONCLUSIONS......................................................................... 35 OPINION ................................................................................. 36 COMMENTS RECEIVED DURING THE PUBLIC CONSULTATION ......... 37 MINORITY OPINION.................................................................. 37 REFERENCES ........................................................................... 38

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ABSTRACT
Triclosan is a biocide used in many product categories, including cosmetics. The information on environmental concentrations of triclosan in the EU is limited and the bioavailability of the triclosan to bacteria in the environment is not known. Although the present mandate concerns the evaluation of a possible association between the use of triclosan in cosmetic products and the development of resistance by certain micro-organisms, the SCCS has taken into account all evidence available from all uses of triclosan to perform its assessment. A number of scientific and technical data gaps about the occurrence and understanding of the resistance profile of triclosan have been identified and should be addressed. At present, several distinct hazards have been identified: (i) the effect of triclosan on the triggering/regulation of resistance genes in bacteria (ii) the existence of defined mechanisms that can promote resistance and cross-resistance to biocides and antibiotics in bacteria, (iii) high concentrations of triclosan (compared to concentrations known to select for resistance in in vitro experiments) have been measured in certain environmental compartments and (iv) bacterial biofilms, which are widespread in the environment and are able to survive exposure to adverse environmental factors. The first two of these hazards have been identified in vitro. The presence of resistance genes in soil bacteria should be investigated further. Based on the six in situ studies and the one meta-analysis quoted in this document and recent data from in vitro investigations (proteomic and genomic analyses), it is not possible to quantify the risk associated with triclosan (including its use in cosmetics) in terms of development of antimicrobial resistance (i.e. selection for less susceptible population), genetic basis for resistance and dessemination of resistance. In view of the concentrations of triclosan reported to trigger resistance in vitro, some of the environmental concentrations found in a number of geographical distinct areas are high enough to suggest that bacterial resistance could be triggered. However, no studies have been conducted on this aspect. The applications of triclosan which contribute to those high environmental concentrations cannot be properly identified nor quantified at present and the presence of other chemicals (e.g. antibiotics, surfactants, other biocides, etc.) in the environment, which may also affect microbial populations, would preclude assessing the effects of triclosan independently. Thus, additional in situ information is needed to provide an answer on the level of risk.

EXECUTIVE SUMMARY
Triclosan is a biocide used in many product categories, including cosmetics. The information on environmental concentrations of triclosan in the EU is limited and bioavailability of the triclosan to bacteria in the environment is not known. Although the present mandate concerns the evaluation of a possible association between the use of triclosan in cosmetic products and the development of resistance by certain micro-organisms, the SCCS has taken into account all evidence available from all uses of triclosan to perform its assessment. Triclosan is the most studied biocide with respect to bacterial resistance. Such a level of information, notably on its activity against bacteria, the identification of mechanisms of microbial resistance including genomic and proteomic aspects, is commendable and should be extended to other biocides. Low concentrations of triclosan can trigger the expression of resistance and cross-resistance mechanisms in bacteria in vitro. In view of the concentrations of triclosan reported to trigger resistance in vitro, some of the environmental concentrations found in a number of 6
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geographical distinct areas are high enough to suggest that bacterial resistance could be triggered. It is however difficult to predict whether microbial resistance would be triggered in these environments. The few in situ studies performed to date did not show any bacterial resistance emerging following triclosan exposure. In addition, the presence of other chemicals (e.g. antibiotics, surfactants, other biocides, etc.) in the environment, which may also affect microbial populations, would preclude assessing the effects of triclosan independently. The emergence of resistance induced/selected by triclosan is related to the genetic control on the resistance gene(s) present on chromosomal and genetic mobile elements. This represents the origin for a hazard about selection and dissemination of cross-resistance with other anti-bacterial molecules including biocides and antibiotics. Triclosan, like any other biocide, contributes to the selection of less susceptible bacteria in a complex microcosm in vitro. The impact of such a selection is unclear, as is the fitness of the selected bacterial species following triclosan exposure. The few in situ studies investigating long-term triclosan exposure (i.e. at least 6 months) did not indicate changes in the resistance susceptibility in the predominant bacteria selected for monitoring, but the changes in the entire flora were not evaluated. Thus additional in situ information is needed to provide a definitive opinion. There are, so far, no epidemiological data linking outbreaks of antimicrobial resistant human and zoonotic pathogens to exposure to triclosan. A number of scientific and technical data gaps about the occurrence and understanding of the resistance profile of triclosan have been identified and should be addressed. In particular, where biocides, including triclosan are used intensely, monitoring for emerging resistance in the microbial flora should be conducted. A more detailed research strategy for investigating the antimicrobial resistance effect of biocides is presented in a separate opinion from the SCENIHR (2010). There is an apparent discrepancy between in situ information that suggests the absence of induction of bacterial resistance and cross-resistance triggered by triclosan, and in vitro studies describing the mechanistic and genetic aspect of triclosan-resistance in bacteria. A better translation of in vitro findings to in situ situations is needed, making full use of molecular tools and environmental conditions used in laboratory investigations. Standardized protocols and similar parameters should be applied to both in vitro and in situ investigations. Although triclosan resistance was not observed in situ, this is not sufficient to conclude that there is no risk. Information is still lacking to provide a risk assessment on the use of triclosan in cosmetic products, including the genetic aspects of resistance, changes in environmental microcosm, maintenance and transfer of virulence and resistance determinants in situ. Due to the limited number of in situ studies of resistance induced by triclosan to date, SCCS can only recommend the prudent use of triclosan, for example in applications where a health benefit can be demonstrated. However, conclusions from in vitro studies cannot be ignored, notably the role of triclosan (and other biocides) in triggering resistance and in the dissemination (horizontal or vertical transfer of) resistance determinants. Research focused on triggering mechanisms of resistance, maintenance of the gene pool and the transfer of resistance and virulence determinants, and improving the translational application of laboratory results to situations in situ are needed. Hence,the SCCS appreciates that research investment from the industry will be maintained to contribute to a better understanding of the potential risks associated with triclosan applications.

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1. BACKGROUND
Triclosan (CAS 3380-34-5) with the chemical name 5-chloro-2-(2,4-dichlorophenoxy)phenol or 2,4,4'-trichloro-2'-hydroxy-diphenyl ether has a long history of use as a preservative in cosmetic products. It is currently regulated in Annex VI, entry 25 with a maximum concentration of 0.3%. An opinion on triclosan (SCCP/1040/06) was adopted by the SCCP at the 9th plenary meeting of 10 October 2006 with the following conclusions to the request: 1. "On the basis of the available data, the SCCP is of the opinion that there is presently no evidence of clinical resistance and cross-resistance occurring from the use of triclosan in cosmetic products. Information is required on consumer exposure to triclosan from all sources, including cosmetic products. 2. For a toxicological assessment of the safe use of triclosan, the SCCP requires a dossier to be submitted in which data is provided to all relevant exposure and toxicological end-points and conforming to currently accepted standards. This should be regarded as a matter of urgency because triclosan has been identified in human milk of some European populations." The dossier provided by Industry consists of an update on the bacterial resistance issue (submission III) and of a toxicological dossier for triclosan (submission IV). Furthermore the Norwegian authority on cosmetics has earlier this year submitted a report "Risk assessment on the use of triclosan in cosmetics; Development of antimicrobial resistance in bacteria II".

2. TERMS OF REFERENCE

Does SCCS consider a continued use of triclosan as a preservative in cosmetic products as safe taking into account the new provided documentation of resistance development by certain micro-organisms and cross-resistance? In parallel, the SCCP/SCCS has been asked to assess the toxicological safety of triclosan when used as a preservative with a maximum concentration of 0.3%. This evaluation has been published as opinion SCCP/1192/08.

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3. INTRODUCTION
Triclosan is an antimicrobial agent that has been used for more than 40 years as an antiseptic, disinfectant or preservative in clinical settings, in various consumer products including cosmetics, plastic materials, toys, etc. It has a broad range of activity that encompasses many, but not all, types of Gram-positive and Gram-negative non-sporulating, bacteria, some fungi (Jones et al. 2000, Schweizer 2001), Plasmodium falciparum and Toxoplasma gondii (McLeod et al. 2001). It has also been shown to be ecotoxic, particularly to algae in aquatic environments (Tatarazako et al. 2004). Additionally, it has been shown to interfere with the cycling of nitrogen in natural systems (Fernandes et al. 2008, Waller and Kookana 2009). Triclosan is bacteriostatic at low concentrations, but higher levels are bactericidal (Suller and Russell 1999, 2000). At sublethal concentrations, it acts by inhibiting the activity of the bacterial enoyl-acyl carrier protein reductase (FabI), a critical enzyme in bacterial fatty acid biosynthesis (Heath et al. 2002, Zhang et al. 2004). At bactericidal concentrations, it is suggested to act through multiple nonspecific mechanisms including membrane damage (Gilbert and McBain 2002). There are concerns that the widespread use of a low concentration of triclosan in various applications might lead to or select for bacterial resistance to antibiotics. Antibiotic resistance has become an increasingly serious problem worldwide, and the continued use of biocides including triclosan may exacerbate this problem. The main cause of antibiotic resistance remains the use and misuse of antibiotics. During the last decade, antibiotic resistance has increased in bacterial pathogens leading to treatment failures in both human and animal infectious diseases (Harbarth and Samore 2005; for reports see: EARSS Annual Report 2005, WHO 2007). The safety of continued use of triclosan in cosmetic products has recently been assessed by the EU Scientific Committee on Consumer Products (SCCP 2009). The SCCP emphasised that this risk assessment concerns only the toxicological profile of triclosan and that before a final conclusion on the safety of triclosan in cosmetic products can be reached, the potential development of resistance to triclosan and cross-resistance by certain microorganisms must be assessed. Earlier evaluations of triclosan, on the basis of available data, EU Scientific Committees concluded that there was no convincing evidence that triclosan poses a risk to humans and environment by inducing or transmitting antibacterial resistance (SSC 2002) as well as there was no evidence of clinical resistance and cross-resistance occurring from the use of triclosan in cosmetic products (SCCP 2006). Further information was sought for an update of these evaluations. The present evaluation of triclosan is based both on the information submitted by COLIPA1 to SCCS and on research published in peer-reviewed scientific journals. It aims at determining whether the continued use of triclosan may be associated to the development of resistance in certain micro-organisms. It also aims at identifying additional research needs. 3.1. Scope Triclosan is used as a preservative in consumer products including cosmetics, where the maximum allowed concentration according to the EU Cosmetics Directive 76/768/EEC is 0.3%. The SCCP has recently performed a risk assessment of the use of triclosan in cosmetic products. Although the present mandate concerns the evaluation of a possible association between the use of triclosan in cosmetic products and the development of resistance by certain micro-organisms, the SCCS has taken into account all evidence available from all uses of triclosan to perform its assessment. This is in line with the SCCP

COLIPA: The European Association of the Cosmetics Industry

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conclusions of 2006 (SCCP/1040/06) and it is scientifically sound as 1) cosmetic uses of triclosan account for most of the total use of this biocide in the EU and 2) it is scientifically impossible at present to assess the use of triclosan in cosmetics only, without taking into account its uses in other applications. In the absence of a clear answer, research needs will be identified. The effect of triclosan on microflora in the environment on the basis of published literature will also be covered, since environmental bacteria represent a pool of antimicrobial resistance genes. Most of the information provided here relates to bacteria, since studies of the effects of triclosan on other micro-organisms are scarce. 3.2. Physico-chemical properties INCI Name: Chemical Name: Synonyms: Triclosan 2,4,4-trichloro-2-hydroxy-diphenylether Phenol, 5-chloro-2-(2,4-dichlorophenoxy)-; Ether, 2'-hydroxy-2,4,4'trichlorodiphenyl; 5-Chloro-2-(2,4-dichlorophenoxy)phenol, Trichloro-2'hydroxydiphenylether Trade Names: CAS Reg. No.: EC: Irgasan DP300, Irgasan PG60, Irgacare MP, Irgacare CF100, Irgacide LP10, ; Cloxifenolum, Irgagard B 1000, Lexol 300, Ster-Zac 3380-34-5 222-182-2

Chemical structure:

CI O

OH

CI

CI

Empirical formula: Molecular weight: Physical form:

C12H7Cl3O2 289.5 White crystalline powder

The purity of batches of triclosan used in personal care products since the 1970s is described in the Table 1 (SCCP 2009). These data were provided by COLIPA. The purity and contaminants might be different in triclosan from other sources.

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Table 1: Purity specifications for triclosan since 1970 Test Point Triclosan Active Substance1
1

Effective from 1970 99.0 100.0%

Effective from 26.09.1985 99% min

Effective from 1.1.1990 99% min

Effective from 31.12.1994 99.0-100%

Effective from 26.6.2000 97.0103.0%

Effective from 06.11.2003 97.0 103.0%

Analysis by gas chromatography.

Impurities / accompanying contaminants: See Table 2. Table 2: Impurities in triclosan Individual related compound (Gas Chromatography) Total related compounds (Gas Chromatography) 2,4 Dichlorophenol Sum of 3- and 4-Chlorophenol 2,3,7,8 Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzo-furan 2,8-Dichlorodibenzo-p-dioxin 1,3,7-Trichlorodibenzo-p-dioxin 2,8-Dichlorodibenzo-furan 2,4,8-Trichlorodibenzo-furan Ash Mercury Arsenic Antimony Lead Cadmium Nickel Copper Chromium Sum of heavy metals as lead sulfide precipitation 0.1% 0.5% 10 mg/kg 10 mg/kg <0.001 g/kg <0.001 g/kg 0.5 mg/kg 0.25 mg/kg 0.25 mg/kg 0.5 mg/kg 0.1% 1 mg/kg 2 mg/kg 10 mg/kg 10 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg 2 mg/kg 20 mg/kg

Partition coefficient: Melting point: Relative density: Vapour pressure: pKa:

Log Pow = 4.8 57 1C 1.55 0.04 g/cm3 4 x 10-6 mmHg (20C) 8.14 (20C)

Stability: Triclosan does not decompose under normal storage conditions over 9 years of storage (information from COLIPA). 11
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The solubility of triclosan is described in Table 3. Table 3: Solubility of triclosan in selected solvents and chemicals Solvent Distilled water (20C) Distilled water (50C) 1 N caustic soda 1 N sodium carbonate 1 N ammonium hydroxide Triethanolamine Acetone Ethanol 70% or 95% Isopropanol Propylene glycol Polyethylene glycol Methyl cellosolve (Union Carbide Corp.) Ethyl cellosolve (Union Carbide Corp.) Dipropylene glycol Glycerine n-Hexane Petroleum jelly (white, USP) Tween 20 (ICI America Inc.) Tween 80 (ICI America Inc.) Triton X-100 (Rohm & Haas) Olive oil Castor oil Solubility at 25C (g Triclosan/100 g solvent) 0.001 0.004 31.7 0.40 0.30 >100 >100 >100 >100 >100 >100 >100 >100 ~40 0.15 8.5 ~0.5 >100 >100 >100 ~60 ~90

3.3. Triclosan in biocidal formulations Biocidal products that contain triclosan as the main antimicrobial are usually complex formulations due to the lack of solubility of this bisphenol. Components of the formulation might affect the activity of triclosan positively (e.g. through synergism) or negatively (e.g. antagonism). There is some information on the effect of formulation components on biocide activity (Alakomi et al. 2006, Ayres et al. 1999, Denyer and Maillard 2002, Maillard 2005b), but by large this information is restricted due to proprietory restrictions, or the lack of understanding on how formulation components work in term of antimicrobial potentiation. In the scientific literature, where triclosan acitivity has been reported, there is little reference to the use of formulation. Instead triclosan is often dissolved in a solvent such as DMSO. 12
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3.4. Mode of action Chemical biocides are generally considered to have multiple target sites against microbial cells, although such interactions are concentration dependent (Russell et al. 1997; Maillard 2005a). The bisphenol triclosan is no exception. At a sub-inhibitory concentration, triclosan was found to profoundly affect bacterial growth, indicating a strong interaction with the bacterial targets, despite the high concentration exponent of triclosan (McDonnell and Russell 1999). At higher concentrations, Gomez Escalada et al. (2005a) observed that triclosan was both rapid-acting and active at all phases of population growth, although some marked differences in its lethality were observed. These observations substantiated earlier findings with Staphylococcus aureus (Regos and Hitz 1974; Suller and Russell 2000). Inhibition of key metabolic pathway and synthesis (Regos and Hitz 1974; McMurry et al. 1998b) might be part of the lethal action mechanisms of triclosan. Indeed, triclosan was found to target specifically fatty acid synthesis with the inhibition of the enzyme enoyl reductase (enoyl-acyl carrier protein reductase, FabI) (McMurry et al. 1998a). It acts as a potent irreversible inhibitor of FabI by mimicking its natural substrate (Heath et al. 1998; Levy et al. 1999) and this inhibition has been described as being slow and competitive (Heath et al. 1999). The propensity of triclosan to inhibit fatty acid synthesis in Plasmodium falciparum and Toxoplasma gondii (McLeod et al. 2001) has led to the development of a number of antimalarial and antibacterial pro-drugs based on triclosan (Mishra et al. 2008; Freundlich et al. 2009). The rapid action of triclosan at a high concentration might be indicative of membrane damage (Villalain et al. 2001) and it is clear that fatty acid synthesis targeting cannot solely explain the lethal effect of triclosan (Gomez Escalada et al. 2005b). Triclosan membranotropic effects result in destabilised structures compromising the functional integrity of cell membranes without inducing cell lysis (Villalain et al. 2001). Intercalation of triclosan into bacterial cell membranes is likely to compromise the functional integrity of those membranes, thereby accounting for some of triclosan antibacterial effects (Guilln et al. 2004). Recently, the first genome-wide transcriptional analysis of Staphylococcus aureus exposed to triclosan (0.05 M), reported that triclosan down regulated primary metabolism-related and carbohydrate transport, the cap operon which is essential for virulence, the clpB chaperone-related genes which might trigger the expression of resistant determinants, genes involved in fatty acid production and utilisation (Jang et al. 2008). A number of factors affect the antimicrobial activity of triclosan. These can be divided into intrinsic factors derived from the biocide and its application (e.g. concentration, contact time, pH) and extrinsic factors which derive from the environment during application (e.g. temperature, soiling). Understanding the complex relationship between concentration and contact time (sometimes referred to as CT concept) is crucial to ensure efficacy (Maillard 2005a). The stability of triclosan in particular environments will also influence efficacy.

4. DEFINITIONS
According to the Directive 98/8/EC of the European Parliament and Council of the 16 February 1998, biocidal products are defined as active substances and preparations containing one or more active substances, put up in the form in which they are supplied to the user, intended to destroy, render harmless, prevent the action of, or otherwise exert a controlling effect on any harmful organism by chemical or biological means. Within the scope of this mandate, the proposition is to apply the following definitions: Antimicrobial: biocide or antibiotic.

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Biocide: an active chemical molecule in a biocidal product to control the growth of or kill micro-organisms (including bacteria, fungi, protozoa and viruses). This includes disinfectants, preservatives and antiseptics. Antibiotic: an active substance of synthetic or natural origin which is used to eradicate bacterial infections in humans or animals. Antimicrobial activity: an inhibitory or lethal effect of a biocidal product or an antibiotic.

The terms employed in the context of this mandate are defined below in order to avoid confusion in the definitions used to describe the level and type of resistance reported. There are several definitions of resistance to antimicrobials biocides or/and antibiotics and several terms used to describe similar phenomena in the literature. A literal/biological definition of resistance is the capacity of bacteria to withstand the effects of a harmful chemical agent. The following definitions are based partly on those put forward by Chapman and colleagues (Chapman 1998, Chapman et al. 1998), Russell and colleagues (Hammond et al. 1987, Russell 2003) and Cloete (2003), and the recent SCENIHR opinion (2009). The practical meaning of antibiotic resistance is to describe situations where (i) a strain is not killed or inhibited by a concentration attained in vivo, (ii) a strain is not killed or inhibited by a concentration to which the majority of strains of that organism are susceptible or (iii) bacterial cells that are not killed or inhibited by a concentration acting upon the majority of cells in that culture. When non-antibiotic agents (i.e. triclosan or other biocides) are considered, the word resistance is used in a similar way where a strain is not killed or inhibited by a concentration attained in practice (the in-use concentration) and in situations (ii) and (iii) described above. These definitions reflect those given by EFSA whereby antimicrobial susceptibility or resistance is generally defined on the basis of in vitro parameters. The terms reflect the capacity of bacteria to survive exposure to a defined concentration of an antimicrobial agent, but different definitions are used depending on whether the objective of the investigation is clinical diagnostics or epidemiological surveillance (EFSA 2008) The term 'Multi-Drug Resistant (MDR) applies to a bacterium that is simultaneously resistant to a number of antibiotics belonging to different chemical classes by using various mechanisms (Depardieu et al. 2007). The term co-resistant is used to denote a strain possessing a biochemical mechanism that inhibits the activity of several antibiotics belonging to the same structural family (e.g. lactamase and -lactams). When the transfer of resistance determinants occurs, coresistance specifically refers to genetic determinants (such as integrons, transposons or plasmids) encoding for unrelated resistance mechanisms, that are transferred in a single event and expressed jointly in a new bacterial host. The term cross-resistant is used to denote a strain possessing a resistance mechanism that enables it to survive the effects of several antimicrobial molecules with mechanism(s) of action that are related or overlap. Other terms such as insusceptibility and tolerance have been used in the published literature. Insusceptibility refers to an intrinsic (innate) property of a micro-organism, such as cell layer impermeability in mycobacteria and Gram-negative bacteria. Tolerance denotes a reduced susceptibility to an antimicrobial molecule characterised by a raised minimum inhibitory concentration (MIC), or a situation in which a preservative system no longer prevents microbial growth.

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5. PRODUCTION, USE AND FATE OF TRICLOSAN


Triclosan is a broad spectrum antimicrobial used as an antiseptic, disinfectant or preservative in clinical settings, various consumer products including cosmetics, household cleaning products, plastic materials, toys, paints, etc. It is also incorporated on the surface of medical devices, plastic materials, textiles, kitchen utensils, etc., from which it might slowly leach for a long period of time during their use, to perform its biocidal action. A detailed list of products containing triclosan is provided by the US Environmental Protection Agency (EPA) (McMahon et al. 2008) and by the Environmental Working Group, a US NGO (http://www.ewg.org/node/26752). According to EU Biocide Directive 1998/8/EC, triclosan is used in product types 1 (human hygiene), 2 (private and public health area), 3 (veterinary hygiene), 7 (film preservative) and 9 (fibre, leather, rubber and polymerised materials preservative). According to the information provided by COLIPA, the quantity of triclosan used within the EU reached approximately 450 tons (as 100% active) in the year 2006. Dye et al. (2007) estimated triclosan production in the EU to be 10-1,000 tonnes per year. It is not clear whether the above information on use or production of triclosan includes the amounts of triclosan which may be imported in the EU or exported from the EU via finished products, such as medical devices, toys, textiles, etc. In the EU, about 85% of the total volume of triclosan is used in personal care products, compared to 5% for textiles and 10% for plastics and food contact materials (usage data reported by COLIPA in 2007). The Danish EPA performed a survey of the use of triclosan in Denmark for the period 20002005 (Borling et al. 2005). This survey showed that the amount of triclosan in products on the Danish market had decreased from approx. 3.9 to 1.8 tonnes (54%) in the period 20002004. Cosmetics were the largest contributor to the amount of triclosan on the Danish market (99% of the total reported amount in the survey). However, this might not be respresentative for the whole EU, as similar data for comparison is not available for the EU as a whole or for any of its Member States. 5.1. Triclosan in cosmetics Triclosan was listed in 1986 in the European Community Cosmetics Directive (76/768/EEC) for use as a preservative in cosmetic products at concentrations up to 0.3%. The recent risk assessment performed by the EU Scientific Committee on Consumer Products (SCCP) concluded that, although its use at a maximum concentration of 0.3% in toothpastes, hand soaps, body soaps/shower gels and deodorant sticks was considered safe on a toxicological point of view in individual products, the magnitude of the aggregate exposure to triclosan from all cosmetic products is not safe. Any additional use of triclosan in face powders and blemish concealers at this concentration was also considered safe, but the use of triclosan in other leave-on products (e.g. body lotions) and in mouthwashes was not considered safe for the consumer due to the resulting high exposures2. Inhalation exposure to triclosan from spray products (e.g. deodorants) was not assessed. In a Danish EPA survey (Borling et al. 2005), the highest amount of triclosan in cosmetics was found in products for dental hygiene, including toothpaste. In this group, the amount had decreased by 37% during 2000-2004. Deodorants were the group of cosmetics with the greatest decrease in amount of triclosan (79%). A recent Danish EPA survey revealed that 15% of the most commonly sold deodorants in the Danish market contained <0.3% triclosan (Rastogi et al. 2007). Triclosan being non-ionic, it can be formulated in conventional dentifrices. However, it does not bind to the oral surfaces for more than a few hours, and therefore does not deliver a sustained level of anti-plaque activity. To increase uptake and retention of triclosan by oral

SCCP opinion on triclosan COLIPA n P32, SCCP/1192/08

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surfaces for the improvement of plaque control and gingival health, triclosan/polyvinylmethyl ether maleic acid copolymer and triclosan/zinc citrate and triclosan/calcium carbonate dentifrice are used (Williams 1998, Davies et al. 2004, Brading et al. 2004, Davies 2007). 5.2. Triclosan in healthcare and medical devices Triclosan has been effectively used clinically to eradicate micro-organisms such as methicillin-resistant Staphylococcus aureus (MRSA) (Brady et al. 1990; Cookson et al. 1991; Webster et al. 1994; Zafar et al. 1995), notably with the recommendation to use 2% triclosan bath. Triclosan is employed as surgical scrubs, and it is widely used in hand washing (Boyce and Pittet 2002) and as a body wash to eradicate MRSA from carriers prior to surgery (Wilcox et al. 2003). Triclosan is used in a number of medical devices, for example ureteral stents (Knudsen et al. 2005), surgical sutures (Ford et al. 2005; Justinger et al. 2009) and might be considered to prevent graft infection (Cakmak et al. 2009). Bojar et al. (2009) did not observe a difference in colonisation between triclosan-coated sutures and regular multifilament suture, although their work concerned five bacteria and is only based on the determination of the zone of inhibition. In ureteral stents, triclosan has been shown to inhibit the growth of common bacterial uropathogens and to reduce the incidence of urinary-tract infections and, potentially, catheter encrustation (Chew et al. 2006, Cadieux et al. 2009). Wignall et al. (2008) have recently demonstrated synergistic effects of triclosan and relevant antibiotics on clinical isolates comprising seven uropathogenic species, and they support the use of the triclosan-eluting stent when necessary, along with standard antibiotic therapy in treating complicated patients. In some further developments, the use of triclosan in urinary Foley catheter was suggested since triclosan successfully inhibited the growth of Proteus mirabilis and controlled encrustation and blockage of the catheter (Stickler et al. 2003, Williams and Stickler 2008). Recently, Darouiche et al. (2009) demonstrated synergistic, broad-spectrum and durable antimicrobial activity of the catheters coated with a combination of triclosan and DispersinB, an anti-biofilm enzyme that inhibits and disperses biofilms (Kaplan et al. 2004, Itoh et al. 2005). 5.3. Triclosan in household and other consumer products The broad-spectrum antimicrobial activity of triclosan has led to its incorporation in an extended range of product formulations intended for home use such as liquid soaps, detergents, chopping boards, childrens toys, carpets and food storage containers (Bhargava and Leonard 1996, McBain et al. 2003, Yazdankhah et al. 2006, Gilbert et al. 2007). A detailed list of consumer products containing triclosan is provided by the US Environmental Protection Agency (EPA) (McMahon et al. 2008) and by the US NGOs "Environmental and "Beyond Pesticides" Working Group" (http://www.ewg.org/node/26752) (http://www.beyondpesticides.org/antibacterial/products.htm). An increasing number of clothing articles are treated with biocides. Triclosan is one of the finishing agents for the production of such textiles (Orhan et al. 2009).The fabrics finished with triclosan are treated with cross-linking agents to provide durable antibacterial properties. On the basis of the available information, 17 products from the Danish retail market were analysed for the content of some selected antibacterial compounds: triclosan, dichlorophen, Kathon 893, hexachlorophen, triclocarban and Kathon CG. Five of the products were found to contain 0.0007% - 0.0195% triclosan (Rastogi et al. 2003). Aiello et al. (2007), in the first systematic review assessing the benefit of soaps containing triclosan, evaluated 27 studies published between 1980 and 2006. One of the key findings is that soaps that contained less than 1% triclosan showed no benefit from non-antimicrobial soaps (the EU limit is 0.3%). Studies that used soap contaning > 1% triclosan showed a significant reduction in bacterial levels on hand, often after multiple applications. The apparent lack of relationship between the use of soap containing triclosan and reduction in 16
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infectious illness was difficult to ascertain in the absence of identification of the biological agents responsible for the illness symptoms. Two recent US studies (Fischler et al. 2007, Fuls et al. 2008) demonstrated that hand washing with antimicrobial soap containing triclosan (0.46%) reduced bacterial load and transfer of bacteria from hands, compared to handwashing with a non-antimicrobial soap.

5.4. Triclosan in food and feed 5.4.1. Triclosan in food production

Triclosan was evaluated by the Scientific Committee on Food (SCF 2000) and the European Food Safety Authority (EFSA 2004) for use in food contact materials and classified in SCF List 33 with a restriction of 5 mg/kg of food. The evaluation was referred to the use of triclosan as surface biocide i.e. as substance intended to inhibit the growth of bacteria on the surface but which is not intended to have an antimicrobial effect on the food itself. Potential uses beyond household articles like cutting boards, kitchen utensils and food storage containers exist (e.g. conveyor belts, machinery, work surfaces and transport containers used in food processing). However, in April 2009 the petitioner has withdrawn the application for these uses. According to a March 2010 Commission Decision4 triclosan shall not be included in the positive list of additives to Directive 2002/72/EC and cannot be used in the manufacture of plastics intended to come into contact with food. In Germany, the use of triclosan in food contact plastics is banned since September 2009. BfR supports the ban on triclosan in food contact materials (BfR Opinion N. 031/2009, 12 June 2009). Triclosan has been identified in drinking water in certain places (Stackelberg et al. 2004, Boyd et al. 2003). Kantiani et al. (2008) found methyl triclosan (12 g/L) in one of the 22 drinking water samples from Barcelona. 5.4.2. Triclosan as disinfectant in food and feed production

Triclosan is not notified in the framework of the European regulations on biocides (Directive 98/8/EC) for use as disinfectant in food and feed production. 5.4.3. Triclosan as food preservative

Triclosan is not approved as food preservative in Europe. Food preservatives are regulated by Directive 95/2/EC on food additives other than colours and sweeteners. In Annex III of this Directive on the permitted preservatives and restrictions for their use, triclosan is not listed. As a result, the use of triclosan in so-called active food contact materials and articles is not allowed. Regarding substances released from such materials in order to extend the shelf-life of food, the Regulation (EC) 1935/2004 on food contact materials refers to the authorisations applicable to their use in foods.

3 Substances for which an Acceptable Daily Intake or Tolerable Daily Intake could not be established, but where the present use could be accepted.

Commission Decision of 19 March 2010 concerning the non-inclusion of 2,4,4-trichloro-2-hydroxydiphenyl ether in the Union list of additives which may be used in the manufacture of plastic materials and articles intended to come into contact with foodstuffs under Directive 2002/72/EC (notified under document C(2010) 1613)

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5.4.4.

Triclosan in animal husbandry

Triclosan is notified in the framework of the European regulations on biocides (Directive 98/8/EC) for use in veterinary hygiene biocidal products. 5.4.5. Triclosan as feed preservative

According to Regulation (EC) 1831/2003 on additives for use in animal nutrition the use of triclosan as preservative in feed is not authorised. The substance is not listed in the corresponding Community Register of Feed Additives (2004/C 50/01). 5.5. Triclosan in the environment The widespread use of triclosan results in the discharge of this compound to wastewater. Incomplete removal of triclosan from wastewater treatment plants (WWTPs) as well as spreading the triclosan laden biosolids into soils, leads to triclosan being distributed in soils and surface waters. Triclosan has been widely detected (see Table 4) in influents, effluents and biosolids of WWTPs, in lakes, rivers and sea water in various countries in Europe (Paxeus 1996, Lindstrm et al. 2002, Adolfsson-Erici et al. 2002, Kanda et al. 2003, Bester 2003, Sabaliunas et al. 2003, Sams-Petersen et al. 2003, Xie et al. 2008, Singer et al. 2002, Tixier et al.2002, van Stee et al. 1999, Kantiani et al. 2008, Dye et al. 2007), in the USA (McAvoy et al. 2009, Coogan et al. 2007, Coogan et al. 2008, US EPA 2009, Cha and Cupples 2009, Fair et al. 2009, Halden and Paull 2005, Chalew and Halden 2009, Kumar et al. 2010), in Canada (Hua et al. 2005), in Australia (Ying and Kookana 2007, Fernandes et al. 2008), in Japan (Okumura and Nishikawa 1996) and in Hong Kong (Chau et al. 2008). 5.5.1. Fate of triclosan in the environment

Bacteria are able to survive triclosan exposure by activating specific or general genetic cascades (see 6.2.4). The environmental concentrations of triclosan may affect bacterial activities. Consequently it is important to evaluate the fate of triclosan in the environment such as in WWTPs, rivers, effluents, etc. Triclosan is transported through the domestic waste stream to WWTPs. Municipal wastewater treatment helps to achieve average removal efficiencies in the range of 5899%, depending on the technical capabilities of sewage treatment systems (McAvoy et al. 2002, Kanda et al. 2003, Bester 2003, Singer et al. 2002, Federle et al. 2002, Lishman et al. 2006, Lindstrm et al. 2002, Lopez-Avila and Hites 1980, Thomson et al. 2005, Ternes et al. 2004). However, mass balance studies have demonstrated that triclosan also exhibits significant persistence, partitioning and sequestration in biosolids (by-product of wastewater treatment). Approximately 50 19% of the incoming mass of triclosan was observed to persist and become sequestered in biosolids produced by a conventional WWTPs employing activated sludge treatment in conjunction with anaerobic biosolid digestion (Heidler and Halden 2007). Thus, important pathways of biocide release into the environment include WWTP effluent discharge into surface waters and the land application of biosolids. Effluent from WWTPs contains a complex mixture of anthropogenic and natural compounds. Soil samples from ten agricultural sites in Michigan previously amended with biosolids, collected over two years, revealed triclosan concentration 0.16-1.02 g/kg (Cha and Cupples 2009). 90 to 7060 g/kg triclosan was found in biosolids from 3 Michigan wastewater treatment plants. Triclosan, along with many other compounds, may have multiplicative or synergistic effects on micro-organisms including bacteria. Environmental concentrations of triclosan reported in the published literature are described in Table 4. 18
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Table 4: Environmental concentrations of triclosan (data from worldwide sources) Environmental matrix Surface water Lake/river/streams with known input of raw wastewater Triclosan concentration 1.4 ng/L-40000 ng/L Reference Kolpin et al. 2002, Lindstrm et al. 2002, Lopez-Avila and Hites 1980, Singer et al. 2002, Remberger et al. 2002, Kolpin et al. 2004, Bendz et al. 2005, Glassmeyer et al. 2005, Zhang et al. 2007, Halden and Paull 2005, Chau et al. 2008, Coogan et al. 2007, Coogan and La Point 2008

Wastewater Influent 20-86161 ng/L Lindstrm et al. 2002, Sams-Petersen et al. 2003, Singer et al. 2002, Remberger et al. 2006, McAvoy et al. 2002, 2009, Halden and Paull 2005, Lishman et al. 2006, Waltman et al. 2006, Heidler and Halden 2007, Kantiani et al. 2008, Fair et al. 2009, Kumar et al. 2009 Lindstrm et al. 2002, Sams-Petersen et al. 2003, Bester 2003; Kanda et al. 2003; Sabaliunas et al. 2003, Bendz et al. 2005, Halden and Paull 2005, Thompson et al. 2005, Ying and Kookana (2007), Fair et al. 2009, Kumar et al. 2009 Xie et al. 2008, Okumura and Nishikawa 1996, Fair et al. 2009

Effluent

23-5370 ng/L

Sea water Sediment Lake/River/other surface water Marine Biosolid from WWTP

<0.001-100 ng/L

<100-53000 g/kg d.w. 0.02-35 g/kg d.w. 20-133000 g/kg d.w.

Fjeld et al. 2004, Remberger et al. 2002, Singer et al. 2002; Morales et al. 2005; Miller et al. 2008
Okumura and Nishikawa 1996, Fjeld et al. 2004

Svensson. 2002; Remberger et al. 2002, 2006; Bester 2003; Morales et al. 2005; Kinney et al. 2006; Chu and Metcalfe 2007, US
EPA 2009, Cha and Cupples 2009, Ying and Kookana 2007

Activated/digested sludge Pore water

580-15600 g/kg d.w. 0.201-328.8g/L (calculated)

McAvoy et al. 2002, 2009, Singer et al. 2002, Chu and Metcalfe 2007, Kumar et al. 2010 Chalew and Halden 2009

d.w.: dry weight

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Photodegradation of Triclosan Despite its high chemical stability, being extremely resistant to high and low pH, triclosan is readily degraded in the environment via photodegradation. Eight photoproducts were tentatively identified, including chlorinated phenols, chlorohydroxydiphenyl ethers, 2,7- and 2,8-dichlorodibenzo-p-dioxin, and a possible dichlorodibenzodioxin isomer or dichlorohydroxydibenzofuran (Tixier et al. 2002; Sanchez-Prado et al. 2006a, 2006b, Canosa et al. 2005; Lores et al. 2005; Aranami and Readman 2007, Prada et al. 2004, Latch et al. 2005, Ingerslev et al. 2003). Some of these products show enhanced toxicity compared to triclosan but have been shown to be degraded in the environment by bacteria such as Pseudomonas, Burkholderia and Sphingomonas (Field et al. 2008a and 2008b). The end products are CO2 and chlorine with chlorocatechols as intermediates. Recently, Son et al. (2009) demonstrated that TiO2-photocatalytic degradation of triclosan is mainly achieved by radicals, and these radicals can further degrade dioxin-type intermediates once they are produced in photocatalysis. The presence of hydrogen peroxide enhanced the oxidation (Yu et al. 2006). Triclosan is hydrolytically stable under abiotic and buffered conditions over the pH 4-9 range based on data from a preliminary test at 50C. Photolytically, triclosan degrades rapidly under continuous irradiation from artificial light at 25C in a pH 7 aqueous solution, with a calculated aqueous photolytic half-life of 41 minutes. One major transformation product was identified, 2,4-dichlorophenol, which was a maximum of 93.8-96.6% of the applied triclosan 240 minutes after treatment. Hydrolysis is not expected to be an important environmental fate process due to the stability of triclosan in the presence of strong acids and bases. However, triclosan is susceptible to degradation via aqueous photolysis, with a half-life of <1 hour under abiotic conditions, and up to 10 days in lake water. An atmospheric half-life of 8 hours has also been estimated based on the reaction of triclosan with photochemically produced hydroxyl radicals. Additionally, triclosan may be susceptible to biodegradation based on the presence of methyl-triclosan following wastewater treatment. Degradation in chlorinated water Triclosan addition to chlorine spiked ultra-pure water or to chlorinated tap water led to the formation of two tetra- and one penta-chlorinated hydroxylated diphenyl ether, as well as 2,4-dichlorophenol. Chlorination of the phenolic ring and cleavage of the ether bond were identified as the main triclosan degradation pathways (Canosa et al. 2005). Free chlorine mediated oxidation of triclosan leads to the formation of chloroform and other chlorinated organics (Rule et al. 2005, Fiss et al. 2007). Ozone treatment Treatment with ozone during municipal sewage treatment was efficient at removal of triclosan (Suarez 2007; Wert et al. 2009; Dodd et al. 2009). The degradation products were however not identified. Biodegradation Aerobic bacterial hydrolysis plays an important role in triclosan degradation. A consortium of bacteria able to partially degrade triclosan was isolated and one consortium member was shown to be a Sphingomonas-like micro-organism (Hay et al. 2001). In a different study, two strains of Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1 were shown to utilise triclosan as sole carbon source (Meade et al. 2001). Zhao (2006) also isolated one strain of triclosan-degrading bacteria (Sphingomonas or Sphingopyxis) from activated sludge. Zhao also found that Nitrosomonas europaea, an important nitrification bacterium in wastewater treatment plants, has the ability to degrade triclosan 20
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through co-metabolism. Triclosan and its chlorinated degradation products can also be degraded by bacteria (Pseudomonas, Sphingomonas, Burkholderia) under aerobic conditions. Very little is known of the biochemistry of the biodegradation of triclosan and nothing is documented in the Minnesota biodegradation database (http://umbbd.msi.umn.edu/). There is a data gap on the degradation pathway of triclosan and its intermediary products. Under anaerobic conditions and in the dark, triclosan is quite stable. Due to its low water solubility, triclosan is readily adsorbed to particles and tends to accumulate in sediments. Digested sludge concentrations of triclosan ranged from 0.5 to 15.6 g/g (dry weight), where the lowest value was from an aerobic digestion process and the highest value was from an anaerobic digestion process. These results suggest that triclosan is readily biodegradable under aerobic conditions, but not under anaerobic conditions (McAvoy et al. 2009). The limited data available indicate that effect levels of triclosan on activated sewage sludge micro-organisms vary depending on the level of acclimation. A concentration of 2 mg/L inhibited activated sludge micro-organisms that had not been acclimated to triclosan; however, the same concentration had no effect on acclimated organisms. Laboratoryderived IC50 values range from 20-239 mg triclosan/L based on carbon dioxide (CO2) evolution and glucose utilisation. Triclosan (2 mg/L) had a slight effect on chemical oxygen demand removal under laboratory conditions, but had a major inhibitory effect on the nitrification process. Anaerobic sludge digestion was significantly inhibited at a concentration of 10 mg/L. A NOEC for sewage microbes was not available (NICNAS 2009). 5.5.2. Effect of triclosan on micro-flora and toxicity of metabolites

Inhibitory effects on micro-organisms were shown to begin at concentrations ranging from 25 to 80,000 gL for triclosan (Federle et al. 2002, Sams-Petersen et al. 2003, Sivaraman et al. 2004, Neumegen et al. 2005, Stasinakis et al. 2007, Farre et al. 2008, Stickler and Jones 2008). It should be noted that the upper range minimum inhibitory concentrations (MICs) reported are well in excess of published solubility limit for triclosan. MIC threshold values for micro-organisms are exceeded by environmental levels of triclosan in several sediments, biosolids, and activated sludge. Lawrence et al. (2009) observed a change in the structure and composition of a river biofilm microcosm following exposure to triclosan (10 g/L) over a 8-week period. Waller and Kookana (2009) studied the effect of triclosan on selected microbiological activity and biochemical parameters in Australian soil. Substrate-induced respiration and nitrification, plus activities of four enzymes relevant for carbon turnover (acid and alkali phosphatase, 3-glucosidase, and chitinase) were measured. The effect of triclosan on enzymatic activity was minimal even at a high concentration (100 mg/kg). Likewise respiration was not affected. However, the study demonstrated that triclosan at concentrations below 10 mg/kg can disturb the nitrogen cycle in some soils. McBain et al. (2003) showed that long-term exposure of domestic-drain biofilms to sublethal levels of triclosan (2-4 g/L, four times daily) did not affect bacterial viability or significantly alter antimicrobial susceptibility. This lack of effect may reflect the biofilm phenotype present in the microcosm, the presence of intrinsically tolerant bacteria and degradation of triclosan by the drain biofilm consortium. However, microbial diversity after exposure to triclosan was profoundly affected. Studies reporting on the effect of repeated exposure of triclosan against complex oral microcosms failed generally to show an increase in resistance determined either by an increase in MIC or in Minimal Bactericidal Concentration (MBC) (Sullivan et al. 2003; McBain et al. 2004). In addition, McBain et al. (2004) did not observe any cross-resistance to other biocides or to some antibiotics (tetracycline and mitrodinazole) in a number of bacterial 21
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species such as Streptococcus sanguis, Sterptococcus oralis and Prevotella nigrescens with a decreased susceptibility to triclosan resulting from exposure to the bisphenol. However, these results contrasted with those obtained with E. coli, for which repeated exposure to increasing concentrations of triclosan led to a 400-fold increase in resistance (MBC from 0.2 to 39.1 mg/L) (McBain et al. 2004). Moreover, bacteria inside biofilms resist better to biocidal agents. For example, reduced susceptibility to triclosan was observed in Salmonella (Tabak et al. 2007) and Proteus/Providencia (Stickler and Jones 2008, Williams and Stickler 2008). 5.6. Triclosan in the human body Triclosan enters the human body orally through toothpaste, mouthwashes and dental treatments. In humans, triclosan is rapidly and completely absorbed from the gastrointestinal tract, while a lower rate of absorption occurs dermally. It has been found in human blood, plasma and milk (Allmyr et al. 2006, 2008, Adolfsson-Erici et al. 2002) in Sweden and Australia. In the USA it was found in human urine (Calafat et al. 2008). A volunteer study in Sweden (Sandborgh-Englund et al. 2006) showed that the accumulated urinary excretion varied between the subjects, with 24 to 83% of the oral dose being excreted during the first 4 days after exposure.

6. MECHANISMS OF RESISTANCE TO TRICLOSAN


6.1. General considerations on biocide resistance in bacteria

Unlike antibiotic resistance, the issues relating to biocide resistance in the healthcare environment are considered to have a very low profile and priority (Cookson 2005). Despite the widespread use of disinfectants and antiseptics in healthcare settings, acquired resistance to biocides in bacteria isolated from clinical specimens or the environment is not routinely characterised. Emerging bacterial resistance to biocides has been well described in vitro, but evidence in practice is still lacking (Russell 2002b, Cookson 2005, Maillard and Denyer 2009). It is widely accepted that biocides have multiple target sites against bacteria (Denyer and Maillard 2002, Lambert 2002, Maillard 2002, Maillard 2007, Poole 2004, Stickler 2004, Gilbert and Moore 2005, Maillard 2005b)with their efficacy depending on a range of intrinsic and extrinsic factors, (Reuter 1984, 1989, 1994, EFSA 2008, SCENIHR 2009). Thus, the emergence of general bacterial resistance is likely to arise from a mechanism/process causing the decrease of the intracellular concentration of a biocide, under the threshold that is harmful to the bacterium (Tattawasart et al. 2000a, Tattawasart et al. 2000b; Braoudaki and Hilton 2005; Maillard 2005a, Maillard and Denyer 2009). Several mechanisms based on this principle (mode of action) have been described including change in cell envelope, change in permeability, efflux and degradation (Table 5). Bacteria in biofilms are also less susceptible to biocides because of a number of factors. It is likely that some of these mechanisms operate synergistically although very few studies investigating multiple bacterial mechanisms of resistance following exposure to a biocide have been performed. Bacterial resistance to biocides is not a new phenomenon and it has been reported since the 1950s (Adair et al. 1971; Russell 2002b; Chapman 2003). To date, bacterial resistance has been described for all the biocides that have been investigated. Resistance often occurs following an improper usage of the formulated biocide, leading notably to a decrease in active concentration (Sanford 1970, Prince and Ayliffe 1972, Russell 2002b).

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Table 5: Mechanisms of bacterial resistance to biocides at the cell level Mechanisms Change in cell permeability References Decrease in concentration (that reaches the target sites) Spores (layers: cortex, spore envelope) Gram-negative (outer membrane) - Lipopolysaccharides - Proteins (porins) - Fatty acid - Phospholipids Russell 1990; Russell et al. 1997; Denyer and Maillard 2002; Lambert 2002; Cloete 2003, Hawkey 2004; Champlin et al. 2005; Munton and Russell 1970; Ayres et al. 1998; McDonnell and Russell 1999; Tattawasart et al. 2000a, b; Denyer and Maillard 2002; Fraud et al. 2003; Stickler 2004; Braoudaki and Hilton 2005 Gandhi et al. 1993; Brzel and Cloete 1994; Winder et al. 2000 Jones et al. 1989; Mchin et al. 1999; Gurin-Mchin et al. 1999, 2000 Boeris et al. 2007 McNeil and Brennan 1991; Broadley et al. 1995; Russell, 1996; Russell et al. 1997; Manzoor et al. 1999; Walsh et al. 2001; Lambert, 2002 Bruinsma et al. 2006 Nikaido, 1996; Paulsen et al. 1996; Schweizer 1998, 2001; Brown et al. 1999; Putman et al. 2000; BorgesWalmsley and Walmsley, 2001; Poole, 2001, 2002a, b; Levy 2002; Chuanchuen et al. 2003; McKeegan et al. 2003; Piddock 2006

Mycobacteria mycoylarabinagalactan

Change in surface properties Efflux mechanisms

Enzymatic modification Target mutation By-pass metabolic blockage

Decrease binding and interaction between biocide and cell surfaces Surface charge Decrease intracellular concentration of a biocide - Small multidrug resistance (SMR) family (now part of the drug/metabolite transporter (DMT) superfamily) - Major facilitator superfamily (MFS) - ATP-binding cassette (ABC) family - Resistance-nodulation-division (RND) family - Multidrug and toxic compound extrusion (MATE) family Decrease intracellular and exocellular concentration of a biocide FabI mutation in Mycobacterium smegmatis Increase in pyruvate synthesis and fatty acid production via an altered metabolic pathway (expression of triclosan resistance network)

Demple 1996; Kummerle et al. 1996; Valkova et al. 2001; Cloete 2003; McMurry et al. 1999; Webber et al. 2008b

It is worth noting that some mechanisms (e.g. efflux, target protection, degradation) can be horizontally transferred to other bacteria (Poole 2002a, Quinn et al. 2006, Roberts and Mullany 2009, Yazdankhah et al. 2006; Hawkey and Jones 2009, Juhas et al. 2009). In addition, Pearce et al. (1999) showed that some biocides, at a sub-lethal concentration, may increase or decrease the frequency of gene transfer by conjugation and transduction. 23
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6.2.

General considerations on the study of triclosan

Triclosan is described as a broad spectrum biocide. However, some bacteria are intrinsically resistant to triclosan, notably P. aeruginosa (Lear et al. 2002) and triclosan is not active against bacterial endospores. This is likely due to the structure of the Gram-negative bacteria and particularly the outer membrane, preventing triclosan to penetrate through the bacterium to reach its target sites. Bacterial resistance mechanisms to triclosan have been widely studied. However, most studies have considered resistance as an increase in MIC and not necessarily as an increase in MBC. Using MICs to measure bacterial resistance is arguable, since much higher concentrations of biocides have usually been used in practice and, therefore, failing to achieve lethality because of elevated MICs is unlikely. Some studies have shown that bacterial strains showing a significant increase in MICs to some biocides, such as cationics, were nevertheless susceptible to higher (in use) concentrations of the same biocide (Thomas et al. 2005) or triclosan (Lear et al. 2006). MRSA showing a 40-fold increase in MIC to triclosan remained susceptible to 1 mg/L (Suller and Russell 1999). Concentration is central to the definition of resistance in practice (Maillard and Denyer 2009). Hence, bacterial resistance based on the determination of MIC does not reflect accurately the true resistance profile of biocides, including triclosan. Concentration is one the most important factors that will affect the activity and efficacy of a biocide (Russell and McDonnell 2000, Maillard 2005a, b 2007). Biocides with a high concentration exponent (Russell and McDonnell 2000) such as triclosan are particularly affected by dilution since a small decrease in concentration will profoundly affect efficacy. Hence, it might not be surprising that products with a low concentration of a phenolic biocide or other biocides with a high concentration exponent (e.g. alcohols) are less effective and might be prone to bacterial contamination and growth. Most laboratory studies have been performed with triclosan dissolved in a solvent such as DMSO, and in some cases alcohol, and did not investigate commercially available formulations. Differences between laboratory (in vitro) investigations and situations in practice have not been addressed to date (Maillard and Denyer 2009). Hence, emerging bacterial resistance to triclosan investigated in vitro conditions might not necessarily reflect such development of resistance in situ. Components of the formulations might have a potentiation effect (or not) on the activity of triclosan, and their role on emerging bacterial resistance to triclosan has not been studied.

6.3.

Mechanisms of bacterial resistance to triclosan

Bacterial resistance against triclosan involves both intrinsic and acquired mechanisms (Yazdankhah et al. 2006), and include: mechanical barrier (altering intracellular concentration), change in target site (mutation of the target site) (Heath et al. 1998), efflux, and by-pass of metabolic pathway (Webber et al. 2008b). These mechanisms have been also described to confer antibiotic resistance (Davin-Regli et al. 2008). Change in enoyl acyl carrier reductase At sub-lethal concentrations, triclosan has been shown to affect specific bacterial targets. Triclosan interacts specifically with an enoyl-acyl reductase carrier protein (ENR) at a low concentration (Heath et al. 1999; Levy et al. 1999, Roujeinikova et al. 1999, Stewart et al. 1999). Triclosan was found to inhibit fatty acid synthesis by targeting FabI in E. coli (Heath et al. 1998) and S. aureus (Heath et al. 2000), and InhA in M. smegmatis (McMurry et al. 1999) and M. tuberculosis (Parikh et al. 2000). 24
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Triclosan resistant mutations in fabI decrease the interaction of triclosan with the ENR-NAD+ binding. Mutation in fabI in E. coli was shown to confer a 60-fold decreased susceptibility to triclosan (Heath et al. 1998). Mutation in fabI has led to an increase in triclosan MIC in a number of bacterial genera (McMurry et al. 1998a, Parikh et al. 2000, Health et al. 2000, Slater-Radosti et al. 2001, Massengo-Tiass and Cronan 2008, Webber et al. 2008b). In Acinetobacter baumannii high-level triclosan resistance could be explained by a Gly95Ser mutation of FabI, whilst wild-type fabI was observed to be overexpressed in low-level resistant isolates (Chen et al. 2009). Likewise in Ps. aeruginosa, high-level resistance to triclosan has been shown to be associated with FabV (Zhu et al. 2010). McMurry et al. (1998b) postulated that mutations at mar and sox in E. coli only conferred a 2-fold increase in resistance presumably by a modest overexpression of AcrAB. This expression is unlikely to decrease the efficacy of triclosan. However such a mutation, together with mutations at other loci such as fabI (increasing resistance to 90-140-fold) could be more significant Efflux of antimicrobials Triclosan is a substrate of AcrAB efflux pump in E. coli, of MexAB-OprM and MexCD-OprJ, MexEF-OprN, MexJK-OprH multidrug efflux pumps in P. aeruginosa, of AcrB in S. enterica serovar Typhimurium, and CmeB in Campylobacter spp. (Piddock 1996; McMurry et al. 1998; Chuanchuen et al. 2001, 2002, 2003; Schweizer 1998). These efflux pumps are similar to other efflux pumps in other Gram-negative pathogens (Piddock 2006) and as such, it is likely that triclosan is a substrate of such pumps in other Gram-negative bacteria. In S. enterica serovar Typhimurium, active efflux via AcrAB-TolC conferred decreased susceptibility to triclosan. The triclosan resistant mutants (MIC 32 mg/L) did not lose any fitness when compared to wild-type strains (Webber et al. 2008a). The pump efflux system of P. aeruginosa has been shown to confer a high level of intrinsic triclosan resistance (Mima et al. 2007). In addition, mutants of E. coli, and S. enterica which overexpress the AcrAB TolC efflux system, have decreased susceptibility to various agents, including triclosan, demonstrating that triclosan is a substrate for efflux pumps (Webber et al. 2008a). As previously reported for antibiotics, the presence of active efflux pumps is required for the acquisition of target mutations, which in turn increase the level of resistance (Webber et al. 2008b). In Acinetobacter baumannii, although active efflux did not appear to be a major reason for triclosan resistance, the acquisition of resistance appeared to be dependent on a background of intrinsic triclosan efflux (Chen et al. 2009). By-pass of metabolic blockage The proteomic analysis of S. enterica serovar Typhimurium triclosan-resistant mutants showed a set of proteins with commonly altered expression in all resistant strains. This triclosan resistance network included 9 proteins involved in production of pyruvate or fatty acid and represents a mechanism to increase fatty acid synthesis by an alternative pathway (Webber et al. 2008b). In addition to the expression of this network, these mutants showed specific patterns of protein expression leading to the conclusion that triclosan resistance was multifactorial and potentially involved a number of mechanisms acting synergistically to attain high-level resistance (32 mg/L) (Webber et al. 2008b). In S. aureus, a modification of the membrane lipid composition associated with the alteration of the expression of various genes involved in the fatty acid metabolism were observed in triclosan resistant strains (Tkachenko et al. 2007). Seaman et al. (2007) studied the appearance of small colony variants in MRSA following exposure to triclosan in vitro. The small colony variants displayed reduced susceptibility (23-60 fold; 1.5-4 mg/L from 0.063 mg/L) to triclosan and resistance to penicillin and gentamicin. Bayston et al. (2009) noted that prolonged exposure (i.e. 72 h) to triclosan25
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impregnated silicone resulted in the induction of small colony variants and a 67-fold increase in triclosan MIC. Recent evidence highlighted that bacterial swarming motility might confer some resistance to triclosan (5 mg/mL in B. subtilis and 0.1 mg/mL in E.coli) when compared to no swarming bacteria. The mechanism(s) by which swarming might confer some resistance is unknown, but is unlikely to be caused by efflux (Lai et al. 2009). Involvement of multiple mechanisms At bactericidal concentrations, triclosan seems to act against multiple and various targets, causing disruption of the bacterial control of cell wall permeability (Villalain et al. 2001; Guilln et al. 2004). One study in particular, investigated the role of both the permeability barrier and efflux in increase resistance to triclosan in E. coli. The MIC of triclosan-resistant E. coli mutants (MIC >1000 mg/L) was reduced to 10-25 mg/L when treated with both ethylene diamine tetra-acetic acid (EDTA; a chelating agent enhancing outer membrane permeability) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; a proton motive force uncoupler), as compared to a MIC of 0.1 mg/L in sensitive E. coli strain, indicating that potentially both permeability and efflux worked together to provide the high level resistance to triclosan. However, neither CCCP nor EDTA reduced the susceptibility of P. aeruginosa to triclosan (Gomez Escalada 2003). In Acinetobacter baumannii, triclosan-resistant isolates were characterized by antibiotic susceptibility, clonal relatedness, fabI mutation, fabI expression, and efflux pump expression (Chen et al. 2009). Yu et al. (2010) described a multiple mechanism response in E. coli following exposure to triclosan. The involvement of a number of mechanisms was shown to confer triclosan resistance up to 80 mg/L. Bacterial biofilms Generally, bacteria are attached to surfaces and associated in a community (termed biofilm) and are rarely present as single cells (planktonic). Bacterial biofilms have been shown to be highly resistant to antimicrobials compared to planktonic cultures. A biofilm-associated phenotype has been described (Brown and Gilbert 1993, Ashby et al. 1994, Das et al. 1998; Gilbert et al. 2003). The mechanisms of resistance involved in a bacterial biofilm include decreased metabolism, quiescence, reduced penetration due to the extracellular polymeric matrix (Pan et al. 2006), enzymatic inactivation of biocides (Sondossi et al. 1985) Giwercman et al. 1991, Huang et al. 1995), and the induction of multi-drug resistant operons and efflux pumps (Maira-Litran et al. 2000). Bacterial biofilm resistance to triclosan has been poorly studied. One study reported that the tolerance to triclosan of Salmonella in biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance both to triclosan and to other antimicrobials (Tabak et al. 2007). McBain et al. (2003) investigated the fate of a complex bacterial biofilm exposed to sublethal concentrations of triclosan (24 g/L) over a 3 month period. The authors identified a change in the composition of the biofilm and an increase in resistance of the complex population as measured by MIC. Interestingly, the composition of the biofilm changed, with a decrease of species diversity. The triclosan tolerant species such as Pseudomonads and Stenotrophomonads were still present, but other triclosan tolerant species (Achromobacter xylosoxidans) demonstrated a clonal expansion. Most importantly, the authors noted that the antibiotic susceptibility profile was not affected. A study investigating the effect of triclosan in the development of bacterial biofilms on urinary catheters highlighted the selectivity of triclosan. While triclosan inhibited P. mirabilis, it had little effect on other common bacterial pathogens (Jones et al. 2006). In addition, the control of P. mirabilis by triclosan resulted in emerging triclosan-resistant strains in vitro. While most of these strains were still susceptible to the triclosan concentration used in the urinary catheter, one strain (MIC = 40 mg/L) was not (Stickler and Jones 2008). Smith and Hunter (2008) showed that recommended concentrations of 26
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three biocidal products used in healthcare (one containing benzalkonium chloride 10 g/L, one containing chlorhexidine gluconate 40 g/L and one containing triclosan 10 g/L), were ineffective in eliminating hospital-acquired MRSA or P. aeruginosa biofilms, highlighting differences in susceptibility between planktonic and biofilm bacteria. It is however interesting to note that Tabak et al. (2009) observed a synergistic action of sequential treatment of triclosan (500 mg/L) followed by ciprofloxacin (500 mg/L) against biofilm of S. enterica serovar Typhimurium. There is little information in the literature about the potentiation of activity between a biocide and an antibiotic and such a study is important and describes an interesting application/effect of triclosan.

6.4.

Mutation rates and transfer of resistance

The development of bacterial resistance through acquired mechanisms such as mutation and the acquisition of resistant determinants are of concern since a bacterium that was previously susceptible can become insusceptible to a compound or a group of compounds (Russell 2002a). In S. enterica serovar Typhimurium, mutation frequency following exposure to triclosan was low (5 x 10-9), lower than mutation frequency observed following antibiotic exposure (Biroov and Mikulov 2009). Cookson et al. (1991) isolated MRSA strains exhibiting triclosan resistance (2-4 mg/L) from patients using mupirocin and triclosan baths. Although in this study the resistance was shown to be transferable in association with the plasmid encoding for mupirocin resistance, this could not be confirmed subsequently by other studies. The transfer of a plasmid encoding for mupirocin resistance to a triclosan sensitive S. aureus strain failed to increase resistance to triclosan (Suller and Russell 2000). Other studies investigating clinical S. aureus isolates resistant to mupirocin also failed to observe this association (Bamber and Neal 1999). Although various genetic mobile elements have been described to be involved in the dissemination of cross-resistance towards biocides-antibiotics (Roberts and Mullany 2009, Schlter et al. 2007) no specific genetic mobile element has been associated with triclosan resistance.

6.5.

Induction of resistance

There are two types of induction. The first corresponds to the trigger of genes governing the genetic cascade (global regulation) which promotes the expression of efflux pumps and/or down regulates membrane permeability (porin synthesis). The second corresponds to the direct activation of the promoter region (local regulation) for example controlling efflux genes (Davin-Regli et al. 2008). The induction of bacterial resistance mechanisms following exposure to a low concentration of a biocide has been reported in a number of studies for a number of biocides (SCENIHR 2009). In some occasions, a specific mechanism has not been established and a phenotypic change leading to the emergence of resistance to several unrelated compounds in vitro has been reported following exposure to a low concentration of a biocide (Moken et al. 1997). It is possible that triclosan induces a stress response followed by, or in addition to, the expression of mechanisms that reduce the deleterious effect of the biocide (McMurry et al. 1998b; Gilbert et al. 2002). A decrease in growth rates in E. coli and P. aeruginosa has been described following exposure to sub-lethal concentrations of triclosan, which indicates the generation of a stress to the organism (Gomez Escalada et al. 2005). Triclosan induces bacterial resistance through the over-expression of efflux pumps via activation of mar and ram (Randall et al. 2007; Webber et al. 2008a; Bailey et al. 2009), over-expression and mutagenesis of fab1, expression of regulatory genes involved in the control of antibiotic resistance cascades (activator of drug efflux, decrease of membrane 27
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permeability) and fatty acid metabolism in a number of bacterial genera (Jang et al. 2008, Webber et al. 2008b, Bailey et al. 2009). These genes are involved in resistance to triclosan, but also in possible cross-resistance and multi-resistance to different antibiotic and biocide classes. In Stenotrophomonas maltophilia, the overexpression of an efflux pump (SmeDEF), involved in antibiotic resistance, was demonstrated in several triclosan-selected mutants (Snchez et al. 2005). In E. coli, overexpression of acrAB or marA or soxS (positive regulator of acrAB) decreased susceptibility to triclosan 2-fold. Deletion of the acrAB locus increased susceptibility to triclosan approximately 10-fold. It was observed that clinical isolates overexpressing acrAB showed enhanced resistance to triclosan. A clinical strain overexpressing marA had a triclosan MIC of 0.27mg/L as compared to susceptible strain with an MIC of 0.090 mg/L. In S enterica serovar Typhimurium overexpressing AcrAB and C. jejuni overexpressing CmeB, triclosan MIC increased to 32 mg/L (Pumbwe et al. 2005; Buckley et al. 2006). Moken et al. (1997) described the induction of the MDR phenotype in E. coli and its relevance to cross-resistance between pine oil, triclosan and multiple antibiotics. Jang et al. (2008) reported that, in S. aureus, exposure to triclosan (0.015 mg/L) resulted in down-regulation of the clpB chaperone-related genes, which might trigger the expression of resistant determinants. A recent study demonstrated that triclosan activates the expression of several groups of genes in E. coli and S. enterica (Bailey et al 2009). Transcriptome analyses (including microarray and RT-PCR experimental approaches) of bacteria exposed to triclosan (0.12 mg/L for 30 minutes) indicated an induction of the expression of various genes involved in drug efflux (e.g. acrB), in the genetic activation of resistance genes (e.g. marA), in the control of oxidative and drug response (e.g. soxS), and in the control of membrane permeability (e.g. ompR). Despite some differences in the response level observed between the two bacterial species, triclosan was shown to induce a rapid and adaptative response including the activation of several regulatory and structural genes involved in antibiotic resistance (Bailey et al. 2009). McBain et al. (2004), however, failed to demonstrate a biologically significant induction of drug resistance in a number of bacterial species exposed to sub-lethal concentrations of triclosan, suggesting that triclosan-induced drug resistance is not generally readily inducible nor is it transferred across bacterial species.

6.6. 6.6.1.

Bacterial cross-resistance to triclosan and antibiotics General considerations

The possibility that the mechanisms involved in triclosan resistance may contribute to reduced susceptibility to clinically important and structurally unrelated antimicrobials is of major concern. It is important to note that antibacterial actions from antibiotics and biocides show some similarities in their mechanisms of action, behaviour and clinical aspects (Poole 2007). Among the similarities, we can mention (i) the penetration/uptake through bacterial envelope by diffusion, (ii) the effect on the membrane integrity and morphology, (iii) the effect on diverse key steps of bacterial metabolism (replication, transcription, translation, transport, various enzymes). Faced with this chemical aggression and stress, bacteria mobilise similar defence mechanisms conferring resistance against structurally non-related molecules (Walsh and Fanning 2008).

6.6.2.

Triclosan and cross-resistance

A number of (but not all) laboratory studies have demonstrated an association between triclosan resistance and resistance to other antimicrobials. However, this link has not been 28
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confirmed in the limited number of in situ studies that have been performed to date. A number of bacterial mechanisms potentially conferring cross-resistance has been identified in laboratory investigations (see Table 6). Table 6 Bacterial mechanisms inducing potential cross-resistance Mechanism Nature Level of susceptibility to other biocides1 no reduced reduced reduced reduced Crossresistance yes yes no2 no3 yes

Change in bacterial envelope (over)Expression of efflux pumps Enzymatic modification Mutation (target site) Phenotypic change
1 2

intrinsic (acquired) intrinsic/acquired acquired/intrinsic acquired Following exposure

to other biocides - level of susceptibility defined according to the concentration of biocides in the case of acquired resistance, co-resistance has been described 3 triclosan cross-resistance with specific antibiotics (e.g. isoniazid) acting against enoyl acyl carrier proteins (e.g FabI) has been described.

Studies on S. enterica and Stenotrophomonas maltophilia described the effect of triclosan on emerging bacterial cross-resistance. In S. enterica, Karatzas et al. (2007) reported that a triclosan-resistant strain overexpressing an efflux pump was less susceptible to antibiotics than the wild type original strain. Another study described the survival of S. enterica serovar Typhimurium following exposure to various disinfectants at a low concentration on the resulting changes in antibiotic profile (Randall et al. 2007). The authors concluded that growth of Salmonella with sub-inhibitory concentrations of biocides favours the emergence of strains resistant to different classes of antibiotics. In Stenotrophomonas, Sanchez et al. (2005) analysed the effect of triclosan on the selection of mutants overexpressing the efflux pump SmeDEF involved in both intrinsic and acquired resistance to antibiotics. The authors demonstrated that triclosan was able to select 5 mutants overexpressing this pump, out of a total of 12 mutants. This overexpression conferred resistance to a number of antibiotics such as tetracycline, chloramphenicol and ciprofloxacin. Similar results have been reported with S. enterica and E. coli (Braoudaki and Hilton 2004). E. coli O157 strains, involved in the "hamburger disease", acquired high- levels of resistance to triclosan after only two sublethal exposures and when adapted, repeatedly demonstrated decreased susceptibilities to various antibiotics, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. Bailey et al. (2009) showed that triclosan triggered the expression of a number of genes (e.g. encoding for efflux pumps, porins) directly involved in antibiotic resistance, and regulatory genes involved in the control of the antibiotic resistance gene cascade (activator of drug efflux, decrease of membrane permeability). Alteration in InhA in M. smegmatis following exposure to triclosan resulted in resistance to isoniazid (McMurry et al. 1999). Likewise, exposure of M. tuberculosis to triclosan led to mutation in inhA causing cross-resistance to isoniazid. However, isoniazid-resistant mutants were still susceptible to triclosan (Parikh et al. 2000). Pycke et al. (2010) observed that triclosan exposure of the environmental proteobacterium Rhodospirillum rubrum led to an increase in triclosan MIC. The extent of this increase as well as the generation of different antibiotic susceptibility profiles was triclosan-concentration dependent, indicating the expression of distinct resistance mechanisms. However, direct linkage between triclosan usage and bacterial resistance to other biocides and antibiotics might not be universal. Cottell et al. (2009) investigated the antibiotic 29
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susceptibility of triclosan tolerant S. aureus, E. coli and Acinetobacter johnsonii and reported that these strains remain susceptible to antibiotics used in clinical settings. In addition, triclosan-tolerant E. coli were found to be significantly more susceptible to aminoglycosides (Cottell et al. 2009). Likewise, triclosan resistant mutants in S. aureus did not show an altered antibiotic susceptibility profile compared to their parent strains (Suller and Russell 2000). Lear et al. (2006) demonstrated that environmental isolates with an increased MIC to triclosan remained susceptible to other biocides and antibiotics. Biroov and Mikulov (2009) reported that continuous exposure of sub-inhibitory concentrations of triclosan did not increase emerging antibiotic resistance in S. enterica serovar Typhimurium but helped to maintain antibiotic-resistant bacteria in the population, notably those showing a mar phenotype. A short-term exposure to triclosan (30 min at 0.5 MIC, i.e. 0.098 mg/L) did not result in the selection of antibiotic resistant mutants.

6.7.

Triclosan resistance in bacteria in situ

Triclosan has been the most studied biocide with respect to its anti-bacterial activity. However, investigations concerned mainly laboratory experiments and only very few studies are available to date on bacterial resistance to triclosan in situ. Furthermore, in most in vitro studies, resistance to triclosan has been measured as an increase in MIC. As mentioned in section 6.2 above, the measurement of resistance based on MIC only, might have little bearing on bacterial survival to concentrations found in situ. Ledder et al. (2006) investigated acquired high-level triclosan resistance in a number of distinct environmental isolates and reported that a relatively small number of strains showed a decrease in triclosan susceptibility (E. coli, Klebsiella oxytoca, Aranicola proteolyticus and S. maltophilia) while the susceptibility of the remaining 35 species remained unchanged. They concluded that repeated exposures to triclosan did not systematically produce high-level triclosan resistance in all bacteria. Furthermore, among the strains with decreased susceptibility, there was no change in antibiotic susceptibility or susceptibility to other biocides. Similarly, another study by the same group on repeated exposure of dental bacteria to triclosan resulted in the same conclusions (McBain et al. 2004). Cole et al. (2003) collected 1238 isolates from the homes of users and non-users of antibacterial product and were unable to demonstrate any cross-resistance to antibiotic and antibacterial agents in target bacteria. In addition, this study showed an increased prevalence of potential pathogens in the homes of non-users of antibacterial products. However, in this study, the isolates were selected based on their antibiotic resistance and were then tested for their insusceptibility to biocides. With our current state of knowledge, it is generally accepted that antibiotic resistance in clinical isolates is not necessarily associated with resistance to biocides. Sullivan et al. (2003) studied the effect of triclosan in toothpaste on some bacterial species from the oral flora of 9 human volunteers over a 14day period. Triclosan usage contributed to a decrease in lactobacilli although this decrease had no clinical significance. Furthermore, the antibiotic susceptibility profile of the oral streptococci investigated did not change following the use of triclosan containing toothpaste. Aiello et al. (2004) did not find any statistical significance between elevated triclosan MICs and antibiotic susceptibility in bacterial isolates taken from the hands of individuals using antibacterial cleaning and hygiene products for a 1-year period. Earlier studies reported no change in the ecology of the oral flora or resistance to triclosan following the use of triclosan-containing toothpaste. Jones et al. (1987) reported no change in the predominant plaque flora in 13 volunteers following the use of triclosan (2 g/L) for seven months. The authors did not observe any increase in triclosan MIC in these bacteria. Similar conclusions were reported by Walker et al. (1994) who reported no changes in the microbial flora in 144 patients following the use of 3 g/L triclosan-containing toothpaste. A meta-analysis of 16 clinical studies of the long-term effect (at least 6 month) of using triclosan toothpaste showed reduction in dental plaques and gingivitis (Davies et al. 2004). 30
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7. TRICLOSAN BIOAVAILABILITY AND FORMULATION EFFECTS


The concentration of triclosan that comes in contact with a micro-organism governs the subsequent effect on that micro-organism (e.g. inhibitory, lethal, adaptation, selection). Hence the bioavailability of triclosan is paramount. As described in Chapter 5, triclosan present in various environmental media is susceptible to degradation by oxidation by ozone, chlorine and sunlight, and to biodegradation by microorganisms. The main route of exposure to soil is expected to be via the application of sewage sludge to agricultural soil. The bioavailability will depend on the sorption, mobility and degradation in soil under various physical conditions. Triclosan is released into surface waters via effluents from WWTP, and the bioavailability of the triclosan to micro-organisms in these media will depend upon sedimentation by binding with the particulate matter and stability of the compound during the exposure period. The US EPA (2008) states on stability of triclosan in the environment that: "Triclosan is hydrolytically stable under abiotic and buffered conditions over the pH 4-9 range based on data from a preliminary test at 50C. Photolytically, triclosan degrades rapidly under continuous irradiation from artificial light at 25C in a pH 7 aqueous solution, with a calculated aqueous photolytic half-life of 41 minutes. Triclosan degrades rapidly in aerobic soils maintained in darkness at 20 2C, with calculated half-lives of 2.9-3.8 days. In aerobic water-sediment systems maintained in darkness at 20 2C, triclosan degraded with calculated nonlinear half-lives of 1.3-1.4 days in the water, 53.7-60.3 days in the sediment, and 39.8-55.9 days in the total system. In soil, triclosan is expected to be immobile based on an estimated Koc of 9,200. Triclosan is not expected to volatilize from soil (moist or dry) or water surfaces based on an estimated Henrys Law constant of 1.5 x 10-7 atm-m3/mole. Triclosan partially exists in the dissociated form in the environment based on a pKa of 7.9, and anions do not generally adsorb more strongly to organic carbon and clay than their neutral counterparts. In aquatic environments, triclosan is expected to adsorb to suspended solids and sediments and may bioaccumulate (Kow 4.76), posing a concern for aquatic organisms. Hydrolysis is not expected to be an important environmental fate process due to the stability of triclosan in the presence of strong acids and bases. However, triclosan is susceptible to degradation via aqueous photolysis, with a half-life of <1 hour under abiotic conditions, and up to 10 days in lake water. An atmospheric half-life of 8 hours has also been estimated based on the reaction of triclosan with photochemically produced hydroxyl radicals. In the laboratory, triclosan degraded via aerobic soil metabolism and aerobic aquatic metabolism, with half-lives of <4 days in soils and half-lives of <1.5 days (water layer) and up to 60 days (sediment and total system) in water-sediment systems." Samse-Petersen et al. (2004) have described that half-life of triclosan for three experimental soils was calculated to be in the range of 17.4 to 35.2 days Some observed concentrations of triclosan in the environment (e.g. Kumar et al. 2010) are high enough to induce changes in the microbial population. However, the bioavailability of triclosan in these environments (WWTP effluents, sludges, sediments, etc.) has not been 31
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determined. It is therefore important that the concentration effects of bioavailable triclosan are measured during the exposure period under study. The presence of other chemicals (e.g. antibiotics, other biocides, surfactants) in the environment may also affect the microbial population. Therefore it may be difficult to assess the effect of triclosan alone against microbial populations in the environment. Triclosan-containing products are complex formulations since triclosan is poorly soluble in water. The role of the formulations is important to ensure the bioavailability of triclosan. Formulations might also enhance biocidal activity and/or reduce microbial aggregation, improving the biocidal activity of the product. The bioavailability of triclosan in surfaces or textiles, etc., is product dependent. Some manufacturers claim that triclosan does not leach out of their product.

8. MEASUREMENT OF RESISTANCE AND CROSS-RESISTANCE


Concentration is central to the definition of bacterial resistance in practice (McDonnell and Russell 1999, Maillard and Denyer 2009). Therefore, the measurement of bacterial lethality rather than the measurement of bacterial growth inhibition is paramount. The determination of the lethality of the in-use concentration of a biocide will indicate, by comparison to a reference strain, whether a bacterial strain is insusceptible (i.e. intrinsically resistant) or has acquired resistance to a biocide or not. The determination of minimum bactericidal concentrations (MBCs) is also an appropriate methodology that allows the comparison of lethality between a reference strain and resistant clinical/environmental isolates. Here, the reference strains represent the population of bacteria which is normally susceptible to the biocide. In addition the determination of the lethality of a biocide must involve the use of a neutralising agent or the removal of the biocide. Failure to do so will provide an over-estimation of the lethality of the biocide. The determination of bacterial growth kinetics in the presence of a low concentration of a biocide can also provide indications to a change in bacterial phenotype (Thomas et al. 2004; Gomez Escalada et al. 2005a; Maillard 2007), but it does not indicate whether bacteria will become resistant to the biocide and cross-resistant to unrelated compounds or not. Likewise, a number of protocols have been used to measure antibiotic susceptibility in bacterial isolates showing resistance, tolerance or increased insusceptibility to biocides or vice versa. The variety of protocols used contributes to the variability of the results reported on antibiotic resistance. For example, some studies based a change in antibiotic susceptibility profile on measurement of zone of inhibition (Tattawasart et al. 1999; Thomas et al. 2005). More meaningfully studies used standardised antibiotic susceptibility methodologies such as those given by the British Society for Antimicrobial Chemotherapy (BSAC) or Clinical and Laboratory Standards Institute (CLSI) to measure a change in antibiotic susceptibility profile. However a limited number of studies have looked at a decrease in antibiotic susceptibility that would be associated with treatment failure (Lear et al. 2006; Cottell et al. 2009).The effect of biocides on antibiotic susceptibility in bacteria has been measured indirectly, whereby a bacterial population is treated first with a biocide and the surviving bacteria then investigated for their susceptibility to antibiotics. However, there are currently no well-referenced criteria or standard protocols for the evaluation of the capability of a biocide to induce or select for resistance to antibiotics. Therefore, tools need to be developed to define for example the "minimal selecting concentration": the minimal concentration of a biocide which is able to select or trigger the emergence/expression of a resistance mechanism that will confer clinical resistance to an antibiotic class in a defined bacterium (SCENIHR 2009). Since cross-resistance can be conferred by a number of distinct mechanisms, it is important to evaluate the propensity of a bacterium to express these mechanisms. Advances in 32
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modern genetic methods (e.g. PCR, -omics) and the development of an assay using specific chemosensitizers or markers (e.g. efflux pumps inhibitors) might allow the development of routine tests to identify resistance mechanisms.

9. DATA GAPS ON SCIENTIFIC KNOWLEDGE


In the course of this work, several important gaps were noted. These can be divided into scientific and technical gaps: 9.1.Scientific gaps: 1. Environmental studies focussing on the identification and characterisation of resistance and cross-resistance to antibiotics following use of triclosan. 2. In vitro studies to demonstrate whether triclosan, used at sub-lethal concentrations, triggers the emergence of antibiotic resistance and/or select bacteria resistant to antibiotics. This has only been demonstrated in a limited number of bacterial genera. Further information for other genera should be obtained. 3. Despite in vitro evidence of the effect of triclosan on the emergence of antibiotic resistance and on the selection of bacteria resistant to antibiotics, epidemiological data indicating public health relevance are lacking. 4. There is no information available on the maintenance and transferability of resistance and virulence markers in the presence of triclosan. 9.2.Technical gaps: 1. Standardisation of methodologies to measure resistance and cross-resistance is needed. 2. Information on production, use volumes is required to assess the exposure of bacteria to triclosan in various matrices. 3. Data on the fate and bioavailability of triclosan in the environment are sparse. Information on environmental concentrations, contact time, microbial population present in the field and bacterial exposure, is insufficient to determine whether expression of resistance actually occurs in situ. 4. No validated methodologies are available for the determination of the dose-response relationships and of the threshold triggering the emergence of antibiotic/biocide resistance and/or the selection of resistant bacteria. 5. The role of bacterial biofilm in resistance to triclosan has been shown. Furthermore, bacterial biofilms are very common in the environment. Yet, most laboratories are not using biofilm tests to assess the efficacy of biocides (Cookson 2005). There are, currently, no European standards for the testing of disinfectants against biofilms for health care applications. A more detailed research strategy for investigating the antimicrobial resistance effect of biocides is presented in a separate opinion from the SCENIHR (2010).

10. RISK ASSESSMENT

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Triclosan is the most studied biocide with respect to antimicrobial resistance. Such a level of information, notably on its activity on bacteria, the identification of mechanisms of microbial resistance, including genomic and proteomic aspects, is commendable. However, in spite of this level of information on mechanisms, information on the interaction between triclosan and microbial cells/communities including data on exposure and bioavailability in situ is lacking. Thus, a full risk assessment of triclosan cannot be performed. However, a number of points can be made: - a hazard has been identified concerning the effect of triclosan on the regulation of resistance genes in bacteria - mechanisms which can promote resistance and cross-resistance to biocides and antibiotics in bacteria have been identified - high concentrations of triclosan (compared to concentrations known to select for resistance in in vitro experiments) have been measured in certain environmental compartments, however a link with cosmetic or other specific product uses could not be made. - bacterial biofilms are widespread in the environment and are able to survive exposure to adverse environmental factors. 10.1. Limitation in activity

Bacteria can be classified according to their intrinsic resistance to biocides. Bacterial endospores are considered to be most resistant, followed by mycobacteria, Gram-negative bacteria and Gram-positive bacteria (Maillard 2005a). Triclosan is not sporicidal. It is not bactericidal against certain bacteria such as P. aeruginosa and Burkholderia sp. (Rose et al. 2009).It might also have limited activity against certain mycobacteria as these microorganisms are considered to be less susceptible to biocides than Gram-negative bacteria. 10.2. Genetic and bacterial point of view

Recent laboratory studies indicate that, during short exposures of mid-logarithmic growth phase to MIC concentrations (30 min at 0.12 mg/L), triclosan can trigger a genetic response in Gram-negative bacteria (e.g. E. coli, S. enterica) inducing expression of genes involved in biocide and antibiotic resistance. In addition, in Listeria monocytogenes triclosan concentrations of 19 mg/L to 150 mg/L activate the expression of virulence factors (Kastbjerg et al. 2010). Concerning the genetic aspects; genetic mobile elements play an important role in bacterial resistance response since they contain resistance genes (coding for pump, enzyme, qnr factors, etc) which can confer resistance to different drug families. The gene pool encoding for various mechanisms that confer resistance to antimicrobials has been shown to be present in soil bacteria (Dantas et al. 2008). Although exposure to some biocides (such as quaternary ammonium compounds) favours the dissemination and maintenance of such genetic mobile elements in bacteria and subsequently may facilitate the exchange of key genes between bacterial species (Paulsen et al. 1998, Pearce et al. 1999, Sidhu et al. 2001 2002, Bjorland et al. 2001, Noguchi et al. 2002), this has not been reported for triclosan. 10.3. Environment point of view

Several recent studies have clearly demonstrated the widespread presence of triclosan in the environment, especially in wastewater, in wastewater treatment plant effluents, in rivers and in sediments. However, there is limited information from the EU. The reported concentrations range from less than 0.001 ng/L (seawater) to 133 mg/kg (biosolids from WWTP) (see Table 4). The following information is also necessary for the risk assessment: a) The bioavailability of triclosan in these environments, 34
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b) The microflora in contact with triclosan in these environmental compartments, c) Whether this microflora contains bacterial species in which triclosan is able to trigger a genetic response. If not, could the environmental bacteria in contact with triclosan transfer genetic elements (containing resistance genes) to "target" bacteria? Regulation cascades and corresponding resistance genes are present in the soil bacteria. These bacteria may both serve as original source/reservoir of genetic mobile elements (horizontal transfer) and as genetic manipulator (exchange between chromosomal and mobile genes) of resistance genes in the presence of a selective pressure. 10.4. Biofilm formation in specific environmental conditions

Bacterial biofilms are widespread in the environment including waters, plants, etc. They deserve a special attention because of three main characteristics: the decrease in bioavailability of antibacterial agents within the biofilm, the presence of dormant/persister bacteria, and in complex biofilms the presence of various bacterial species in close contact that facilitate exchange of genetic material.

11. CONCLUSIONS
Triclosan is the most studied biocide with respect to bacterial resistance. Such a level of information, notably on its activity against bacteria, the identification of mechanisms of microbial resistance including genomic and proteomic aspects, is commendable and should be extended to other biocides. This information allows better understanding of triclosan interactions with bacterial cells and should be applied to ensure that its use is sustainable for human health. Based on the available scientific information, it is not possible to quantify the risk of development of antimicrobial resistance induced by triclosan applications, including its use in cosmetics. However, there are environmental concentrations in a number of geographically distinct areas high enough to suggest that triggering of bacterial resistance could also occur in the environment. The applications of triclosan which contribute to those high environmental concentrations cannot be properly identified nor quantified at present. This should be taken into account when considering the current and future uses of triclosan in all applications so as to ensure that the demonstrable benefits for human health in certain applications are not compromised. Low concentrations of triclosan can trigger the expression of resistance and cross-resistance mechanisms in bacteria in vitro. Some environmental concentrations reported in a number of geographically distinct areas are high enough to give plausibility to this scenario occurring outside of the laboratory and warrant further investigation. The presence of other chemicals (e.g. antibiotics, surfactants, other biocides, etc.) in the environment, which may also affect microbial populations, would preclude assessing the effects of triclosan alone. The emergence of resistance induced/selected by triclosan is related to the genetic control on the resistance gene(s) present on chromosomal and genetic mobile elements in vitro. This represents the origin for a hazard about selection and dissemination of cross-resistance with other anti-bacterial molecules including biocides and antibiotics. Bacterial biofilms are widespread in the environment including waters, plants, etc. They deserve special attention because of three main characteristics: the decrease in bioavailability of antibacterial agents within the biofilm, the presence of dormant/persister bacteria, and in complex biofilms the presence of various bacterial species in close contact that facilitates some genetic exchange. Triclosan, like any other biocide, contributes to the selection of less susceptible bacteria in a complex microcosm in vitro. The impact of such a selection is unclear, as is the fitness of the selected bacterial species following triclosan exposure. The few in situ studies 35
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investigating long-term triclosan exposure (i.e. at least 6 months) did not indicate changes in resistance susceptibility in the predominant bacteria selected for monitoring but the changes in the entire flora were not evaluated. There is so far no epidemiological data linking outbreaks of antimicrobial resistant human and zoonotic pathogens following exposure to triclosan from cosmetics and other products. When used appropriately, biocides, including triclosan, have an important role to play in disinfection, antisepsis and preservation. Information on the expression/triggering of bacterial resistance mechanisms should be considered to (re-)assess the uses of triclosan in order to preserve its efficacy. Where biocides, including triclosan, are used intensely, monitoring for emerging resistance in the microbial flora should be conducted.

12. OPINION
Does the SCCS consider a continued use of triclosan as a preservative in cosmetic products as safe taking into account the new provided documentation of resistance development by certain micro-organisms and cross-resistance? At present, several distinct hazards have been identified: (i) the effect of triclosan on the triggering/regulation of resistance genes in bacteria (ii) the existence of mechanisms which can promote resistance and cross-resistance to biocides and antibiotics in bacteria, (iii) high concentrations of triclosan (compared to concentrations known to select for resistance in in vitro experiments) have been measured in certain environmental compartments and (iv) bacterial biofilms are widespread in the environment and are able to survive exposure to adverse environmental factors. The first two of these hazards have been identified in vitro. The presence of resistance genes in soil bacteria should be investigated further. The six in situ studies and the one meta-analysis quoted in this document have failed to demonstrate an increase in antibiotic resistance following triclosan use. While these results are at first sight reassuring, the differences of methodologies used to measure resistance and to analyse the data make it premature at this stage to conclude that triclosan exposure never leads to developing microbial resistance. These studies were state-of-the art at the time they were performed but they did not have the modern tools (e.g. proteomic or genomic analysis) available today to investigate the complete bacterial population and the bacterial response to biocides. These useful in situ studies do not provide information on expression of genes involved in resistance, maintenance of resistance and virulence genes and transfer of resistance determinants. Thus the SCCS strongly recommends performing additional in situ studies looking at these aspects and bacterial phenotypes where known concentrations of triclosan have been found in the environment. This opinion concerns the safety of triclosan in terms of microbiology, i.e. generation of bacterial resistance harmful for human health. Based on the available scientific information including recent data from in vitro investigations (proteomic and genomic analyses), it is not possible to quantify the risk associated with triclosan (including its use in cosmetics) in terms of development of antimicrobial resistance (i.e. selection for less susceptible population), genetic basis for resistance and dessemination of resistance. In view of the concentrations of triclosan reported to trigger resistance in vitro, some of the environmental concentrations found in a number of geographical distinct areas are high enough to suggest that bacterial resistance could be triggered. However, no studies have been conducted on this aspect. The applications of triclosan which contribute to those high environmental concentrations cannot be properly identified nor quantified at present and the presence of other chemicals (e.g. antibiotics, surfactants, other biocides, etc.) in the environment, which may also affect microbial populations, would preclude assessing the effects of triclosan independently. 36
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Due to the limited number of in situ studies of resistance induced by triclosan to date, SCCS can only recommend the prudent use of triclosan, for example in applications where a health benefit can be demonstrated. However, conclusions from in vitro studies cannot be ignored, notably the role of triclosan (and other biocides) in triggering resistance and in the dissemination (or lack of) resistance determinants. Hence, the SCCS appreciates that research investment from industry will be maintained to contribute to a better understanding of the potential risks associated with triclosan applications. Research in triggering mechanisms of resistance, maintenance of the gene pool and the transfer of resistance and virulence determinants, and improving the translational application of laboratory results to situations in situ are needed.

13. COMMENTS RECEIVED DURING THE PUBLIC CONSULTATION


A public consultation on this opinion was opened on the website of the EU non-food scientific committees from 29 March to 26 May 2010. Information about the public consultation was broadly communicated to national authorities, international organisations and other stakeholders. In total, 10 contributions were received of which 5 were from public authorities, 3 from industry and two from individuals with professional links to this issue. Each submission to the public consultation was carefully considered by the Working Group and responses were formulated for each.The opinion has been revised to take account of all the relevant comments and the literature has been updated with relevant publications. The scientific rationale and the opinion were clarified and strengthened in certain respects. The overall opinion, however, remains unchanged.

14. MINORITY OPINION


None

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15. REFERENCES
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Biroov L, Mikulov M. (2009) Development of triclosan and antibiotic resistance in Salmonella enterica serovar Typhimurium. J Med Microbiol 58:436-441. Bjorland J, Sunde M, Steinar Waage. (2001) Plasmid-borne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus. J Clin Microbiol 39: 3999-4004 Boeris PS, Domenech CE, Lucchesi GI. (2007) Modification of phospholipid composition in Pseudomonas putida A ATCC 12633 induced by contact with tetradecyltrimethylammonium. J Appl Microbiol 103:1048-1054. Borges-Walmsley MI, Walmsley AR. (2001) The structure and function of drug pumps. Trends Microbiol 9:71-79. Bojar W, Kazmierska K, Szalwinski M, Zareba T. (2009) Triclosan-Coated Sutures in Oral Surgery. Adv Clin Exp Med 18:401-405. Borling P, Engelund B, Srensen H. (2005) Kortlgning af triclosan Kortlgning af kemiske stoffer i forbrugerprodukter. Nr. 732006. Danish EPA: Copenhagen. Borling P, Engelund B, Srensen H. (2005) Kortlgning af triclosan Kortlgning af kemiske stoffer i forbrugerprodukter. Nr. 732006. Danish EPA: Copenhagen. Boyce JM, Pittet D. (2002) Guideline for hand hygiene in health-care settings: recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Infect Control Hosp Epidemiol 23:S340. Boyd GR, Reemtsma H, Grimm DA, Mitra S. (2003) pharmaceuticals and personal care products (PPCPs) in surface and treated waters of Louisiana, USA and Canada. Sci Total Environ 311:135-149. Brading M G, Cromwell V J, Green A K, DeBrabander S, Beasley T. (2004) The role of triclosan in dentifrice formulations, with particular reference to a new 0.3% triclosan calcium carbonate-based system. Int Dental J 54:291-298. Brading MG, Cromwell VJ, Green AK, DeBrabander S, Beasley T, Marsh PD. (2004) The role of Triclosan in dentifrice formulations, with particular reference to a new 0.3% Triclosan calcium carbonate-based system. Int Dent J 54:291-298. Brading MG, Marsh PD. (2003) The oral environment: the challenge for anti-microbials in oral care products. Int Dent J 53:353-362. Brady LM, Thomson M, Palmer MA, Harkness JL. (1990). "Successful control of endemic MRSA in a cardiothoracic surgical unit". Med J Aust 152:240245. Braoudaki M, Hilton AC. (2004) Adaptive resistance in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents. J Clin Microbiol 42:73-78. Braoudaki M, Hilton AC. (2005) Mechanisms of resistance in Salmonella enterica adapted to erythromycin, benzalkonium chloride and triclosan. Int J Antimicrob Agents. 25:3137 Broadley SJ, Jenkins PA, Furr JR, Russell AD. (1995) Potentiation of the effects of chlorhexidine diacetate and cetylpyridinium chloride on mycobacteria by ethambutol. J Med Microbiol 43:458-460. Brown MH, Paulsen IT, Skurray RA. (1999) The multidrug efflux protein NorM is a prototype of a new family of transporters. Mol Microbiol 31:393-395. Brown MRW, Gilbert P. (1993) Sensitivity of biofilms to antimicrobial agents. J Appl Bacteriol 74:S87-97. Brzel VS, Cloete TE. (1994) Resistance of Pseudomonas aeruginosa to isothiazolone. J Appl Bacteriol 76:576-582.

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Bruinsma GM, Rustema-Abbing M, van der Mei HC, Lakkis C, Busscher HJ. (2006) Resistance to a polyquaternium-1 lens care solution and isoelectric points of Pseudomonas aeruginosa strains. J Antimicrob Chemother 57:764-766. Bryskier, A. (2002) Viridans group streptococci: cavities. Clin Microbiol Infect 8:65-69. a reservoir of resistant bacteria in oral

Buckley A, Webber MA, Cooles S, Randall L, La Ragione RM, Woodward M, Piddock LJV (2006) The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis. Cell Microbiol, 8:847-856. Cadieux PA, Chew BH, Nott L, Szeney S, Elwood CN, Wignall GR, Goneau LW, Denstedt JD. (2009) Use of triclosan-eluting ureteral stents in patients with long-term stents. J Endourol 23:1187-1194. Cakmak A, Cirpanli Y, Bilensoy E, Yorganci K, Calis S, Saribas Z, Kaynaroglu V. (2009) Antibacterial activity of triclosan chitosan coated graft on hernia graft infection model. Int J Pharm 381:214-219. Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL. (2008) Urinary concentrations of triclosan in the U.S. population: 2003-2004. Environ Health Perspec 116:303-307. Canosa P, Morales S, Rodriguez I, Rubi E, Cela R, Gomez M. (2005) Aquatic degradation of triclosan and formation of toxic chlorophenols in presence of low concentrations of free chlorine. Anal Bioanal Chem 383:1119-1126. Capdevielle M, Van Egmond R, Whelan M, Versteeg D, Matthias Hofmann-Kamensk M, Inauen J, Cunningham V, Woltering D. (2008) Consideration of Exposure and Species Sensitivity of Triclosan in the Freshwater Environment Integrated Environ Assess Manag 4:15-23. Cha J, Cupples AM. (2009) Detection of the antimicrobials triclocarban and triclosan in agricultural soils following land application of municipal biosolids. Water Res 43:25222530. Chalew TEA, Halden R (2009) Environmental exposure of aquatic and terrestrial biota to triclosan and triclocarban. J Am Water Resources Assoc 45:4-13. Champlin FR, Ellison ML, Bullard JW, Conrad RS. (2005) Effect of outer membrane permeabilisation on intrinsic resistance to low triclosan levels in Pseudomonas aeruginosa. Int J Antimicrob Agents. 26:159-164. Chapman JS. (2003) Disinfectant resistance mechanisms, cross-resistance, and coresistance. Int Biodeter Biodegrad 51:271-276. Chau WC, Wu JL, Zongwei Z (2008) Investigation of levels and fate of triclosan in environmental waters from the analysis of gas chromatography coupled with ion trap mass spectrometry. Chemosphere 73:S13-S17 Chen Y, Pi B, Zhou H, Yu Y, Li L. (2009) Triclosan resistance in clinical isolates of Acinetobacter baumannii. J Med Microbiol 58:1086-1091 Chew BH, Cadieux PA, Reid G, Denstedt JD. (2006) In-Vitro Activity of Triclosan-Eluting Ureteral Stents against Common Bacterial Uropathogens. J Endourol 20:949-958. Chu S, Metcalfe CD. (2007) Simultaneous determination of triclocarban and triclosan in municipal biosolids by liquid chromatography tandem mass spectrometry. J Chromatogr A 1164:212-218. Chuanchuen R, Beinlich K, Hoang TT, Becher A, Karkhoff-Schweizer RR, Schweizer HP (2001) Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nxfB mutants overexpressing MexCD-OprJ. Antimicrob Agents Chemother 45:428-432. 40
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Chuanchuen R, Murata T, Gotoh N, Schweizer HP (2005) Substrate-dependent utilization of OprM or OpmH by the Pseudeomonas aeruginosa MexJK efflux pump. Antimocrob Agents Chemother 49:2113-2136. Chuanchuen R, Narasaki CT, Schweizer HP (2002) The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for effect of triclosan. J bacteriol 184:5036-5044. Chuanchuen R, Karkhoff-Schweizer RR, Schweizer HP. (2003) High-level triclosan resistance in Pseudomonas aeruginosa is solely a result of efflux. Am J Infect Control 31:124-127. Cloete TE. (2003) Resistance mechanisms of bacteria to antimicrobial compounds. Int Biodet Biodegrad 51:277-282. Cole EC, Addison RM, Dulaney PD, Leese KE (2006) Investigation of antibiotic and antibacterial resistance in Staphylococcus from the skin of users and non-users of antibacterial wash products in home environments. Annual Conference on Antimicrobial Resistance, June 26-28, Bethesda, MD. Cole EC, Addison RM, Rubino JR, Leese KE, Dulaney PD, Newell MS, Wilkins J, Gaber DJ, Wineinger T, Criger DA (2003) Investigation of antibiotic and antibacterial agent crossresistance in target bacteria from homes of antibacterial product users and nonusers. J Appl Microbiol 95:664676. Coogan MA, La point TW (2008) Snail bioaccumulation of triclocarban, triclosan, and methyltriclosan in a North Texas, USA, stream affected by wastewater treatment plant runoff. Environ Toxicol Chem 27:1788-1793. Coogan MA, Edziyie RE, La Point TW, Venables BJ (2007) Algal bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater treatment plant receiving stream. Chemosphere 67:1911-1918. Cookson B. (2005) Clinical significance of emergence of bacterial antimicrobial resistance in the hospital environment. J Appl Microbiol 99:989-996. Cookson BD, Farrelly H, Stapleton P, Garvey RPJ ,Price MR. (1991) Transferable resistance to triclosan in MRSA. Br Med J 337:15481549. Cottell A, Denyer, SP, Hanlon GW, Maillard J-Y. (2009) Triclosan-tolerant bacteria: Changes in susceptibility to antibiotics. J Hosp Infect 72:71-76. Dantas, G., Sommer, MO, Oluwasegun, RD, Church, GM. (2008) Bacterial subsisting on antibiotics. Science 320:100-103. Darouiche RO, Mansouri1 MD, Gawande PV, Madhyastha S (2009) Antimicrobial and antibiofilm efficacy of triclosan and DispersinBw Combination. J Antimicrobial Chemother 64:8893. Das JR, Bhakoo M, Jones MV, Gilbert P (1998) Changes in the biocide susceptibility of Staphylococcus epidermidis and Escherichia coli cells associated with rapid attachment to plastic surfaces. J Appl Microbiol 84:852-858. Davies R M, Elwood RP, Davies GM. (2004) Effectiveness of a toothpaste containing Triclosan and polyvinyl-methyether maleic acid copolymer in improving plaque control and gingival health. A systematic review. J Clin Periodontol 31:1029-1033. Davies RM. (2007) The clinical efficacy of triclosancopolymer and other common therapeutic approaches to periodontal health. Clin Microbiol Infect 13:25-29. Davies RM, Ellwood RP, Davies GM. (2004) The effectiveness of a toothpaste containing triclosan and polyvinyl-methyl ether maleic acid copolymer in improving plaque control and gingival health. J Clin Periodont 31:1029-1033. Davin-Regli A, Bolla JM, James C, Lavigne JP, Chevalier J, Garnotel E, Molitor A, Pags JM (2008) Membrane permeability and regulation of drug "influx and efflux" in enterobacterial pathogens, Current Drug Targets 9:750-759. 41
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DeLorenzo ME, Keller JM, Arthur CD, Finnegan MC, Harper HE, Winder VL, Zdankiewicz DL. (2008) Toxicity of the antimicrobial compound triclosan and formation of the metabolite methyl-triclosan in estuarine systems Environ Toxicol 23:224 232. Demple B. (1996) Redox signaling and gene control in the Escherichia coli soxRS oxidative stress regulon - a review. Gene 179:53-57. Denyer SP, Maillard J-Y. (2002) Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria. J Appl Microbiol 92:S35-45. Depardieu F, Podglajen I, Leclercq R, Collatz E, Courvalin P. (2007), Modes and modulations of antibiotic resistance gene expression, Clin Micobiol Rev 20:79-114 Dodd MC, Kohler HP, von Gunten U. (2009) Oxidation of antibacterial compounds by ozone and hydroxyl radical: elimination of biological activity during aqueous ozonation process. Environ Sci Technol 43:2498-2504. Doyle MP. (2006) Antimicrobial Resistance: Implications for the Comprehensive Reviews in Food Science and Food Safety 5:71-137. Food System.

Dye C, Schlabach M, Green J, Remberger M, Kaj L, Palm-Cousins A, Brorstrm-Lundn E. (2007) Bronopol, resorcinol, m-cresol and triclosan in the Nordic environment. Nordic Council of Ministers, Copenhagen.TemaNord: 585. Dynes JJ, Lawrence JR, Korber DR, Swerhone GD, Leppard GG, Hitchcock AP. (2009) Morphological and biochemical changes in Pseudomonas fluorescens biofilms induced by sub-inhibitory exposure to antimicrobial agents. Can J Microbiol 55:163-178. EARSS (European Antimicrobial Resistance Surveillance System). Annual Report 2005. Available from http://www.rivm.nl/earss/ EFSA, Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the Commission related to a 3rd list of substances for food contact materials adopted on 15 March 2004, The EFSA Journal (2004) 37, 1-7 EFSA, Assessment of the possible effect of the four antimicrobial treatment substances on the emergence of antimicrobial resistance, Scientific Opinion of the Panel on Biological Hazards, Adopted on 6 March 2008, The EFSA Journal (2008) 659, 1-26 Eley BM. (1999) Antibacterial agents in the control of supragingival plaque a review. Brit Dent J 186:286-296. Escalada GM, Russell AD, Maillard J-Y, Ochs D. (2005) Triclosan-bacteria interactions: single or multiple target sites? Lett Appl Microbiol 41:476-481. Fair PA, Lee HB, Adams J, Darling C, Pacepavicius G, Alaee M, Bossart GD, Henry N. Muir D (2009) Occurrence of triclosan in plasma of wild atlantic bottlenose dolphins (Tursiops truncates) and in their environment. Environ Pollut 157:2248-2254. Fan F, Yan K, Wallis NG, Reed S, Moore TD, Rittenhouse SF, DeWolf WE Jr, Huang J, McDevitt D, Miller WH, Seefeld MA, Newlander KA, Jakas DR, Head MS, Payne DJ. (2002) Defining and combating the mechanisms of triclosan resistance in clinical isolates of Staphylococcus aureus. Antimicrob Agents Chemother 46:33433347. Farre M, Asperger D, Kantiani L, Gonzalez S, Petrovic M, Barcelo D. (2008) Assessment of the acute toxicity of triclosan and methyl triclosan in wastewater based on the bioluminescence inhibition of Vibrio fischeri. Anal Bioanal Chem 390:1999-2007. Federle TW, Kaiser SK, Nuck. BA (2002) Fate and effects of triclosan in the activated sludge. Environ Toxicol Chem 2:1330-1337. Fernandes M, Shareef A, Karkkainen M and Kookana R. (2008) The occurrence of endocrine disrupting chemicals and triclosan in sediments of Barker Inlet, South Australia. A Report prepared for the Adelaide and Mount Lofty Ranges Natural Resources Management 42
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Board. SARDI Publication Number F2008/001026-1. South Australian Research & Devlopment Institute (Aquatic Sciences), Adelaide. http://www.sardi.sa.gov.au Field JA, Sierra-Alvarez R. (2008a) Chemosphere 71:1015-1018. Microbial degradation of chlorinated dioxins.

Field JA, Sierra-Alvarez R. (2008b) Microbial degradation of chlorinated benzenes, Biodegradation 19:463-480. Fine, DH, Furgang D, Markowitz K, Sreenivasan PK, Klimpel K, DeVizio, W. (2006) The antimicrobial effect of a triclosan/copolymer dentifrice on oral micro-organisms in vivo. J Am Dent Assoc 137:1407-1414. Fischler GF, Fuls JL, Dail EW, Duran MH, Rodgers, Waggoner AL (2007) Effect of Hand Wash Agents on Controlling the Transmission of Pathogenic Bacteria from Hands to Food. J Food Protection 70:28732877. Fiss EM, Rule KL, Vikesland PJ. (2007) Formation of chloroform and other chlorinated byproducts by chlorination of triclosan-containing antibacterial products. Environmental science & technology 41:2387-2394. Fjeld E, Schlabach M, Berge J.A, Eggen T, Snilsberg P, Kllberg G, Rognerud S, Enge EK, Borgen A, Gundersen H (2004) Screening of selected new organic contaminants brominated flame retardants, chlorinated paraffins, bisphenol-A and Triclosan. ISBN 82577-4488-3. Ford HR, Jones P, Gaines B, Reblock K, Simpkins DL. (2005) Intraoperative handling and wound healing: controlled clinical trial comparing Coated VICRYL Plus Antibacterial Suture (coated polyglactin 910 suture with triclosan) with Coated VICRYL Suture (coated polyglactin 910 suture). Surg Infect 6:31321. Fraud S, Hann AC, Maillard J-Y, Russell AD. (2003) Effects of ortho-phthalaldehyde, glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and M. abscessus strains with modified permeability. J Antimicrob Chemother 51:575-584. Freundlich JS, Wang F, Vilcheze C, Gulten G, Langley R, Schiehser GA, Jacobus DP, Jacobs WR, Sacchettini JC. (2009) Triclosan derivatives: towards potent inhibitors of drug sensitive and drug resistant Mycobacterium tuberculosis. Chem Med Chem 4: 241-248. Fuls JL, Rodgers ND, Fischler GE, Howard JM, Patel M, Weidner PL, Duran MH. (2008) Alternative hand contamination technique to compare the activities of antimicrobial and nonantimicrobial soaps under different test conditions. Appl Environ Microbiol 74:37393744. Gandhi PA, Sawant AD, Wilson LA, Ahearn DG. (1993) Adaptation and growth of Serratia marcescens in contact lens disinfectant solution containing chlorhexidine gluconate. Appl Environ Microbiol 59:183-188. Gilbert P, Allison DG, McBain AJ. (2002) Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance? J Appl Microbiol 92:S98110. Gilbert P, McBain AJ. (2002) Literature-based evaluation of the potential risks associated with impregnation of medical devices and implants with triclosan. Surg Infect (Larchmt) 3(suppl 1):S55S63. Gilbert P, McBain AJ, Rickard AH. (2003) Formation of microbial biofilm in hygienic situations: a problem of control. Inter Biodeter Biodegrad 51:245-248. Gilbert P, McBain A, Sreenivasan P (2007), Common therapeutic approaches for the control of oral biofilms: microbiological safety and efficacy. Clin Microbiol Infect 13 (Suppl. 4): 1724. Giwercman B, Jensen ET, Hoiby N, Kharazmi A, Costerton JW. (1991). Induction of betalactamase production in Pseudomonas aeruginosa biofilm. Antimicrob Agents Chemother 35:1008-1010. 43
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Gjermo P, Saxton CA. (1991) Antibacterial dentifrices. Clinical data and relevance with emphasis on zinc/ Triclosan. J Clin Periodontol 18:468-472. Glassmeyer ST, Furlong ET, Kolpin DW, Cahill JD, Zaugg SD, Werner SL, Meyer MT, Kryak DD. (2005) Transport of chemical and microbial compounds from known wastewater discharges: potential for use as indicators of human fecal contamination. Environ Sci Technol 39:5157-5169. Gomez Escalada M, Russell AD, Maillard J-Y, Ochs D. (2005a) Triclosan-bacteria interactions: single or multiple target sites? Lett Appl Microbiol 41:476-481. Gomez Escalada M, Harwood JL, Maillard J-Y, Ochs D (2005b). Triclosan inhibition of fatty acid synthesis and its effect on growth of E. coli and Ps. aeruginosa. J Antimicrob Chemother 55:879-882. Gomez Escalada, M. (2003) Studies on the mechanisms of action of and resistance to a phenylether, triclosan. PhD Thesis, Cardiff University. Gurin-Mchin L, Dubois-Brissonnet F, Heyd B, Leveau JY. (2000) Quaternary ammonium compounds stresses induce specific variations in fatty acid composition of Pseudomonas aeruginosa. Inter J Food Microbiol 55:157-159. Gurin-Mchin L, Dubois-Brissonnet F, Heyd B, Leveau JY. (1999). Specific variations of fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by quaternary ammonium compounds and relation with resistance to bactericidal activity. J Appl Microbiol 87:735-742. Gilbert P and Moore LE. (2005) Cationic antiseptics: diversity of action under a common epithet. J ApplMicrobiol 99: 703-715. Guilln J, Bernabeu A, Shapiro S, Villalan J. (2004) Location and orientation of Triclosan in phospholipid model membranes. Eur Biophys J 33:448-453. Gunsolley JC. (2006) A meta-analysis of six-month studies of antiplaque and antigingivitis agents. J Am Dent Assoc. 137:1649-1657. Halden RU and Paull DH. (2005) Co-occurrence of triclocarban and triclosan in U.S. water resources. Environ Sci Technol 39:1420-1426. Hammond SA, Morgan JR, Russell AD. (1987) Comparative susceptibility of hospital isolates of Gram-negative bacteria to antiseptics and disinfectants. J Hosp Infect 9:255-264. Harbarth S, Samore MH. (2005) Antimicrobial resistance determinants and future control. Emerg Infect Dis 11:794-801. Hawkey PM. (2004) Mycobactericidal agents. In: Fraise AP, Lambert PA, Maillard J-Y, editors. Principles and Practice of Disinfection, Preservation and Sterilization 4th edn. Oxford: Blackwell Scientific Publication; p. 191-204. Hawkey PM, Jones AM. (2009) The changing epidemiology of resistance. J AntimicrobChemother 64 (Suppl. 1): i3-10 Hay AG, Dees PM, Sayler GS. (2001) Growth of bacterial consortium on triclosan. FEMS Microbiol Ecol 36:105-112. Heath RJ, Li J, Roland GE, Rock CO. (2000) Inhibition of the Staphylococcus aureus NADPHdependent Enoyl-acyl carrier protein reductase by triclosan and hexachlorophene. J Biol Chem 275:4654-4659. Heath RJ, Su N, Murphy CK, Rock CO. (2000). The enoyl-[acyl-carrier-protein] reductases FabI and FabL from Bacillus subtilis. J Biol Chem 275:40128-40133. Heath RJ, White SW, Rock CO (2002) Inhibitors of fatty acid synthesis as antimicrobial chemotherapeutics. Appl Microbiol Biotechnol 58:695703.

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Heath RJ, Yu YT, Shapiro MA, Olson E, Rock CO. (1998) Broad spectrum antimicrobial biocides target the FabI component of fatty acid synthesis. J Biol Chem 273:3031630320. Heath, R, Rubin J, Holland D, Zhang EL, Snow ME, Rock CO. (1999) Mechanism of triclosan inhibition of bacterial fatty acid biosynthesis. J Biol Chem 274:1111011114. Heath RJ, Yu Y-T, Shapiro S, Olson E, Rock CO. (1998) Broad spectrum antimicrobial biocides target the FabI component of fatty acid biosynthesis. J Biol Chem 273:30316 30320. Heidler J, Halden RU. (2007) Mass balance assessment of triclosan temoval during conventional sewage treatment. Chemosphere 66:362-369. Hon HS, Ko G and Zoh KD. (2009) Kinetics and mechanism of photolysis and TiO2 photocatalysis of triclosan. J Hazardous Materials 166:954-960. Hua W, Bennet ER and Letcher RJ. (2005) Triclosan in waste and surface waters from the upper Detroit River by liquid chromatography-electrospray-tandem quadrupole mass spectrometry. Environ Intern 31:621-630 Huang CT, Yu FP, McFeters GA, Stewart PS. (1995) Nonuniform spatial patterns of respiratory activity within biofilms during disinfection. Appl Environ Microbiol 61:22522256. Ingerslev F, Vaclavik E, Halling-Sorenson B. (2003) Pharmaceutical and personal care products: a source of endocrine disruption in the environment? Pure Appl Chem 75: 1181-1893 Itoh Y, Wang X, Hinnebusch BJ, Preston JF, Romeo T. (2005) Depolymerization of b-1,6-Nacetyel-D-glucosamine disrupts the integrity of diverse bacterial biofilms. J Bacteriol 187: 382387. Jang H-J, Chang MW, Toghrol F, Bentley WE. (2008) Microarray analysis of toxicogenomic effects of triclosan on Staphylococcus aureus. Appl Microbiol Biotechnol 78:695-707. Jones CL, Ritchie JA, Marsh PD, Van der Ouderaa F. (1987) The effect of long term use of a dentifrice containing zinc citrate and a non-ionic agent on the oral flora. J Dent Res 67:46-50. Jones CL, Saxton CA, Ritchie JA. (1990) Microbiological and clinical effects of a dentifrice containing zinc citrate and Triclosan in the human experimental gingivitis model. J Clin Periodontol 17 570-574. Jones GLl, Muller CT, OReilly M, Stickler DJ. (2006) Effect of triclosan on the development of bacterial biofilms by urinary tract pathogens on urinary catheters. J Antimicrob Chemother 57:266-272. Jones MW, Herd TM, Christie HJ. (1989) Resistance of Pseudomonas aeruginosa to amphoteric and quaternary ammonium biocides. Microbios 58:49-61. Jones R D, Jampani H B, Neman J L et al. (2000) Triclosan: a review of effectiveness and safety in health care settings. Am J Infect Control 28:184196. Juhas M, van der Meer JR, Gaillard M, Harding RM, Hood DW, Crook DW. (2009) Genomic islands: tools of bacterial horizontal gene transfer and evolution. FEMS Microbiol Rev 33:376-393. Justinger C, Moussavian MR, Schlueter C, Kopp B, Kollmar O, Schilling MK. (2009). Antibiotic coating of abdominal closure sutures and wound infection. Surgery 145:330334. Kanda R, Griffin P, James HA, Fothergill J. (2003) Pharmaceutical and personal care products in sewage treatment work. J Environ Monit 5: 823-830.

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Kantiani L, Farr , Asperger D, Rubio F, Gonzlez S, Alda MJL, Petrovi M, Shelver WL, Barcel D. (2008) Triclosan and methyl-triclosan monitoring study in the northeast of Spain using a magnetic particle enzyme immunoassay and confirmatory analysis by gas chromatographymass spectrometry. J Hydrol 361:1-9. Kaplan JB, Ragunath C, Velliyagounder K, Fine DH, Ramasubu N. (2004) Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 48: 26332636. Karatzas KA, Webber MA, Jorgensen F, Woodward MJ, Piddock LJ, Humphrey TJ. (2007) Prolonged treatment of Salmonella enterica serovar Typhimurium with commercial disinfectants selects for multiple antibiotic resistance, increased efflux and reduced invasiveness, J Antimicrob Chemother 60:947-955 Kastbjerg VG, Halberg Lardsen M, Gram L, Ingmer H. (2010). Influence of sublethal concentrations of common disinfectants on expression of virulence genes in Listeria monocytogenes. Appl Environ Microbiol 76:303-309. Kim JW, Jang HS, Kim JG, Ishibashi H, Hirano M, Nasu K, Ichikawa N., Takao Y, Shinohara R and Arizono K. (2009) Occurrence of Pharmaceutical and personal care products in surface water from Mankyung River, South Korea. J Health Sci 55: 249-258. Kinney CA, Furlong ET, Zaugg SD, Burkhard MR, Werner SL, Cahill JD, Jorgensen GR. (2006) Survey of organic wastewater contaminants in biosolids destined for land application. Environ Sci Technol 40: 7207-7215. Knudsen BE, Chew BH, Denstedt JD. (2005) Drug-eluting biomaterials in urology: the time is ripe. BJU Int 95:726727. Kolpin DW, Skopec M, Meyer MT, Furlong ET, Zaugg SD. (2004) Urban contribution of pharmaceuticals other organic wastewater contaminants to streams during differing flow conditions. Sci Total Environ 328:119-130. Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT. (2002) Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: A National Reconnaissance. Environ Sci Technol 36:1202-1211. Kumar KS, S. Priya M, Peck AM, Sajwan KS. (2010) Mass Loadings of Triclosan and Triclocarban from Four Wastewater Treatment Plants to Three Rivers and Landfill in Savannah, Georgia, USA. Arch Environ Contam Toxicol 58:275-285 Kummerle N, Feucht HH, Kaulfers PM. (1996) Plasmid-mediated formaldehyde resistance in Escherichia coli: characterization of resistance gene. Antimicrob Agents Chemother 40:2276-2279. Lai S, Tremblay J, Deziel E (2009). Swarming motility: a multicellular behaviour conferring antimicrobial resistance. Environ Microbiol 11:126-136. Lambert PA. (2002) Cellular impermeability and uptake of biocides and antibiotics in Grampositive bacteria and mycobacteria. J Appl Microbiol 92(Suppl.):S46-S55. Larson EL, Lin SX, Gomez-Pichardo C, Della-Latta P. (2004) Effect of antibacterial home cleaning and handwashing products on infectious disease symptoms: a randomized, double-blind trial. Ann Intern Med 140:321-329. Latch DE, Packer JL, Stender BL, VanOverbeke J, Arnold WA, McNeill K. (2005) Aqueous Photochemistry of Triclosan: Formation of 2,4-Dichlorophenol, 2,8-Dichlorodibenzo-pDioxin, and Oligomerization Products. Environ Toxicol Chem 24:517-525. Lawrence JR, Zhu B, Swehone GDW, Roy J, Wassenar LI, Topp E, Korber DR. (2009) Comparative microscale analysis of the effects of triclosan and tricocarban on the structure and function of river biofilm communities. Sci Total Environ 407:3307-3316.

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Lear JC, Maillard J-Y, Dettmar PW, Goddard PA, Russell AD. (2006) Chloroxylenol- and triclosan-tolerant bacteria from industrial sources susceptibility to antibiotics and other biocides. Inter BiodeterBiodegrad 57:51-56. Lear CJ, Maillard, J-Y, Dettmar PW, Goddard PA, Russell AD. (2002) Chloroxylenol- and triclosan- tolerant bacteria from industrial sources. J Indus Microbiol Biotechnol 29:238242. Ledder RG, Gilbert P, Willis C and McBain AJ. (2006) Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates. J Appl Microbiol 100:1132-1140. Levy CW, Roujeinikova A, Sedelnikova S, Baker PJ, Stuitje R, Slabast AR, Rice DW, Rafferty JB. (1999) Molecular basis of triclosan activity. Nature 398:384385. Lindstrm A, Buerge IJ, Poiger T, Bergqvist PA, Mller MD, Buser HR. (2002) Occurrence and environmental behavior of the bactericide triclosan and its methyl derivative in surface waters and in wastewater. Environ Sci Technol 36:2322-2329. Lishman L, Smyth SA, Sarafin K, Kleywegt S, Toito J, Peart T, Lee B, Servos M, Beland M, Seto P. (2006) Occurrence and reductions of pharmaceuticals and personal care products and estrogens by municipal wastewater treatment plants in Ontario, Canada. Sci Total Environ 367:544-558. Lopez-Avila V, Hites RA (1980) Organic compounds in an industrial wastewater. Their transport into sediments. Environ Sci Tech 14:1382-1390. Lores M, Llompart M, Sanchwz-Prado L, Gracia-Jares C and Cela R. (2005) Confirmation of the formation of dichlorodibenzo-p-dioxin in the photodegradation of triclosan by photoSPME. Anal Bioanal Chem 381:1294-1298 Maillard J-Y, Denyer SP. (2009) Emerging bacterial resistance following biocide exposure: should we be concerned? Chemica Oggi 27:26-28. Maillard J-Y. (2002) Bacterial target sites for biocide action. J Appl Microbiol 92: S16-27. Maillard J-Y. (2005a) Usage of antimicrobial biocides and products in the healthcare environment: efficacy, policies, management and perceived problems. Ther Clin Risk Manag 1:340-370. Maillard J-Y. (2005b) Biocides: Health care application. Pharml J 275:639-642. Maillard J-Y. (2007) Bacterial resistance to biocides in the healthcare environment: shall we be concerned? J Hosp Infect 65 (suppl 2): 60-72. Maira-Litrn T, Allison DG, Gilbert P. (2000). An evaluation of the potential of the multiple antibiotic resistance operon (mar) and the multidrug efflux pump acrAB to moderate resistance towards ciprofloxacin in Escherichia coli biofilms. J Antimicrob Chemother 45:789-795. Manzoor SE, Lambert PA, Griffiths PA, Gill MJ, Fraise AP. (1999) Reduced glutaraldehyde susceptibility in Mycobacterium chelonae associated with altered cell wall polysaccharides. J Antimicrob Chemother 43:759-765. Marsh PD, Bradshaw DJ. (1993) Microbiological effects of new agents in dentifrices for plaque control. Int Dent J 43:399-406. Marsh PD. (1991) Dentifrices containing new agents for the control of plaque and gingivitis: microbiological aspects. J Clin Periodontol 18:462-467. Massengo-Tiass RP, Cronan JE. (2008) Vibrio cholerae FabV defines a new class of enoylacyl carrier protein reductase. J Biol Chem 283:1308-1316. McAvoy DC, Schatowitz B, Jacob M, Hauk A, Eckhoff WS. (2002) Measurement of triclosan in wastewater treatment systems. Environ Toxicol Chem 21:1323-1329. 47
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McAvoy DC, Schatowitz B, Jacob M, Hauk A, Eckhoff WS (2009) Measurement of triclosan in wastewater treatment systems. Environmental Toxicol Chem 21:13231329. McBain AJ, Ledder RG, Sreenivasan P, Gilbert P. (2004) Selection for high-level resistance by chronic triclosan exposure is not universal. J Antimicrob Chemother 53:772-777. McBain AJ, Bartolo RG, Catrenich CE, Charbonneau D, Ledder RG, Price BB, Gilbert P. (2003) Exposure of Sink Drain Microcosms to Triclosan: Population Dynamics and Antimicrobial Susceptibility. Appl EnvironMicrobiol 69:5433-5442. McDonnell G, Russell AD. (1999) Antiseptics and disinfectants: activity, action and resistance. Clin Microbiol Rev 12:147-179. McKeegan KS, Borges-Walmsley MI, Walmsley AR. (2003) The structure and function of drug pumps: an update. Trends Microbiol 11:21-29. McLeod R, Muench SP, Rafferty J B et al. (2001) Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicompexan FabI. Int J Parasitology 31:109113. McMahon T, Shamim N, Gowda S, Angle G, Leighton T. 5-Chloro-2-(2,4-dichlorophenoxy) phenol (Triclosan): Risk Assessment for the Reregistration Eligibility Decision (RED) Document. Case No 2340. PC Code: 054901. DP Barcode: 373535. US EPA, Washington DC, 17 April 2008 McMurry LM, McDermott PF, Levy SB. (1999) Genetic evidence that InhA of Mycobacterium smegmatis is a target for triclosan. Antimicrob Agent Chemother 43:711-713. McMurry LM, Oethinger M, Levy SB. (1998a) Triclosan targets lipid synthesis. Nature 394: 531532. McMurry LM, Oethinger M, Levy SB. (1998b) Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli. FEMS Microbiol Lett 166:305309. McNeil MR, Brennan PJ. (1991) Structure, function and biogenesis of the cell envelope of mycobacteria in relation to bacterial physiology, pathogenesis and drug resistance; some thoughts and possibilities arising from recent structural information. Res Microbiol 142:451-463. Meade MJ, Waddell RL, Callahan TM. (2001) Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. dentrificans inactivate triclosan in liquid and solid substrates. FEMS Microbiol Lett 204:45-48 Mchin L, Dubois-Brissonnet F, Heyd B, Leveau JY. (1999). Adaptation of Pseudomonas aeruginosa ATCC 15442 to didecyldimethylammonium bromide induces changes in membrane fatty acid composition and in resistance of cells. J Appl Microbiol 86:859-866. Miller T, Heidler J, Chillrud S, DeLaquil A, RitchieJ, Mihalic J, Bopp R, Halden RU. (2008) Fate of triclosan and triclocarban in estuarine sediment. Environ Sci Technol 42: 45704576. Mima T, Joshi S, Gomez Escalada M, Schweizer HP. (2007) Identification and characterization of TriABC-OpmH, a triclosan efflux pump of Pseudomonas aeruginosa requiring two membrane fusion proteins. J Bacteriol 189:7600-7609 Mishra S, Karmodiya K, Parasuraman P, Surolia A, Surolia N. (2008) Design, synthesis, and application of novel triclosanprodruges as potential antimalarial and antibacterial agents. BioorgMed Chem 16:5536-5556. Moken MC, McMurry LM, Levy SB. (1997) Selection of multiple-antibiotic-resistant (Mar) mutants of Escherichia coli by using the disinfectant pine oil: Roles of the mar and acrAB loci. Antimicrob Agents Chemother 41:2770-2772. Morales S, Canosa P, Rodriguez I, Rubi E, Cela R. (2005) Microwave assisted extraction followed by gas chromatography with tandem mass spectrometry for the determination 48
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of triclosan and two related chlorophenols in sludge and sediments. J Chromatogr A 1082:128-135. Munton TJ, Russell AD. (1970) Effect of glutaraldehyde on protoplasts of Bacillus megaterium. J Gen Microbiol 63:367-370. National Industrial Chemicals Notification and Assessment Scheme, Australia. Priority Existing Chemical Assessment Report No. 30. Triclosan. 2009 Neumegen RA, Fernandez-Alba AR, Chisti Y. (2005). Toxicities of triclosan, phenol, and copper sulfate in activated sludge. Environ Toxicol 20:160-164. NICNAS (National Industrial Chemicals Notification and Assessment Scheme), Australia. Priority Existing Chemical Assessment Report No. 30, Triclosan. 2009. http://www.nicnas.gov.au/publications/car/pec/pec30.asp Niederman R. (2005) Triclosan-containing toothpastes reduce plaque and gingivitis. Based Dent 6: 33. Evid

Nikaido H. (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593-656. Noguchi N, Tamura M, Narui K, Wakasugi K, Sasatsu M. (2002) Frequency and genetic characterization of multidrug-resistant mutants of Staphylococcus aureus after selection with individual antiseptics and fluoroquinolones. Biol Pharm Bull 25:1129-1132. Okumura T, Nishikawa Y (1996) Gas chromatographymass spectrometry determination of triclosans in water, sediment and fish samples via methylation with diazomethane. Anal Chim Acta 325:175184. Orhan M, Kut D and Gunesoglu C. (2009) Improving the Antibacterial Activity of Cotton Fabrics Finished with Triclosan by the Use of 1,2,3,4-Butanetetracarboxylic Acid and Citric Acid. J Appl Polymer Sci 111:1344-1352. Pan Y, Breidt F, Kathariou S. (2006) Resistance of Listeria monocytogenes biofilms to sanitizing agents in a simulated food processing environment. Appl Environ Microbiol 72:7711-7717. Parikh SL, Xiao G, Tonge PJ. (2000). Inhibition of InhA, enoyl reductase from Mycobacterium tuberculosis by triclosan and isoniazid. Biochemistry 39:7645-7650. Paulsen IT, Brown MH, Skurray RA. (1996) Proton-dependent multidrug efflux systems. Microbiol Rev 60:575-608. Paulsen IT, Brown MH,Skurray RA. (1998) Characterization of the earliest known Staphylococcus aureus plasmid encoding a multidrug efflux system. J Bacteriol 180:3477-3479. Paxeus N. (1996) Organic pollutants in the effluents of large wastewater treatment plants in Sweden. Water Res 30:1115-1122. Pearce H, Messager S, Maillard J-Y. (1999) Effect of biocides commonly used in the hospital environment on the transfer of antibiotic-resistance genes in Staphylococcus aureus. J Hos Infect 43:101-108. Peter S, Nayak DG, Philip P, Bijlani NS. (2004) Antiplaque and antigingivitis efficacy of toothpastes containing Triclosan and fluoride. Int Dent J 54:299-303. Phan TN, Marquis RE. (2006) Triclosan inhibition of membrane enzymes and glycolysis of Streptococcus mutans in suspensions and biofilms. Can J Microbiol 52:977-983. Piddock LJV. (2006) Clinically relevant chromosomally encoded multidrug resistance efflux pump in bacteria. Clin Microbiol Rev 19:382-402. Poole K. (2007) Efflux pumps as antimicrobial resistance mechanisms. Ann Med 39:162176. 49
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Poole K. (2001) Multidrug resistance in Gram-negative bacteria. Curr Opin Microbiol 4:500508. Poole K. (2002a) Mechanisms of bacterial biocide and antibiotic resistance. J Appl Microbiol 92:S55-64. Poole K. (2002b) Outer membranes and efflux: the path to multidrug resistance in Gramnegative bacteria. Curr Pharm Biotechnol 3:77-98 Prada D, Mezuca M, Gmez MJ, Cerda V, Ferrer I, Farre F, Townshend A, Aguera A, Hernando MD, Fernndez-Alba AR. (2004) Evidence of 2,7/2,8-dibenzodichloro-p-dioxin as a photodegradation product of triclosan in water and wastewater samples Anal Chim Acta 524: =241-247 Prince J, Ayliffe GAJ. (1972). In-use testing of disinfectants in hospitals. J Clin Pathol 25:586-589. Pumbwe L, Randall LP, Woodward MJ, Piddock LJV. (2005) Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiply antibiotic-resistant Campylobacter spp. J Antimicrob Chemother 54:341-347. Putman M, van Veen HW, Degener JE, Konings WN. (2009) Antibiotic resistance: era of the multidrug pump. Mol Microbiol 36:772-773. Quinn T, O'Mahony R, Baird AW, Drudy D, Whyte P, Fanning S. (2006). Multi-drug resistance in Salmonella enterica: efflux mechanisms and their relationships with the development of chromosomal resistance gene clusters. Curr Drug Targets7:849-860. Rabih O, Darouiche RO, Mansouri MD, Purushottam V. Gawande PV, Madhyastha S. (2009) Antimicrobial and antibiofilm efficacy of triclosan and DispersinBw combination J Antimicrob Chemother 64:8893. Randall LP, Cooles SW, Coldham NG, Penuela EG, Mott AC, Woodward, MJ, Piddock LJV, Webber MA. (2007) Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence. J Antimicrob Chemother 60:1273-1280. Rastogi SC, Jensen GJ and Johansen JD. Survey and risk assessment of chemical substances in deodorants. Survey of Chemicals in Consumer Products No. 86,2007. Danish EPA, Copenhagen, Rastogi SC, Krongaard T, Jensen GH. Antibacterial compounds in clothing articles. Survey of Chemicals in Consumer Products No. 24, 2003, Danish EPA, Copenhagen. Regos J, Zak O, Solf R, Vischer WA, Weirich EG. (1979) Antimicrobial spectrum of triclosan, a broad-spectrum antimicrobial agent for topical application. II. Comparison with some other antimicrobial agents. Dermatologica 158:7279. Regos J, Hitz HR. (1974) Investigations on the mode of action of triclosan, a broad spectrum antimicrobial agent. Zentralbl Bakteriol Hyg I Abt Orig 226:390401. Remberger M, Sternbeck J, Strmberg K. (2002) Screening av Triclosan och vissa bromerade fenoliska mnen i Sverige. IVL Rapport B1477. In Swedish Remberger, M, Woldegiorgis A, Kaj L, Andersson J,Palm Cousins A, Dusan B, Ekheden Y, Brorstrm-Lundn, E Results from the Swedish Screening 2005., 2006, Subreport 2. Biocides. IVL Rapport B1700. Reuter G. (1989) Requirements on the efficacy of disinfectants in the food-processing area, 1 Zentralblatt fr Bakteriol Mikrobiol u Hyg, Serie B-Umwelthygiene Krankenhaushygiene Arbeitshygiene Prventive Med 187:564-577. Reuter G. (1984) Cleaning and disinfection in food hygiene. Fleiswirtschaft 64:668-672. Reuter G. (1994) The effectiveness of cleaning and disinfection during meat production and processing Influence factors and use recommendations. Fleiswirtschaft 74:808-813. 50
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Roberts AP, Mullany P. (2009) A modular master on the move: the Tn916 family of mobile genetic elements. Trends Microbiol 17:251-8. Rose H, Baldwin A, Dowson CG. (2009) Mahenthiralingam E. Biocide susceptibility of the Burkholderia cepacia complex. J Antimicrob Chemother 63:502-510. Roujeinikova A, Levy CW, Rowsell S, Sedelnikova S, Baker PJ, Minshull CA, Mistry A, Colls JG, Camble R, Stuitje AR, Slabas AR, Rafferty JB, Pauptit RA, Viner R, Rice DW. (1999) Crystallographic analysis of triclosan bound to enoyl reductase. J Mol Biol 294:527-535. Rule KL, Ebbett VR, Vikesland PJ. (2005) Formation of chloroform and chlorinated organics by free-chlorine-mediated oxidation of triclosan, Environ Sci Tech 39:3176-3185. Russell AD, Furr JR, Maillard J-Y. (1997) Microbial susceptibility and resistance to biocides: an understanding. ASM News 63:481-487. Russell AD, McDonnell G. (2000). Concentration: a major factor in studying biocidal action. J Hosp Infect 44:1-3. Russell AD. (2003) Biocide use and antibiotic resistance: the relevance of laboratory findings to clinical and environmental situations. Lancet Infect Dis 3:794803. Russell AD. (2004) Whither triclosan? J Antimicrob Chemother 53: 693695. Russell AD. (1996) Activity of biocides against mycobacteria. J Appl Bacteriol 81:87-101. Russell AD. (2002a) Antibiotic and biocide resistance in bacteria: comments and conclusion. J Appl Microbiol 92:S171-173. Russell AD. (2002b) Introduction of biocides into clinical practice and the impact on antibiotic-resistant bacteria. J Appl Microbiol 92:S121-135. Sabaliunas D, Webb SF, Hauk A, Jacob, M, Eckhoff WS. (2003) Environmental fate of triclosan in the River Aire basin, UK. Water Res 37:3145-3154. Samos-Petersen L, Winther-Nielsen M, Madsen T. (2003) Fate and effects of triclosan. Danish Environmental Protection Agency, Project Number 861, Copenhagen, Denmark, 47 pp. Snchez P, Linares JF, Moreno E, Martinez JL. (2005) The biocide triclosan selects Stenotrophomonas maltophilia mutants that overproduce the SmeDEF multidrug efflux pump. Antimicrob Agents Chemother 49:781-782. Sanchez-Prado L, Llompart M, Lores M, Garca-Jares C, Bayona JM, Cela R. (2006a) Monitoring the photochemical degradation of triclosan in wastewater by UV light and sunlight using solid-phase microextraction. Chemosphere 65:1338-1347. Sanchez-Prado L, Llompart M, Lores M, Fernndez-Alvarez M, Garca-Jares C, Cela R. (2006b) Further research on photo-SPME of triclosan. Anal Bioanal Chem 384:15481457. Sandborgh-Englund G, Adolfsson-Erici M, Odham G, Ekstrand J. (2006) Pharmacokinetics of triclosan following oral ingestion in humans. J Toxicol Environ Health A 69:1861-1873. Sanford JP. (1970) Disinfectants that dont. Ann Intern Med 72:282-283. Saxton CA, Svatun B, Lloyd AM. (1988) Antiplaque effects and mode of action of a combination of zinc citrate and a nonionic antimicrobial agent. Scand J Dent Res 96: 212217. Saxton CA, van der Ouderaa FJG. (1989) The effect of a dentifrice containing zinc citrate and Triclosan on developing gingivitis. J Periodontol Res 24:75-80. Saxton CA. (1986) The effect of a dentifrice containing zinc citrate and 2, 4, 4Tricholo- 2Hydroxydiphenyl Ether. J Periodontol 57:555-561. Saxton CA. (1989) Maintenance of gingival health by a dentifrice containing zinc citrate and Triclosan. J Dent Res 68 (Spec. Iss):1724-1726. 51
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SCCP. (2006) Scientific Committee on Consumer Products, Opinion on: Triclosan (SCCP/1040/06). Adopted by the SCCP during the 9th plenary meeting of 10 October 2006. SCCP. (2009) Scientific Committee on Consumer Products opinion on: Triclosan (SCCP/1192/08) Adopted by the SCCP during the 19th plenary meeting on 21 January 2009 . SCENIHR, Scientific Committee on Emerging and Newly Identified Health Risks, The antibiotic resistance effect of biocides, adopted by the SCENIHR on January 19, 2009 SCENIHR, Scientific Committee on Emerging and Newly Identified Health Risks, Research strategy to address the knowledge gaps on the antimicrobial resistance effects of biocides, adopted by the SCENIHR on March 17, 2010 Schaeken MJM, van der Hoeven JS, Saxton CA, Cummins D. (1994) The effect of mouthrinses containing zinc and Triclosan on plaque accumulation and development of gingivitis in a 3-week clinical test. J Clin Periodontol 21:360-364. Schmid MB, Kaplan N. (2004) Reduced triclosan susceptibility in methicillin-resistant Staphylococcus epidermidis. Antimicrob Agents Chemother 48:13971399. Schlter A, Szczepanowski R, Phler A, Top EM. (2007) Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool. FEMS Microbiol Rev 31:449-477. Schweizer HP. (2001) Triclosan: a widely used biocide and its link to antibiotics. FEMS Microbiol Lett 202:17. Schweizer HP. (1998) Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems. Antimicrob Agents Chemother 42:394-398. Seaman PF, Ochs D, Day, MJ. (2007) Small-colony variants: a novel mechanism for triclosan resistance in methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 59:43-50. Sidhu MS, Heir E, Srum H, Holck A. (2001) Genetic linkage between resistance to quaternary ammonium compounds and beta-lactam antibiotics in food-related Staphylococcus spp. Microb Drug Resist 7:363-371. Sidhu MS, Heir E, Leegaard T, Wiger K, Holck A. (2002). Frequency of disinfectant resistance genes and genetic linkage with beta-lactamase transposon Tn552 among clinical staphylococci. Antimicrob Agents Chemother 46:2797-2803. Singer H, Mller S, Tixier C, Pillonel. (2002) Triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments. Environ Sci Technol 36:4998-5004. Sivaraman S, Sullivan TJ, Johnson F, Novichenok P, Cui G, Simmerling C, Tonge PJ. (2004). Inhibition of the Bacterial Enoyl Reductase Fabi by Triclosan: A Structure-Reactivity Analysis of FabI inhibition by triclosan analogues. J Med Chem 47:509-518. Slater-Radosti C, Van Aller G, Greenwood R, Nicholas, R, Keller PM, DeWolf WE, Fan F, Payne DJ, Jaworski DD. (2001) Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus. J Antimicrob Chemother 48:1-6. Smith K, Hunter IS. (2008) Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates. J Med Mirobiol 57:966-973. Son HS, Gwangpyo Ko G, KD. (2009) Kinetics and mechanism of photolysis and TiO2 photocatalysis of triclosan. J Hazardous Materials 166:954-960.

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Sondossi M, Rossmoore HW, Wireman JW. (2002) Observations of resistance and crossresistance to formaldehyde and a formaldehyde condenstae biocide in Pseudomonas aeruginosa. Int Biodeter 21:105-106. SSC. (2002) Opinion on Triclosan Resistanvce Adopted by the Scientific Steering Committee at its meeting on 27-28 June 2002 Stackelberg PE, Furlong ET, Meyer MT, Zaugg SD, Hendersen AK, Reissman DB. (2004) persistence of pharmaceutical and other organic wastewater contaminants in a conventional drinking-water-treatment plant. Sci Total Environ 329:99-113. Stasinakis AS, Petalas AV, Mamais D, Thomaidis NS, Gatidou G, Lekkas TD. (2007) Investigation of triclosan fate and toxicity in continuous-flow activated sludge systems. Chemosphere 68: 375-381. Stephen KW, Saxton CA, Jones CL, Ritchie JA, Morrison T. (1990) Control of gingivitis and calculus by a dentifrice containing a zinc salt and Triclosan. J Periodontol 61 674-679. Stewart MJ, Parikh S, Xiao G, Tonge PJ, Kisher C. (1999) Structural basis and mechanisms of enoyl reductase inhibition by triclosan. J Mol Biol 290:859-865. Stickler DJ, Jones GL, Russell AD. (2003). Control of encrustation and blockage of Foley catheters. Lancet 361:14351437 Stickler DJ, Jones GL. (2008) Reduced Susceptibility of Proteus mirabilis to triclosan. Antimicrob Agents Chemother 52:991-994. Stickler DJ. (2004) Intrinsic resistance of Gram-negative bacteria. In: Fraise AP, Lambert PA, Maillard J-Y, editors. Principles and Practice of Disinfection, Preservation and Sterilization 4th edn. Oxford: Blackwell Scientific Publication; p.154-69. Suarez S, Dodd MC, Omil F and von Gunten U. (2007) Kinetics of triclosan oxidation by aqueous ozone and consequent loss of antibacterial activity: relevance to municipal wastewater ozonation. Water Res 41:2481-2490. Suller MTE, Russell AD. (1999) Antibiotic and biocide resistance in methicillin-resistant Staphylococcus aureus and vancomycin resistant Enterococcus. J Hosp Infect 43:281 291. Suller MTE, Russell AD. (2000) Triclosan and antibiotic resistance in Staphylococcus aureus. J Antimicrobial Chemother 46:1118. Sullivan A, Wretlind B, Nord CE. (2003) Will triclosan in toothpaste select for resistant oral streptococci? Cli Microbiol Infect 9:306-309. Svatun B, Saxton CA, Huntington E, Cummins D. (1993a) The effects of a silica dentifrice containing Triclosan and zinc citrate on supragingival plaque and calculus formation and the control of gingivitis. Int Dent J 43:431-439. Svatun B, Saxton CA, Huntington E, Cummins D. (1993b) The effects of three silica dentifrices containing Triclosan on supragingival plaque and calculus formation and on gingivitis. Int Dent J 43:441-452. Svatun B, Saxton CA, Rolla G, van der Ouderaa F. (1989a) One year study of the efficacy of a dentifrice containing zinc citrate and Triclosan to maintain gingival health. Scand J Dent Res 97:242-246. Svatun B, Saxton CA, Rolla G, van der Ouderaa F. (1989b) A one year study on the maintenance of gingival health by a dentifrice containing a zinc salt and non-anionic antimicrobial agent. J Clin Periodontol 16:75-80. Svatun B, Saxton CA, Rolla G. (1990) Six month study of the effect of a dentifrice containing zinc citrate and Triclosan on plaque, gingival health and calculus. Scand J Dent Res 98:301-304.

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Svatun B, Saxton CA, van der Ouderaa F, Rolla G. (1987) The influence of a dentifrice containing a zinc salt and nonionic anti-microbial agent on the maintenance of gingival health. J Clin Periodontol 14:457-461. Svensson A. (2002) Ecotoxic substances in sewage sludge A study of 19 WWTPs in Vastra Gotaland, Sweden. Lansstyrelen I Vastra Gotaland, Report 2002:39. In Swedish. Tabak M, Scher K, Hartog E, Romling U, Matthews KR, Chikindas ML, Yaron S. (2007) Effect of triclosan on Salmonella typhimurium at different growth stages and in biofilms. FEMS Microbiol Lett 267:200-206. Tatarazako N, Ishibashi H, Teshima K, Kishi K, Arizono K. (2004) Effects of triclosan on various aquatic organisms. Environ Sci 11:133-1140 Tattawasart U, Hann AC, Maillard J-Y, Furr JR, Russell AD. (2000a) Cytological changes in chlorhexidine-resistant isolates of Pseudomonas stutzeri. J Antimicrob Chemother; 45:145-152. Tattawasart U, Hann AC, Maillard J-Y, Furr JR, Russell AD. (2000b) membrane changes in Pseudomonas stutzeri strains resistant to chlorhexidine diacetate and cetylpyridinium chloride. Inter J Antimicrob Agents 16:233-238. Ternes TA, Joss A, Siegrist H (2004). Scrutinizing pharmaceuticals and personal care products in wastewater treatment. Environ Sci Technol 38:393A-399A Tkachenko O, Shepard J, Aris VM, joy A, Bello A, Londono I, Marku J, Soteropoulos P, Peteroy-Kelly MA. (2007) A triclosan-ciprofloxacin cross-resistant mutant strain of Staphylococcus aureus displays an alteration in the expression of several cell membrane structural and functional genes. Res Microbiol 158:651-658. Thomas L, Russell AD, Maillard, J-Y. (2005). Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues. J Appl Microbiol 98:533-543. Thompson A, Griffin P, Stuetz R, Cartmell E. (2005) The fate and removal of triclosan during wastewater treatment. Water Environ Resh 77:63-67. Thorrold CA, Letsoalo ME, Dus AG, Marais E. (2007) Efflux pump activity in fluoroquinolone and tetracycline resistant Salmonella and E. coli implicated in reduced susceptibility to household antimicrobial cleaning agents. Int J Food Microbiol 113:315-320. Tixier C, Singer HP, Canonica S, Stephan R. (2002) Phototransformation of Triclosan in Surface Waters: A Relevant Elimination Process for This Widely Used Biocide Laboratory Studies, Field Measurements, and Modeling. Environ Sci Technol 36:34823489 U.S. EPA. 2009. Targeted National Sewage Sludge Survey: Sampling and Analysis Technical Report. Statistical Analysis Report. Available: : http://www.epa.gov/waterscience/biosolids/tnsss-overview.html#results Targeted National Sewage Sludge Survey Statistical Analysis Report ("Statistical Report") (PDF) Valkova N, Lepine F, Valeanu L, Dupont M, Labrie L, Bisaillon J-G, Beaudet R, Shareck F, Villemur R. (2001) Hydrolysis of 4-hydroxybenzoic acid esters (parabens) and their aerobic transformation into phenol by the resistant Enterobacter cloacae strain EM. Appl Environ Microbiol 67:2404-2409. Van der Ouderaa FJG. (1991) Anti-plaque agents Rationale and prospects for prevention of gingivitis and periodontal disease. J Clin Periodontol 18:447-454. van Stee LLP, Leonards PEG, Vreuls RJJ,Brinkman UAT. (1999) Identification of non-target compounds using gas chromatography with simultaneous atomic emission and mass spectrometric detection (GCAED/MS): analysis of municipal wastewater. Analyst 124: 15471552.

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Villalain, J, Mateo, CR, Aranda FJ, Shapiro, S, Micol, V. (2001) Membranotropic effects of the antibacterial agent triclosan. Arch Biochem Biophys 390:128136. Walker C, Borden LC, Zambon JJ, Bonta Cy, DeVizio W, Volpe AR. (1994) The effects of 0.3% triclosan-containing dentiftrice on the microbial composition of supragingival plaque. J Clin Periodontol 21:334-341. Waller NJ, Kookana RS. (2009) Effect of triclosan on microbiological activity in Australian soils. Environ Toxicol Chem 28:65-70 Walsh SE, Maillard J-Y, Russell AD, Hann AC. (2001) Possible mechanisms for the relative efficacies of ortho-phthalaldehyde and glutaraldehyde against glutaraldehyde-resistant Mycobacterium chelonae. J Appl Microbiol 91:80-92. Walsh C, Fanning S. (2008) Antimicrobial resistance in foodbornbe pathogens a cause for concern? Current Drug Targets 9:808-815 Waltman EL, Venables BJ, Waller WZ. (2006) Triclosan in a North Texas wastewater treatment plant and the influent and effluent of an experimental constructed wetland. Environ Toxicol Chem 25:367-372. Webber MA, Randall LP, Cooles S, Woodward MJ, Piddock JV. (2008a) Triclosan resistance in Salmonella enterica serovar Typhimurium. J Antimicrob Chemother 62:83-91. Webber MA, Coldham NG, Woodward MJ and Piddock LJV. (2008b). Proteomic analysis of triclosan resistance in Salmonella enterica serovar Typhimurium. J Antimicrob Chemother 62:92-97. Weber DJ, Rutala WA. (2006) Use of Germicides in the Home and the Health care Setting: Is There a Relationship Between Germicide Use and Antibiotic Resistance? Infect Control Hosp Epidemiol 27:1107-1119. Webster J, Foagali JL, Cartwright D. (1994) Elimination of methicillin-resistant Staphylococcus aureus from a neonatal intensive care unit after hand washing with triclosan. J Paediatr Child Health 30:5964. Wert EC, Rosario-Ortiz FL, Snyder SA. (2009) Effect of ozone exposure on the oxidation of trace organic contaminants in wastewater. Water Res 43:1005-1014. WHO, the world health report 2007 - A safer future: global public health security in the 21st century; available at http://www.who.int/whr/2007/en/index.html Wignall GR, Goneau LW, Chew BH, Denstedt JD, Cadieux PA. (2008). The effects of triclosan on uropathogen susceptibility to clinically relevant antibiotics, J Endourol 22:2349-2356 Wilcox MH, Hall J, Pike R Templeton PA, Fawley WN, Parnell P, Verity P. (2003) Use of perioperative mupirocin to prevent methicillin-resistant Staphylococcus aureus (MRSA) orthopaedic surgical site infections. J Hosp Infect 54:196201. Williams C, McBride S, Mostler K et al. (1998) Efficacy of a dentifrice containing zinc citrate for the control of plaque and gingivitis: a six-month clinical study in adults. Comp Cont Educ Dent; 19 (Special Issue):415. Williams GJ and Stickler DJ. (2008) Effect of triclosan on the formation of crystalline biofilms by mixed communities of urinary tract pathogens on urinary catheters. J Med Microbiol 57:1135-1140. Winder CL, Al-Adham IS, Abdel Malek SM, Buultjens TE, Horrocks AJ, Collier PJ. (2000) Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1. J Appl Microbiol 89:289-295. Wisniewska K, Piechowicz L, Galinski J. (2006) Reduced susceptibility to triclosan in methicillin-resistant Staphylococcus aureus. Med Dosw Mikrobiol 58:11-17. Xie Z, Ebinghaus R, Flser G, Caba A and Ruck W. (2008) Occurrence and distribution of triclosan in the German Bight (North Sea). Environ Poll 156:1190-1195. 55
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Yazdankhah SP, Scheie AA, Hoiby EA, Lunestad BT, Heir E, Fotland TO, Nartestad K, Kruse H. (2006) Triclosan and antimicrobial resistance in bacteria: an overview. Microb Drug Resist 12:83-90 Yu JC, Kwong TY, Luo Q, Cai Z. (2006) Photolytic oxidation of triclosan. Chemosphere 65: 390-399 Ying GG, Kookana. (2007) Triclosan in wastewaters and biosolids from Australian wastewater treatment plants. Environ Int 33:199-205. Zafar AB, Butler RC, Reese DJ, Gaydos LA, Mennonna PA (1995) Use of 0.3% triclosan Bacti-Stat) to eradicate an outbreak of methicillin-resistant Staphylococcus aureus in a neonatal nursery. Am J Infect Control 23:200208. Zhang YM, Lu YJ Rock CO. (2004) The reductase steps of the type II fatty acid synthase as antimicrobial targets. Lipids 39:10551060. Zhang S, Zhang Q, Darisaw S, Ehie O, Wang G. (2007) Simultaneous quantification of polycyclic aromatic hydrocarbons (PAHS), polychlorinated biphenyls (PCBS), and pharmaceuticals and personal care products (PPCPS) in Mississippi River water, in New Orleans, Louisiana, USA. Chemosphere 66:1057-1069. Zhao F. (2006) Biodegradation of triclosan by a triclosan-degrading isoalate and an ammonia-oxidizing bacterium. A Thesis for the Master of Science, Office of Graduate Studies of Texas A&M University. Zhu L, Lin J, Ma J, Cronan JE, Wang H. (2010). The Triclosan Resistance of Pseudomonas aeruginosa PA01 is Due to FabV, a Triclosan-Resistant Enoyl-Acyl Carrier Protein Reductase. Antimicrob Agents Chemother 54:689-698.

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Personal Care

Products Council
Committed to Safety, Quality & Innovation

Memorandum
TO: F. Alan Andersen, Ph.D. Director COSMETIC INGREDIENT REVIEW (CIR)
-

FROM:

Triclosan Review Subcommittee of the CJR Science and Support Committee Mayll,2010 Comments on the Scientific Literature Review on Triclosan

DATE: SUBJECT:

The Triclosan Review Subcommittee of the CR Science and Support Committee is concerned that this draft of the Triclosan report is still incomplete. The attached comments should be addressed before the CIR Expert Panel is provided a draft of the Triclosan report to review. Therefore, please delay the review of this report until at least the August 30-3 1, 2010 CR Expert Panel meeting. References that still need to be added to the report: 2009 SCCP Opinion on Triclosan (available from the On-Line) Ailmyr M, Panagiotidis G, Sparve E, et al. 2009. Human exposure to triclosan via toothpaste does not chinge CYP3A4 activity or plasma concentrations of thyroid hormones. Basic Clin Pharmacol Toxicol. 105(5):339-44. Bagley D, Lin Y. 2000. Clinical evidence for the lack of triclosan accumulation from daily use in dentifrices. Am J Dent 13: 148-152. (attached) Calafat A, Ye X, Wong L, Reidy J, Needham L. 2008. Urinary concentrations of triclosan in the U.S. population: 2003-2004. Envion Health Perspect 116: 303-307. Crofton, K; Paul K, DeVito M, Hedge J. 2007. Short-term in vivo exposure to the water contaminant triclosan: Evidence for disruption of thyroxine. Env. Tox. Pharm 24: 194-197. Dayan A. 2007. Risk assessment of triclosan [Irgasan] in human breast milk. Food Chem Toxicol 45: 125-128. (attached) Rodricks JV, Swenberg JA, Borzelleca JF, et al. 2010. Triclosan: A critical review of the experimental data and development of margins of safety for consumer products. Critical Reviews in Toxicology 40(5): 422-484. More information on exposure to Triclosan can be found in the 2009 SCCP opinion on Triclosan, the Rodricks et al. (2010) review, and the CDC biomonitoring study (Calafat et al., 2008). The expectation is that the CR Expert Panel will limit Triclosan use in cosmetics to USP grade material. The Rodricks et al. (2010) review provides an overview of the Triclosan carcinogenicity data. Endocrine activity of Triclosan is reviewed in the following unpublished review (attached).

1101 17th Street, N.W., Suite 3OO Washington, D.C. 20036-4702

202.331.1770

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www.personalcarecouncil.org

CIR Panel Book Page 293

Environ International Corporation. 2009. Investigation of potential endocrine activity of Triclosan. Assessing the potential for Triclosan when used in cosmetic products to result in bacterial resistance is beyond the scope of the CIR Expert Panels mission to review and assess the safety of ingredients used in cosmetics. If bacterial resistance continues to be an issue considered in the CIR report, the 2010 SCCS Preliminary opinion on triclosan antimicrobial resistance (available from the On-Line) needs to be added to the report. p.3 Laundry detergents and fabric softeners are not regulated by FDA. These product categories are regulated by the Consumer Product Safety Commission (CPSC) when they do not contain antimicrobial ingredients, and by the Environmental Protection Agency (EPA) when they contain antimicrobial ingredients. Facial tissues are only regulated by FDA when they make cosmetic or drug claims (see: http ://www.fda. gov/lCECllComplianceManuals/CompliancePolicyGuidanceManual/ucmO73 82 5.htm). p.3 The heading Tricolsan in Cosmetics needs to be corrected to Triclosan in Cosmetics. p.3 The statement in Japan, Triclosan in cosmetics is limited to a maximum concentration of 0.1% should be moved to the previous sentence with the other information about Japan. This information is currently in a sentence about Triclosan regulations in Europe. p6 It is possible for a substance to show receptor binding activity in in vitro studies but not have activity in vivo. Therefore, please state and thus may act as an endocrine disruptor. p.7 It is not necessary to include trade names and trade name mixtures in the report, as these are likely to change by the time the report is published. If they are left in the report Pretameen needs to be changed to Protameen. p.10, p.30 Table 5 At the April CIR Expert Panel meeting, the Cifi Expert Panel clearly indicated that they did not want product names from EWG to be included in CIR reports. Please remove the brand name of the dry shampoo and the lip color. The dry shampoo is a product that does not use water to clean hair so it is formulated quite differently than traditional shampoos. It would more appropriately be in the other hair preparations (noncoloring) category. p.10 In the last paragraph of the Cosmetic aerosol section, please change particle diameters to aerodynamic diameters or da as used in the previous paragraph. p.11 Please provide some quantitative information about the dermal penetration and toxicokinetics of Triclosan. For example, additional information can be found in the 2009 SCCP opinion, Bagley and Lin (2000), and Rodricks et al. (2010). p.11 What concentrations of Triclosan are used in hand washes? The sentence: Leave-on cosmetics, however, are reported to contain only 0.1-0.3% Triclosan, a factor of 3-10 less than seen in handwashes. suggests that up to 3% Triclosan is used in hand washes, although earlier on this page it says that hand wash contained 1%. p.12 The NOAEL for baboons is from a 4 or 13 week study. It should not be presented in the Acute Exposure section. p.12 Please provide the concentrations of Triclosan considered a moderate dermal irritant. It would be helpful if the data on which Australia concluded that Triclosan is a weak sensitizer in guinea pigs were summarized in this report.
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p.12 In the paragraph on Repeat dose exposure, it says summaries are presented in Table 8 it should be Table 7. p.13 Please provide the route of exposure of the two-generation reproductive toxicity study reviewed by EPA. This study should be referred to as the rat reproductive toxicity study rather than the rat reproductive study. p.13 Please provide the routes of exposure for the studies summarized in the Developmental Toxicity section. p.13 Please move the Fort et al. (2010) study to the endocrine disruption section. p.13 In the third paragraph in the Endocrine Disruption section, it should be stated that Triclosan estrogenic antagonist activity is very weak. As there are no data to support that Triclosan is an androgen receptor antagonist, please delete the following. While the authors stated that Triclosan was a potent antagonist in the AR assay, no data were provided. p.13-15 In the Endocrine Disruption section, it would be helpful to have subheadings for In Vitro Studies and In Vivo Studies. p.14 It should be made clear that three doses (5, 10, 20 mg/kg/day) were used in the Kumar et al. (2009) (ref. 33) study. Rather than stating Endpoints studied included decreased body weights (no-effect level of 20 mg/kg/day), it should state Triclosan did not induce changes in body weight at any of the test doses. p.14-15 A brief summary of the Crofton et al. (2007) study should also be added to the report. It should be noted that no clinical signs of toxicity were observed in either the Crofton et al. (2007) or the Paul et al. (2010) studies. p.14-15 What was the route of exposure used in the Paul et al. (2010) study? p.15 In the first complete paragraph, please revise the following as it does not make sense: with decreases at 100, 300, and 1000 mg/kg/day producing significant decreases. p.16 What was the route of exposure in the rat oral carcinogenicity study evaluated by EPA (a concentration is given but it does not state if this is a dietary or drinking water study)? p.18 The second paragraph of the summary notes that Triclosan uses are likely under reported to the VCRP. Why is the under reporting being mentioned for Triclosan while under reporting is not specifically mentioned in most other CW reports? p.20 Please provide the concentrations at which Triclosan is a moderate dermal and eye irritant. p.21, Figure 2 Was there any information on how high the temperature needs to be to convert Triclosan to triclorodibenzo-p-dioxin? Please provide a reference for this figure. p.22, Table 1 It would be better to cite that Triclosan is under consideration as an acne therapeutic to the Federal Register notice that was published in December 2005. p.22, Table 1 Deodorants are not regulated under CDER. The 1994 tentative monograph on antibacterial products should be added to this table. The FDA request for Cifi to review Triclosan should not be in this table as it is not a Regulatory Decision. p.22, Table 1 not to be used by children <12 years of age and labeled do not swallow is a restriction in Canada and should not be in the Comment field. p.31, Table 6 It is not clear where the dermal absorption value came from. As there are no rat plasma levels reported, that column should be deleted. As there are no data in the Ocular row, that row should be deleted.
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p.32-33 Please provide more details of these studies, e.g., the actual duration of the study, numbers of animals, more details about the oral route of exposure (gavage, diet, drinking water). p.36, Reference 35 Please correct thyrozine tothyroxine?
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CIR Panel Book Page 296

Personal Care

Products Council
Committed to Safety, Quality & Innovation

Memorandum
TO: F. Alan Andersen, Ph.D. Director COSMETIC ThGREDIENT REVIEW (CIR)
-

FROM:

John Bailey, Ph.D. Industry Liaison to the CW Expert Panel


May 17, 2010

J__. I

( (9.i(b

DATE:
SUBJECT:

Comments on the Scientific Literature Review on Triclosan

Enviorn International Corporation. 2010. Comments on the Scientific Literature Review Safety of Triclosan as a Preservative in Cosmetics.

1101 17th Street, N.W., Suite 300

Washington, D.C. 20036-4702

202.331.1770

202.331.1969 (fax)

www.personalcarecouncil.org

CIR Panel Book Page 297

Comments on the Scientific Literature Review Safety of Triclosan as a Preventative in Cosmetics Dated: April 9, 2010

Prepared by: ENVIRON International Corporation May 11, 2010

General Comment:
The Cosmetic Ingredient Review group has released entitled, Scientific Literature Review on the Safety of Triclosan as a Preventative in Cosmetics. As noted, preparation of this Scientific Literature Review (SLR) document relied on reviews prepared by various government agencies, especially that prepared by the Australian Government Department of Health and Ageing (NICNAS), the United States Environmental Protection Agency (USEPA) and US National Toxicology Program (NTP). A more recent, comprehensive review of the triclosan toxicity and pharmacokinetic data, which relied on a review of the prime literature, should be considered (Rodricks et al. 2010). While the SLR clearly states that the CW has relied extensively on reviews already available from various governmental sources, some review of primary literature is needed to insure that study results have been summarized correctly.

Specific Comments:
Comments on CIR Toxicokinetic section (Section IV) Both in vivo and in vitro studies have been conducted in human volunteers and in animals to evaluate the potential dermal absorption of triclosan following single or repeated applications of various products and formulations, as reviewed by Rodricks et al. (2010). In vitro dermal absorption studies with human skin to which several triclosan containing products, e.g., body lotion, were used to provide estimates of the amount of triclosan delivered from dermal application (Rodricks et al. 2010). These results were compared to triclosan levels reported in the latest NHANEs study. In this SLR, reliance on this single study to derive an estimate of systemic exposure, expressed in igIkgJday, is likely to result in an overestimate of that exposure. p. 11, Absorption/Toxicokinetics While a single plasma level as high as 229 ng/ml was reported in one volunteer, this level was the maximum concentration measured in one out of 13 volunteers on day 20 of a pilot study designed to evaluate dermal absorption of triclosan from a specific use pattern of a handwash

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product (Ciba Specialty Chemicals Corporation 2002). The mean plasma concentration for the entire group of volunteers was approximately 73 ng/ml (mean value included the one outlier value of 229 ng/ml); the minimum concentrations were betow the limit of quantitation. p. 1, Absorption/Toxicokinetics The estimate of plasma levels associated with 1 leave-on cosmetics (1.15 to 3.82 igIml) appears to be based on a factor of 3 to 10 less than the estimated plasma concentration from whole body exposure to a handwash (11.45 pg/mI), rather than 3 to 10% less than seen in handwashes (<229 ng/ml). This clearly is an overestimate of the amount of triclosan levels in plasma resulting from leave-on cosmetics, which would be expected to cover only a fraction of the body. In addition, these triclosan plasma levels would be expected to be similar to that measured from contact with a handwash product. This is critical if these plasma levels were to be compared with plasma levels from toxicity studies in an attempt to evaluate the potential for health effects from leave-on cosmetics.
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p. 1, Distribution The available information from rodent radioactivity studies 1 suggests similar distribution patterns in the hamster and the rat, with no evidence of accumulation in tissues. In contrast to this, distribution of triclosan in the mouse is different, with evidence of accumulation in the liver observed (Rodricks et al. 2010).
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P.11, Metabolism For most species, including humans, the glucuronide metabolite is the predominant metabolite at lower concentrations; however, in the case of the mouse and the dog, the sulfate conjugate is the dominant metabolite formed. The metabolism shift from the generation of predominantly glucuronide conjugates to sulfate conjugates depending upon triclosan concentrations is not observed in the mouse (van Dijk 1995; Rodricks et al. 2010).

Comments on the Reproductive and Developmental Toxicity Section (Section V):


On p. 12, the document states that triclosans potential reproductive/developmental effects indicate that triclosan is not highly toxic for these endpoints. This statement is misleading and may imply to the reader that triclosan has reproductive and developmental effects at some high dose. The reproductive and developmental studies conducted with triclosan do not support such a conclusion. On p. 13, under Reproductive Toxicity, the SLR states that in the two generation rat study, triclosan produced both reproductive and systemic effects at the highest dose tested, 150 mg/kg/day. That statement is inaccurate. This study was reviewed in detail in Rodricks et al. (2010).

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No effects on reproductive function were reported in either the FO or Fl parental generations nor were consistent or dose-related indices of pup health and survival in the Fl and F2 offspring. Decreases in pup body weight in offspring of dams receiving the highest dose of triclosan 150 mg/kg/day were noted in the Fl generation pups after Post-natal day 14 but returned to control levels at later time point. Pup body weights were not affected at in any group in the F2 generation. Transient changes in body weight not accompanied by any changes in reproductive competence should not be classified as a systemic effect.

On p. 13 under Developmental Toxicity, the SLR cites the USEPA review for information on developmental endpoints. o No discussion of developmental toxicity studies is provided in the EPA (2008) document cited in the SLR. The EPA (2008) document cites a separate EPA document entitled 5-Chloro2-(2,4dichlorophenoxy)phenol (triclosan): Toxicology Chapterfor the Reregistration Eligibility Decision (RED) document and dated August 29, 2008 as providing this information. However, this document could not be readily located on the EPA website (www.epa.gov). The basis for the NOAEL of 50 mg/kg/day cited by NICNAS (2009) is unclear. CIR incorrectly suggests that it is based on a mouse study that showed maternal toxicity, but not developmental toxicity; however, this is also incorrect. The study discussed is a mouse developmental study conducted at Argus Research Laboratories (1992) in which pregnant CD-i mice were given triclosan in the diet on gestation days (GD) 6 through 15 at concentrations resulting in doses of 0, 10, 25, 75, or 350 mg/kg/day; therefore this study does not provide the basis for a maternal NOAEL of 50 mg/kg/day.

Comments on the Endocrine Disruption Section (Section V)


On pgs.14 and 15, the SLR has a section on the potential endocrine disruption action of triclosan. The section relied heavily on two in vitro studies and did not integrate the full toxicity data based, especially the data generated in chronic bioassays in mice, rats, and hamsters with the results from short-term studies of changes in thyroid hormone levels. The SLR relied on the in vitro studies conducted by Gee et al. (2008) and Ahn et al. (2008) that investigated triclosan interaction with estrogenic and androgenic receptors; however, a number of issues must be considered. o While these in vitro studies suggest potential activity of triclosan at certain receptor sites, it must be considered that the cells used in some of these experiments were conducted in immortalized or cancer cell lines that are 3
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not expected to respond in a similar manner to normal or primary cells (Gentry et a!. 2010).
o Tn aclclition cc!I lines, such as those drrvcc1 from the kidney, are not the target organ of concern and may not behave in a manner similar to cells from the appropriate organ.

The concentrations of tnclosan and endogenous steroids used in some of these assays (i.e., Chen et al. 2007) were selected by the authors to achieve a maximum response and are not similar to those expected in vivo. It should be noted that no effects on reproduction or development were reported in the battery of tests for tnclosan conducted in vivo in rats at doses of tnclosan ranging from 15 to 150 mg/kg in a two-generation reproductive study (Morseth 1988) or at doses of 15 to 300 mg/kg/day in one-generation developmental studies (Schroeder and Daly 1992a; Denning et al. 1992). In addition, no developmental effects have been noted in mice administered doses up to 25 mg/kg/day (Christian and Hoberman 1992), or in rabbits administered doses up to 50 mg/kg/day (Schroeder and Daly 1992b) or in hamsters administered doses up to 80 mg/kg/day (Piekacz 1978). Therefore, these in vitro results must be interpreted with caution when extending them to the whole animal.

The SLR also discusses the potential effects of triclosan on thyroid hormone levels. Again a number of issues must be considered before drawing conclusions as to the relevance of these short-term studies reporting changes in thyroid hormone levels in the absence of evidence of toxicity.

With regard to the potential effects on the thyroid: The short-term studies conducted in rats report decreases in one of the three key thyroid hormone levels (T4) when triclosan was administered at very high doses (up to 300 to 1000 mg triclosan/kg body weight/day) (Crofton et al. 2007; Paul et al. 2009, 2010; Zorrilla et al. 2009). Levels of TSH were not affected in any of these studies, indicating a lack of direct effect on centrally-mediated control of thyroid function but rather likely to be the result of altered levels of liver enzymes. No signs of altered thyroid function was seen in any of the toxicity studies conducted in several species for durations up to two years: o No overt signs of thyroid-related toxicity in experimental animals or human volunteers,

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No signs of continued pressure on thyroid homeostasis, such as the production of thyroid hyperplasia or neoplasia, and No signs of altered thyroid homeostasis during critical windows of development from in utero exposure.

It should be noted that changes in thyroid hormone levels in rats do not necessarily mean that thyroid function in rats will be altered or that physiological systems in rats will be adversely affected. Nor do these changes in serum thyroid hormone levels in rats translate quantitatively to similar changes in humans because of the differences between rats and humans in both the capacity to keep thyroid hormones within normal physiological ranges but also because of the decreased sensitivity of humans to these changes. Further, no changes in thyroid hormone levels were measured in human volunteers who brushed twice daily with a triclosan-containing toothpaste (triclosan concentration was 0.3% w/w) for 14 days resulting in an estimated intake typical for the use of triclosan-containing toothpaste of 0.01 mg/kg/day).

With regard to the potential from reproductive/developmental effects: As noted above for the changes in serum thyroid hormone concentrations, changes in levels of circulating hormones associated with male or female reproductive systems in rats serve as a screen for the potential of a xenobiotic to have an adverse impact on reproductive/developmental competency in humans. Such changes in these screening assays should be viewed in parallel with in vivo tests for reproductive/developmental effects, when conducted as they have for triclosan, to determine if changes in hormone levels measured in these screening studies were sufficient to affect function. Two studies were conducted in rats with considerably different results reported (Kumar et a!. 2009; Zorrilla et al. 2009). Both Kumar et al. (2009) and Zomlia et al. (2009) evaluated changes in LH and testosterone concentrations, both indicators of a potential impact on steroidogenesis. o Kumar et al. (2009) reported significant decreases in LH, testosterone, and testes weight, while Zorrilla et al. (2009) did not. Zorrilla et al. (2009) did not see any histopathological changes in the reproductive tissues examined. More importantly, Zomlla et al. (2009) reported no change in the age of onset of preputial separation, an indicator of the onset of puberty, which would have been expected if the changes in serum hormones, i.e., testosterone were impacted.

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Several methodological flaws in the Kumar et al. (2009) raise questions about these results and their application to humans.

While Kumr ct al. (2009) measured channes in endocrine levels associated cith normal functioning of the reproductive/developmental system, no evidence of an impact on this organ system in animals has been demonstrated after chronic exposure in rats (Yau and Green 1986), in a two-generation reproductive study in rats (Morseth 1988), or in developmental studies in rats (Schroeder and Daly 1992a; Denning et al. 1992) or in mice (Christian and Hoberman 1992), hamsters (Piekacz 1978) or rabbits (Schroeder and Daly 1992b). The lack of reproductive/developmental effects reported in several tnclosan studies in experimental animals do not support the potential for triclosan to result in adverse reproductive effects in rats or humans. More importantly, humans have high levels of naturally occurring estrogen and testosterone. As with thyroid hormones, humans have a critical buffering capacity to bind these hormones and act as a reserve to release free biologically active estrogen or testosterone, as well as critical feedback loops involving the pituitary-hypothalamic axis to respond to decreased or increased levels of estrogen or testosterone. Both the capacity in humans to bind and release these hormones, with a much higher capacity in humans than in rodents, and the feedback loops act to maintain homeostasis. As with the thyroid, changes in hormone status that could occur with exposure to a xenobiotic can be compensated for in humans by these active systems that maintain homeostasis, especially at environmentally relevant exposures to triclosan.

Comments on the Carcinogenicity Section (Section V)


On p. 16 the SLR correctly stated that triclosan is not considered likely to be a human carcinogen. On p. 16, first paragraph, the SLR states that the FDA considered the data inadequate to address the question of carcinogenicity by the oral route of exposure. It is clear, however, that tnclosan was not carcinogenic when administered to rats and hamsters in the diet resulting in internal doses that would exceed by possibly orders of magnitude that received by the dermal route from the use of tnclosan-containing products. The SLR correctly reports that USEPA has concluded that triclosan when administered in the rat and hamster 2-year dietary studies was not carcinogenic. Further the USEPA concluded that increase in the incidence of liver cancer in the mouse 2-year dietary study occurred by a PPARCL mode of action and was not likely to be carcinogenic in humans.

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The SLR also correctly states that USEPA noted that the PPARct mode of action is theoretically plausible in humans. However, the SLR failed to mention the USEPAs final conclusion that due to pharmacokinetic and pharmacodynamic responses, ic.. the response to PPARa cornpunds in humans is unlikc thet in the rodent, such a response leading to cancer in humans is highly unlikely. PPARct compounds have been used therapeutically for generations as cholesterollowering agents without such effects manifested.

Comments on the Antibacterial/Antimicrobial Resistance (Section VI) The CW SLR reviewed the safety of triclosan as a preservative in cosmetics from the perspective of antibacterial/antimicrobial resistance. The review concluded that although improvements can be made on analytical methods to assess antimicrobial resistance and that laboratory studies have demonstrated triclosan resistance, studies have not indicated triclosan resistance occurring in situ. The CW SLR provides a summary of laboratory-elucidated mechanisms of triclosan resistance, and indicates that differing responses occur depending on the dose (bactericidal compared bacteriostatic). The CIR SLR also concluded that bacteria resistance to triclosan can be developed in laboratory studies. The CIR SLR conclusions were based on the SCCS 2002 report and Lambert (2004). While there is little doubt that triclosan selection/resistance can be derived in the laboratory (Russell, 2003), the value in relying on laboratory-derived data in predicting likely in situ responses has been questioned (Russell, 2003; and SCCS, 2010). Limitations in the laboratory studies for predicting in situ responses have been identified including the use of E. coli and several other enteric species as models for biocide resistance assessments (Bailey et al. 2009; Ledder et al. 2006; McBain, et al. 2004); the solubility threshold of triclosan in laboratory studies compared to environmental exposures (Aiello, et al. 2007; and Russell, 2003); and the use of minimum inhibitory concentrations as opposed to minimum bactericidal concentrations (SCCS, 2010). The CIR SLR has not included several limitations of laboratory studies in its review and conclusions. It is unclear from the laboratory studies if the results reflect true acquired resistance, especially due to genetic changes, or because of the use of bacterostatic concentrations, the selection of subpopulations in the bacterial colony that may already have, for example, an enhanced efflux pump or decreased cell permeability. Triclosan has been extensively studies in mutagenicity and genotoxicity studies (as reviewed in Rodncks et al. 2010) that clearly demonstrate that triclosan has been classified as non-mutagenic in bacterial species and non genotoxic in mammalian species. The CIR SLR refers to the very few targeted studies of resistance to triclosan in situ. At least six in situ studies (Cole et al. 2003; Jones et al. 1987; Ledder et a].

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2006; McBain et al. 2004; Sullivan et al. 2003; and Walker et aT. 1994); and one meta-analysis (Aiello et al. 2004) have failed to demonstrate an increase in antibiotic resistance following triclosan use. Although drawing similar conchisions to the seven studies iclcntificr! hove. the CIR SLR only rites the studies by Aioello et aT. (2004), Cole et al. (2003) and Walker et aT. 1994), the CIR SLR should review and cite the additional in situ studies as there is a limited number available. There is a reference to2009 SCENIHR, further in the summary a reference to a more recent opinion by SCENIIIR, both refer to the same reference. The second reference is probably the 2010 Draft Preliminary Opinion. The reference should be checked and revised accordingly.

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References
Aiello, A. E., E. L. Larson, and S. B. Levy. 2004. Relationship between triclosan and susccp lit les or bacteria isoatcd rrum hands in the community. nit; cmhial Agents chemotherapy. 48: 2973-2979. Aiello, A. E., E. L. Larson, and S. B. Levy. 2007. Consumer antibacterial soaps: Effective or just risky? Clinical Infectious Diseases. 45 :S 137-47. Bailey, A.M., C. Constantinidou, A. Ivens, M.I. Garvey, M.A. Webber, N. Coidham, J.L. Hobman, I. Wain, M.J. Woodward, and L.J.V. Piddock. 2009. Exposure of Escherichia coli and Salmonella enterica serovarTyphimurium to triclosan induces a species-specific response, including drug detoxification. Journal of Antimicrobial Chemotherapy. 64, 973-985. Chen, J., Ahn, K., Gee, N., Gee, S., Hammock, B., and B. Lasley. 2007. Antiandrogenic properties of parabens and other phenolic containing small molecules in personal care products. Toxicology and Applied Pharmacology 22 1(3) 278-284. Christian, M., and Hoberman, A. 1992. Developmental Toxicity (Embryo-Fetal Toxicity and Teratogenic Potential) Study of C-P Sample No. 38326 Administered Orally Via the Diet to Crl:CD-1(ICR)BR Presumed Pregnant Mice. Argus Research Laboratories,. Protocol number 403-0 10. Sponsors Study number 92-001. Ciba Speciality Chemicals Corporation. 2002. A pilot study for the in vivo evaluation of the percutaneous absorption of triclosan. Unpublished report No: CIBA03-01-013 1. Arizona Clinical Research Centre, Tuscon, Arizona, USA. Cole, E. C., R. M. Addison, J. R., Rubino, K. E. Leese, P. D. Dulaney, M. S. Newell, J. Wilkins, D. J. Gaber, T. Wineiger, and D. A. Criger. 2003. Investigation of antibiotic and antibacterial agent cross-resistance in target bacteria from homes of antibacterial product users and non users. Journal of Applied Microbiology. 95, 664676. Crofton, K., Paul, K., DeVito, M., and J. Hedge. 2007. Short-term in vivo exposure to the water contaminant triclosan: Evidence for disruption of thyroxine. Environmental Toxicology and Pharmacology 24 194-197. Denning, H., Sliva, S., and Wilison, G. 1992. Tnclosan: Effects on Pregnancy and Post-Natal Development in Rats. Environmental Safety Laboratory. Unilever Research. Document Reference 92-105. Gentry, P., McDonald, T., Sullivan, D., Shipp, A., Yager, J., and H.J. Clewell, III. 2010. Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicity. Environmental and Molecular Mutagenesis 5 1:1-14.

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Jones, R. D., H. B. Jampani, J. L. Neman, and A. S. Lee. 2000. Triclosan: a review of effectiveness and safety in health care settings. American Journal of Infection Control. 28, 184-196. Kumar, V., Chakraborty, A., Kural, M., and P. Roy. 2009. Alteration of testicular steroidogenesis and histopathology of reproductive system in male rats treated with triclosan. Reproductive Toxicology 27(2): 177-185. Lambert, R. J. W. 2004. Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000. Journal of Applied Microbiology. 97:699-711. Ledder, R.G., P. Gilbert, C. Willis, and A.J. McBain. 2006. Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates. Journal of Applied Microbiology. 100.1132-1140. McBain, A.J., R.G. Ledder, P. Sreenivasan, and P. Gilbert. 2004. Selection for highlevel resistance by chronic triclosan exposure is not universal. Journal of Antimicrobial Chemotherapy. 53, 772-777. Morseth, S. 1988. Two-Generation Reproduction Study in Rats Fat 80023 A. CibaGeigy Corporation. Hazleton Laboratories America, Inc. HLA Study No. 2386-100. March 18. Paul, K., Hedge, J., DeVito, M., and K. Crofton. 2010. Short-term exposure to triclosan decreases thyroxine in vivo via upregulation of hepatic catabolism in young Long-Evans rats. Toxicological Sciences 113(2):367-379. Paul, K., Hedge, J., DeVito, M., and K. Crofton. 2009. Triclosan disrupts thyroxine mechanisms and life-stage susceptibility. Toxicologist 108(1) No. 163. Piekacz, H. 1978. Effects of certain preservative agents on the course of pregnancy and fetal development in experimental animals with preliminary toxicological characters. Roczn Pzh. 29(5) 469-48 1. Rodncks,J.V., Swenberg, J.A., Borzelleca, J.F., Maronpot, RR., and A.M. Shipp. 2010. Triclosan: a critical review of the experimental data and development of margins of safety for consumer products. Critical Reviews in Toxicology 40(5):422484. Russell, A. D. 2003. Biocide use and antibiotic resistance: the relevance of laboratory findings to clinical and environmental situations. Lancet Infectious Diseases. 3, 794-803.

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Schroeder, R., and Daly, I. 1992a. A Segment II Teratology Study in Rats with Irgacare MP (C-P Sample No. 38328). Bio/Dynamics. Project No. 9 1-3665. ColgatePalmolive Study No. 91-005. April 16. Schroeder, R., and Daly, I. 1992b. A Segment II Teratology Study in Rabbits with Irgacare MP (C-P Sample No. 38328). Bio/Dynamics. Project No. 91-3666. Colgate-. Palmolive Study No. 9 1-006. April 16. Scientific Committee on Consumer Safety. 2010. Preliminary Opinion on triclosan th antimicrobial resistance. Approved for public consultation at its 6 plenary of 23 March, 2010. Sullivan, A., B. Wretlind, and C.E. Nord. 2003. Will triclosan in toothpaste select for resistant streptococci? Clinical Microbiology and Infection. 9:306-309. Van Dijk, A. 1995. 14C-Triclosan: Absorption, Distribution Metabolism and Elimination after Single/Repeated Oral and Intravenous Administration to Mice. RCC Umweltchemie AG. RCC Project 337781. March 1. Walker, C., L. C. Borden, J. J. Zambon, C. Y. Bonta, W. DeVizio, A. R. Volpe. 1994. The effects of 0.3% triclosan-containing dentrifice on the microbial composition of supragingival plaque. Journal of Clinical Periodontology. 21:334-41. Yau, E., and Green, J. 1986. Fat 80023 2-Year Oral Administration to Rats (MIN 833005). Ciba-Geigy Limited. April 28. Zorrilla, L., Gibson, E., Jeffay, S., Crofton, K., Setzer, W., Cooper, R., and T. Stoker. 2009. The effects of triclosan on puberty and thyroid hormones in male Wistar rats. Toxicological Sciences 107(1): 56-64.

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