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NATURAL AND ENVIRONMENTAL SCIENCES 241
Cytotoxicity and Spermatozoa Motility Inhibition Resulting from Reactive Dyes and Dyed Fabrics
Doctoral dissertation To be presented by permission of the Faculty of Natural and Environmental Sciences of the University of Kuopio for public examination in Auditorium ML1, Medistudia building, University of Kuopio on Friday 17 th October 2008, at 12 noon
Department of Biosciences University of Kuopio
JOKA KUOPIO 2008
Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 40 355 3430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html Professor Pertti Pasanen, Ph.D. Department of Environmental Science Professor Jari Kaipio, Ph.D. Department of Physics
Savonia University of Applied Sciences Kuopio Academy of Design P.O. Box 98 FI-70101 KUOPIO FINLAND Tel. +358 17 308 111 Fax +358 17 308 222 E-mail: email@example.com Docent Pirjo Lindström-Seppä, Ph.D. Faculty of Medicine University of Kuopio Professor Jyrki Liesivuori, Ph.D. Department of Pharmacology, Drug Development and Therapeutics University of Turku
Professor Hanna Tähti, Ph.D. Faculty of Medicine, Medical School University of Tampere Professor Pertti Nousiainen, Ph.D. Department of Materials Science Tampere University of Technology
Docent Eero Priha, Ph.D. Finnish Institute of Occupational Health Tampere
ISBN 978-951-27-0979-3 ISBN 978-951-27-1094-2 (PDF) ISSN 1235-0486 Kopijyvä Kuopio 2008 Finland
Klemola, Kaisa. Textile toxicity: Cytotoxicity and spermatozoa motility inhibition resulting from reactive dyes and dyed fabrics. Kuopio University Publications C. Natural and Environmental Sciences 241. 2008. 67 p. ISBN 978-951-27-0979-3 ISBN 978-951-27-1094-2 (PDF) ISSN 1235-0486 ABSTRACT The textile industry utilises chemicals in the production of ﬁbres, to reﬁne materials in different processes and to produce better quality textile products. Although the chemical itself may be toxic, there is limited data relating to the toxicity of the ﬁnal textile product. This information is of clear importance for consumers. The aim of this study was to investigate the toxicity of textile substances by using cell tests in vitro. These tests have been found to be useful when materials containing unknown chemicals need to be evaluated. Boar semen, mouse hepatoma cell line (hepa-1) and a human keratinocyte cell line (HaCaT cells) were exposed to different concentrations of three reactive dyes (Reactive Yellow 176, Reactive Red 241 and Reactive Blue 221) and to the extracts of cotton fabrics dyed with these dyes. The viability of the cell cultures was evaluated. The concentrations IC50 and IC20 to decrease cell protein concentrations in Hepa-1 and HaCaT cell cultures were calculated. These values represent the concentration of the test sample where the protein content in the wells is 50% (IC50) or 80 % (IC20) compared to that of non-exposed cells. The IC20 values were taken to represent the limit of toxicity for fabric extracts. The IC50 and IC20 values were estimated when the dyes were studied. The spermatozoa motility inhibition test was considered to show evidence of toxicity, if at least 25% of the cells were not motile by microscopic observation (50% was set as maximal value of viability). Thus in the spermatozoa test only IC50 value was estimated. After 24 hours exposure of spermatozoa cells to reactive dyes, the IC50 values were 135 µg/ml (yellow), 124 µg/ml (red) and 127 µg/ml (blue). After 72 hours exposure, the blue dye was most toxic to the spermatozoa cells. In hepa-1 cells, no statistical signiﬁcant difference in the toxicity between blue, red and yellow was found, the IC50 values being as follows: 392 µg/ml (yellow), 370 µg/ml (red), 361 µg/ml (blue). The IC20 values were 176 µg/ml (yellow), 108 µg/ml (red), 158 µg/ml (blue). In HaCaT cells, IC50 values were 237 µg/ml (yellow), 155 µg/ml (red), 278 µg/ml (blue). HaCaT cells exhibited toxicity with low concentrations of the dyes, with the red dye being the most toxic. The IC20 values in the HaCaT cell line were 78 µg/ml (yellow), 28 µg/ml (red), 112 µg/ml (blue). However, the dyed fabrics were not toxic to all studied cells. The fabric extracts were not toxic to hepa-1 and HaCaT cells since the measured protein content was over 80% of control. In the spermatozoa test compared to control, more than 50% of the test spermatozoa cells showed motility. In addition to reactive dyes and dyed fabrics, the effects of industrial dyed and ﬁnished cotton fabrics were investigated in cell tests. All of the studied raw fabric materials (untreated) were non- toxic. The reactive dyed and press shrunk fabric was not toxic. The ﬂame retarded cotton fabric caused little toxicity to the spermatozoa cells. Most of the knitted cotton fabrics were toxic to hepa-1 and HaCaT cells with the exception that the yellow fabric extract was not toxic to HaCaT cells neither was the red fabric extract toxic to the hepa-1 cells. The other knitted fabric extracts affected the viability of the cells less than 80% compared to control. These results show that cell tests are suitable for studies into the toxicity of textile dyes and fabrics but different cell models should be used in these evaluations. The in vitro bioassays provide information which will help in the development of less harmful textile processes and products.
Universal Decimal Classiﬁcation: 667.281, 677.027.423.5 National Library of Medicine Classiﬁcation: QV 235, QV 602, QV 627, QV 663, WA 465, QY 95 Medical Subject Headings: Textiles/toxicity; Cotton Fiber; Coloring Agents/toxicity; Azo Compounds/toxicity; Flame Retardants/toxicity; Spermatozoa; Sperm Motility; Toxicity Tests; Cell Line; Cells, Cultured; Cell Survival; Inhibitory Concentration 50; Biological Assay; In Vitro
I owe my warmest thanks to Virve Kärkkäinen. for discussions. This work was also supported by a grant from the Lisa Andström Fund (International Zonta District 20). for their care. Mrs. Magnus Ehrnrooth Foundation and Juho Vainio Foundation. University of Kuopio during 20022008. Helena Kauttonen. During this work. I am particularly grateful to Ulla Honkalampi-Hämäläinen. for revising the language of this thesis. for providing the facilities and position for my work in his department. I wish to express my gratitude to Professor Hanna Tähti and Professor Pertti Nousiainen. my husband Paavo and our son Väinö. Mrs. Ph. September 2008 Kaisa Klemola .Sc. the referees of this thesis. M. This work was conducted mainly with the support of Finnish Concordia Fund. Kuopio. their patience and friendship has been valuable. The encouragement and support of my friends and relatives are deeply appreciated. I am deeply grateful to my co-author Professor John Pearson. Raili Mähönen. I express my sincere thanks to Ewen MacDonald. I am deeply indebeted for her kindness. for his advice and encouragement. Jouni Heikkinen. I wish to thank all those persons who have made this series of studies possible by helping me either in the ﬁeld of laboratory work or by providing technical assistance. M. I express my thanks to Mrs. I thank Professor Osmo Hänninen for giving encouragement and his belief to me.ACKNOWLEDGEMENTS This study was carried out in the Department of Biosciences. Mr.Sc. Riitta Junnila-Savolainen. all her advice and support to Docent Pirjo Lindström-Seppä. the principal supervisor of my work. encouragement and her friendship. Eeva Kontturi and Mrs. I am delighted to have had the change to enjoy such a pleasant working atmosphere... Väinö Klemola.D. Head of the Department of Biosciences. My warmest thanks belong to my family. I owe my thanks to Professor Atte von Wright. Riitta Venäläinen for guiding me with cell cultures. Mr. for their constructive comments on my work. My sincere thanks are also due to my supervisor Professor Jyrki Liesivuori. I greatly appreciate his efforts in scientiﬁc research of textiles and for his pleasant collaboration. Mrs. Mrs. Mirja Rekola. I thank warmly my colleagues in the Kuopio Academy of Design.. Marke Iivarinen. patience and understanding. and Mrs. Tuomo Jalkanen and Mr.
2-bis(p-chlorophenyl)ethane dimethylolhydroxyethyleneurea Dulbecco`s Modiﬁed Eagle`s Medium dimethylsulphoxide 2. required to kill 50% of animals in the acute toxicity test lowest adverse effect level mitogen-activated protein kinase The Multicenter Evaluation of In Vitro Cytotoxicity Minimum Essential Medium (3-[4. adenosine triphosphate bovine serum albumin coefﬁcient of variation Colour Index carboxymethylcellulose a subfamily of cytochrome P450 1.1. Evaluation and Authorisation of Chemicals ATP BSA C of V CI CMC CYP1A DDT DMDHEU DMEM DMSO DNF EC50 ECVAM EPA EROD FDA GLP HaCaT Hepa-1 IARC IC20 IC50 INVITTOX ISO LD50 LOAEL MAP MEIC MEM MTT NOAEL OECD PBDE PBS REACH .4-dinitrophenol effective concentration for 50% of maximal effect The European Centre for the Validation of Alternative Methods Environmental Protection Agency 7-ethoxyresoruﬁn O-deethylase Food and Drug Administration Good Laboratory Practice human keratinocyte cell line hepa-1 mouse hepatoma cell line International Agency for Research on Cancer inhibitory concentration decreasing response to 80% compared to control inhibitory concentration decreasing response to 50 % compared to control data bank on the use of in vitro techniques in toxicology and toxicity testing International Standard Organization lethal dose.ABBREVIATIONS ASA Syöpäsairauden vaaraa aiheuttaville aineille ja menetelmille ammatissaan altistuvien rekisteri. Vuosittainen tilasto. Työterveyslaitos.5-diphenyl tetrazolium bromide) test no adverse effect level Organization for Economic Cooperation and Development polybromide diphenylether phosphate buffered saline The Registration.1-trichloro-2. The Finnish Register of occupational exposure to carcinogens. Finnish Institute of Occupational Health. Helsinki.5-dimethylthiazol-2-yl]-2.
chloride tetrakis-hydroxymethyl-phosphonium.phosphonium.sulphate volatile organic compounds World Health Organization Water-soluble tetrazolium assay .SD THP THPC THPS VOC WHO WST-1 standard deviation tetrakis-hydroxymethyl-phosphonium tetrakis-hydroxymethyl.
Pearson J. Liesivuori J. von Wright A. I Klemola K. Pearson J. 224-230. in press. Evaluating the toxicity of reactive dyes and fabrics with the spermatozoa motility inhibition test. Evaluating the toxicity of reactive dyes and dyed fabrics with the hepa-1 cytotoxicity test. Lindström-Seppä P. Evaluating the toxicity of fabric extracts using the hepa-1 cytotoxicity test. 182-190. Evaluating the toxicity of reactive dyes and dyed fabrics with the HaCaT cytotoxicity test. AUTEX Research Journal 2006 6(3). . Pearson J. AUTEX Research Journal 2007 7(3). 217-223. Honkalampi-Hämäläinen U. AUTEX Research Journal 2007 7(3). the HaCaT cytotoxicity test and the spermatozoa motility inhibition test. Pearson J. Klemola K. Liesivuori J. Lindström-Seppä P. The Journal of Textile Institute. Liesivuori J. Klemola K.LIST OF ORIGINAL PUBLICATIONS This thesis is based on following original publications referred to in the text by their Roman numerals I-IV. Klemola K. II III IV The original papers in this thesis have been reproduced with the permission of the publishers. Lindström-Seppä P. Lindström-Seppä P.
2 2.1 2.2.1 2.CONTENTS 1.4 2.3 2.1 2.2 2.2 2.2.2 184.108.40.206.5.4.3 2. MATERIALS AND METHODS 4.1.1 4.1 2.1 2. INTRODUCTION 2.1.4 Textile ﬁbres Classiﬁcation of textile ﬁbres Cellulose in cotton Textile dyes Dye molecule Classiﬁcation of textile dyes Reactive dyes Finishing of cellulosic textiles Adverse effects of textile substances Adverse effects of chemicals caused by the production of cellulosic ﬁbres Adverse effects of reactive dyes Adverse effects of ﬁnishing chemicals used for cellulosic textile materials Toxicity tests Mechanisms of toxicity Testing for toxicity The use of cells in vitro Endpoints used in the evaluation of toxicity in in vitro cell tests 13 15 15 15 16 17 17 19 20 22 23 23 24 25 26 26 27 29 30 32 33 33 33 33 33 33 34 34 220.127.116.11. OBJECTIVES 4.1 4.5 2.5.2 18.104.22.168 2.3 4.2 4.4.3 4.2 2. REVIEW OF LITERATURE 2.3 2.1 Dyes and fabric samples Reactive dyes Fabric Commercial fabrics Origin of the cells Procedures for sample preparation and toxicity testing Procedure for dyeing the fabrics .
4 4.2 5.3.3. DISCUSSION 6.4 6.4.1 6.3. RESULTS 5.5 Reactive dyes in the cell tests The fabric extracts in the cell tests In vitro cell tests for assaying textile substances The reliability of the tests The possibilities for utilizing cell-based tests for studying textile substances 7.3 4.5 Preparation of fabric extracts The spermatozoa motility inhibition test Cytotoxicity test with hepa-1 mouse hepatoma cells and with human keratinocyte HaCaT cells Statistical methods 34 34 35 36 37 37 39 40 41 42 42 44 45 46 47 49 50 5.3 6.4 The IC50 and IC20 values for three reactive dyes The toxicity of the fabric extracts Reliability of the results Statistical signiﬁcance 6. REFERENCES .2 4.3 5. CONCLUSIONS 8.3.2 6.1 5.
In a study of 72 textile workers in North Carolina. 1998. which can cause adverse effects (IARC 2004). 2004. many textile chemicals are organic compounds and not easily extracted from water. there is limited information about the overall toxicity of dyed and ﬁnished materials. However. 1993. For example. organic solvents. INTRODUCTION The manufacture and processing of textiles utilises many different chemical reagents. Although a reagent itself may be toxic. Niven et al. Some textile dyes have been assessed for potential mutagenicity (Przybojewska et al. Ofﬁcial patient organizations concerned with asthma and allergies as well as consumer organisations provide some information about the safety of different consumer products (http://www. (2002//61/ EY. C. in Jaipur. http://www. 1997. Dincer et al. Mathur and Bhatnagar 2007. A globally used Kuopio Univ. 2007). 1996. 2005a). have a high mutagenic activity due to the presence of chemicals released from the textile industry (Mathur et al. There is some information available about the toxic and other effects of the individual reagents on textile workers. A signiﬁcant number of these are harmful to the environment.) and genotoxicity (Sharma and Sobti 2000). However. Finland has set the limiting values (100mg – 300mg/kg) for the amounts of formaldehyde permitted in textiles (KTMa 210/1988). in Finland. its presence in the ﬁnished material may cause no adverse effects. Zuskin et al. http://www. Nat. a high incidence of bladder cancer was detected in Mataro. The study revealed positive results for about 28% of the dye products investigated (Schneider et al. such as acids. Nilsson et al. e. 2008 13 .g. There is some information about the possible toxic effects of textile chemicals. Currently. The United Kingdom Health and Safety Executive has 2 ppm workplace exposure limit for formaldehyde.kuluttajavirasto. In a European Union EU-funded research project. However.Introduction 1. water softeners. dyes and a range of ﬁnishes (Trotman 1984). 1989. Jäger et al. Sci. a mere 26 work-related diseases were recorded in the 25. salts.ﬁ). although the toxic effects of many of the reagents used in their manufacture are known. bases. 2004.allergia. ASA): this may be due to good working conditions. It has been shown that some surface waters in India. Publ. It is well-known that certain textile ﬁnishing compounds are able to release formaldehyde. and Environ. 2004.com. VNa694/2003). 281 textile dyes were assessed for potential mutagenic properties using Salmonella typhimurium strains TA98 and TA100.000 workers in the textile industry during 2001 (Karjalainen 2002. there is little information available about possible toxic effects of textile products. Spain among the textile workers using reactive dyes (Gonzales et al. 241:1-67. contact dermatitis developed in 24 of them after ﬁve years’ exposure to textile chemicals (Soni and Sheretz 1996). Schneider et al. Järvholm 2000). to the people working in textile processing and potentially to consumers.efanet. Allergic reactions and irritation to the skin and respiratory tract have been found to be the most common occupational diseases in workers in the textile industry (Hatch 1984. There have been many studies conducted on environmental problems of wastewaters due to the presence of toxic textile chemicals and techniques for decolourisation of dyes and removal of textile chemicals are under development (Choudhary et al. In contrast. the EU has set the limiting values for the amounts of carcinogenic aromatic amines (30 ppm) allowed to evaporate from textiles. 2004). org/. 1988). 2003/3/EY. Khan and Husain 2007.
for instance heavy metals (Öko-Tex Standard 100. mouse hepatocyte cell line (Hepa-1) and human keratinocyte cell line (HaCaT). 14 Kuopio Univ. However. Three different types of cells were used: boar spermatozoa cells. Sci. Information about product safety is still limited. in vitro cell tests were used to evaluate the potential toxicity of textile dyes and dyed fabrics. 2008 . 241:1-67. These cell tests may be useful in the development of safer working conditions in the textile industry and healthier textile products for consumers. Many chemical analyses have been performed on these eco-labelled fabrics. this standard does not require any biological tests to evaluate the adverse effects of textile materials. Cotton was selected because it is the most widely used natural textile ﬁbre and reactive dyes since they are widely used for dying cotton. In the present study. 1997). Nat. Publ. and Environ.Kaisa Klemola: Textile Toxicity Öko-Tex-100 textile standard assesses whether textile products with this eco-label contain harmful amounts of certain compounds. Cell tests were selected because of their sensitivity to chemicals. C. Reactive dyes used to dye cotton were used as the test material.
man-made ﬁbres regenated ﬁbres viscose modal lyocell acetate triacetate synthetic ﬁbres polyester polyamide acrylic modacrylic elasthane inorganic ﬁbres glass metallic ceramic About 49 million tonnes of all textile ﬁbres were produced during 1994 in the whole world about 38 per cent of this being cotton. 1988). and Environ. a. REVIEW OF THE LITERATURE 2. Since the production of cellulose ﬁbres is so extensive. Nat. b= man-made ﬁbres. 2008 15 . The classiﬁcation of the textile ﬁbres (modiﬁed from ISO 6938 1984. fur alpaca angora camel’s hair cashmere lama mohair vicuna wool silk asbestos fibres b.Review of the literature 2. The ﬁbres can be classiﬁed according to their chemistry or according to their origin.1 Textile ﬁbres 2. Kuopio Univ. Sci.1. ISO 2076 1999).1 Classiﬁcation of textile ﬁbres Textile ﬁbres contain natural and man-made ﬁbres. In fact. (Table1: a and b). the need for chemicals for the industrial treatments of the cellulose ﬁbres is also widespread. Sundquist 1987. 241:1-67. a= natural ﬁbres. Publ. The man-made ﬁbres consist of raw oil based synthetic ﬁbres and natural polymers regenerated ﬁbres. cotton is the mostly produced natural ﬁbre in the world (Worldwide Textile Production 1980-2003). mineral plant fibres seed fibres cotton kapok bast fibres ﬂax hemp jute ramie fruit fibres coconut leaf fibres abaca sisal henequen animal fibres wool. ISO 2076 1999. the latter being the most commonly used (ISO 6938 1984. Table 1: a and b. C. The natural ﬁbres include cellulose ﬁbres and protein ﬁbres.
Van der Waals` bonds are also present but compared with hydrogen bonding.Kaisa Klemola: Textile Toxicity 2.8 nm thick. Hydrogen bonds are formed between the polar groups on adjacent polymer chains in crystalline areas of the ﬁbres. Nat. Gordon 2006). which links the two glucose units to form the cellobiose unit. Sci. However. The important reactive groups on cellulose are the highly polar hydroxyl (–OH) and methylol (–CH2OH) groups: reactive dye molecules react with these groups. Oxidising bleaches leave the ﬁbre polymer system largely intact if the process is done carefully. Cellulose ﬁbres are relatively resistant to alkalis. Figure 1. C.2 Cellulose in cotton Cotton cellulose is a linear. correspondingly. cellulose polymer. The cotton ﬁbre is hygroscopic owing to the polar –OH groups in its polymers. Acidic conditions hydrolyse the polymer at the glucoside oxygen atom. 16 Kuopio Univ. Amorphous and crystalline regions of the polymer system (Gohl & Vilensky 1983). they are of little signiﬁcance. 241:1-67. the polymer system is about 65 to 70% crystalline and. The DP in ﬂax is higher and lower in viscose. Figure 2. The degree of polymerisation (DP) is about 5000 and the polymer is about 5000 nm long and about 0. the repeat unit (monomer) being cellobiose which consists of two glucose units (Figure 1). Cellulose ﬁbres are weakened and destroyed by strong acids. about 30-35% amorphous (Figure 2). This enables cotton to avidly absorb the polar dye and pigment molecules. 2008 .1. Cotton cellulose consists of cellobiose units. and Environ. In cotton. Publ. water and dye molecules can only enter the polymer system in its amorphous regions since the inter-polymer spaces in the crystalline regions are too small to accommodate these molecules (Gohl & Vilensky 1983.
2. This also intensiﬁes and deepens the hue of the dye molecule. and Environ. *In the formula of NO2 the bond should be drawn as -O-. which causes these electrons to absorb and reﬂect incident light energy only of speciﬁc wavelengths. Table 2. C. not as . and are thus useful dye molecules. Loosely held electrons in the conjugated system of the chromophores are able to absorb certain incident light waves (Gohl & Vilensky 1983).fastness properties of the dyed ﬁbre (Gohl & Vilensky 1983). Chromophores are unsaturated organic radicals. 2008 17 . Auxochromes also increase the overall polarity of the dye molecule and make it more readily soluble in water and more readily attracted to the ﬁbre polymer (Figure 3) which improves the colour. Their speciﬁc state of unsaturation enables them to absorb and reﬂect incident electromagnetic radiation within a very narrow band of visible light. The auxochromes inﬂuence the orbitals of the loosely held electrons of the chromophores. Broadbent 2001). Sci.1 Dye molecules Organic molecules become coloured. (Gohl & Vilensky 1983.Review of the literature 2. Chromophores. (Gohl & Vilensky 1983). 241:1-67.2 Textile dyes 2. Nat. if they contain at least one of the following radicals called chromophores (which provide colour) and auxochromes (which intensify and deepen the colour) which can selectively absorb and reﬂect incident light (Tables 2-3). Kuopio Univ. Publ.
44075 – an acid dye. Structural formula of a textile dye molecule.I. Figure 3. Nat. 18 Kuopio Univ. C. C. Sci. (Gohl and Vilensky 1983). 2008 . and Environ. Publ. 241:1-67. Acidic Blue 86.Kaisa Klemola: Textile Toxicity Table 3 Auxochromes (Gohl & Vilensky 1983).
Therefore. the classiﬁcation according to application class is more signiﬁcant. direct dyes. Sci. cationic (basic) dyes. metal-complex dyes. n=1 (red). The CI classiﬁes the dyes according to their application class and to their chemical structure (Table 5). modiﬁed from Aalto et al. sulphur dyes and vat dyes as well as pigments and ﬂuorescent brighteners (Sundquist 1985. Publ. there is a clearly deﬁned shift of the light absorbed towards red. As the number (n) of double bonds increases. About 8000 compounds of them are textile dyes and they give rise to about 40000 commercial names which are used as textile dyes.2 Classiﬁcation of textile dyes There are over 13. C.Review of the literature The majority of dyes can be regarded as resonance hybrids with the colours obtained depending on the energy states of the orbitals. the longer the conjugated chain. reactive dyes. azoic (naphthol) dyes. 2. When n = 0 (yellow). The classes are: acidic dyes. and Environ. disperse dyes. mordant dyes. n=2 (greenish blue).000 different compounds classiﬁed as dyes in the Colour Index (CI) 2001. This can be seen in the properties of carbocyanine dye (Trotman 1984) (Figure 4). Each dye has a ﬁve-digit CI-number. 241:1-67. 1994. Figure 4. the less energy will be required to excite the electrons and the greater will be the wavelength of the absorbed light. Talvenmaa 1998. Nat. 2008 19 . For the textile dyer. with a corresponding relative increase in the proportion of blue reﬂected (Trotman 1984). Lengthening the conjugated chains increases the number of double bonds and decreases the energy gaps between the π-orbitals.2. Carbocyanine dye. n=3 (blue) (Trotman 1984). Colour Index 2001). Kuopio Univ. developed dyes.
These dyes are widely used in dyeing cotton and other cellulose-based ﬁbres.3 Reactive dyes Reactive dyes are named according to their chemical reactivity with ﬁbre polymers. modiﬁed from Aalto et al. means that reactive dyes. Textile dyes according to their chemistry.Kaisa Klemola: Textile Toxicity Table 5. Nat. Broadbent 2001). The vinyl sulphate radical of a reactive dye molecule and its bonding to the cellulose polymer. C. despite their relatively high cost. (Colour Index 2001. and Environ. 2. Publ.and indophenol dyes azine dyes oxanthine dyes thiazine sulfur dyes amino developed dyes hydroxyketone dyes anthracinone dyes indigo dyes phtalocyanine dyes organic natural dyes oxidation developed dyes inorganic pigments Synthetic textile dyestuffs contain many different chemicals with different applications. 241:1-67. Reactive dyes form a covalent bond between the dye molecule and the ﬁbre polymer (Figures 5-7). This produces a stable covalent bond which results in excellent colour fastness.000 tonnes are produced per year accounting for over 60% of all dyes for cellulosic ﬁbres (Holme 2004).2. to improve water solubility and to reduce dusting. This property as well as the simplicity of the dyeing process. Thus commercial dyestuffs contain many other molecules in addition to dye molecules themselves (Talvenmaa 1997. are widely used on cellulosic ﬁbres. Around 120. 1994) nitroso dyes nitro dyes azoic dyes azoic developed dyes stilbene dyes carotenoid dyes diphenylmethane dyes triarylmethane dyes xanthene dyes arcidine dyes cinoline dyes methine and polymethine dyes thiazole dyes indamine. for instance to increase shelf life. Figure 5. Sci. 2008 . (Gohl & Vilensky 1983) 20 Kuopio Univ.
A dichlorotriazinyl reactive dye and its bonding to the cellulose polymer. A monochlorotriazinyl reactive dye. and Environ. 2008 21 . 241:1-67. Sci. (Trotman 1984) Kuopio Univ. C.Review of the literature Figure 6. Publ. Nat. (Gohl & Vilensky 1983) Figure 7.
. Fabrics can also be made water-repellent by incorporation of thermo-setting silicone resins. 22 Kuopio Univ. Durable ﬂame retardancy is typically obtained by the use of organophosphorous compounds (Lewin and Sello 1983). and pyridinium.3 Finishing of cellulosic textiles Cellulose ﬁbres ignite and burn easily and therefore it has been important to develop ﬂame-retardant treatments for those materials. rain and perspiration: they provide only temporary protection. metal complexes. Sci.tetrakis-hydroxymethyl phosphonium chloride (THPC)-urea) are used as durable ﬂame retardants for cotton. residues of which may be found in treated fabrics. such as cetyl trimethyl ammonium chloride being used to achieve this goal. graft polymerisation of N-halamide monomers. Chemical modiﬁcation of the cellulose polymers is possible: reaction of ﬁbres with acrylonitrile produces durable protection (Gohl and Vilensky 1983. There are numerous chemicals used to make fabrics water repellent.Kaisa Klemola: Textile Toxicity Typically reactive dyes are applied using salt and subsequently ﬁxation is brought about by the addition of soda ash to the dye bath. 241:1-67. Cotton textiles can be protected against attack by micro-organisms with quaternary ammonium compounds. chloromelamine derivatives and cross-linked chitosan to produce durable effects. cellulose and cellulose-blend clothing fabrics. C. and Environ. and non-ionic softeners. Nat. The dye can also undergo hydrolysis with water and this decreases the colour yield of the dye as well as lessening the colour fastness due to reduced number of covalent bonds within the ﬁbres (Gohl & Vilensky 1983. The reaction between the ﬁbre and the dye molecule takes place under alkaline conditions. Trotman 1984. Schindler and Hauser 2004). Modern developments include the use of nanotechnology. Broadbent 2001). 2. Schindler and Hauser 2004). Water-repellency combined with oil. wax like substances. The use of methylol stearamide with partially formed urea-formaldehyde provides a good water-repellent ﬁnishes with adequate fastness properties. A number of durable easy-care ﬁnishes have been developed to reduce creasing of cellulose ﬁbres with most of these based on dimethyloldihydroxy-ethyleneurea (DMDHEU) (Priha 1988. Schindler and Hauser 2004). waxes. WHO 2000. Many ﬁnishing formulations require the use of catalysts.compounds. Pyrovatex CP (Ciba Speciality Chemicals) is a phosphonoalkylamide used to confer durable ﬂame retardancy on cellulosic fabrics being used in furnishings (Gohl & Vilensky 1991. These include aluminium and zirconium soaps. Boron (polybromide diphenylether PBDE) and phosphorous compounds are widely used. Schindler and Hauser 2004). Publ. Tetrakis-hydroxymethyl-phosphonium-salts such as Proban (Rhodia.and methylol. Many of these compounds are based on water-soluble inorganic salts that are easily removed by water. 2008 .and soilrepellency can also be obtained by the application of ﬂuoropolymers such as Scotchguard (3M) and Teﬂon (Du Pont) (Kissa 1984. Chun & Gamble (2007) reported the use of silver nanoparticles.
com) which is now used globally to indicate that the textile product has been tested for the presence of harmful substances. Sci.4. especially for sensitized consumers. It has been found that patients who were exposed to organophosphates and carbamates had low levels of cholinesterase activity in their blood (De Peyester 1993). 1992). It is known that harmful chemicals may have been used. However. Öko-Tex Standard 100. It has been stated that women working with organophosphates are at risk of developing non-Hodgkins lymphoma (Zahm et al. The Austrian Textile Research Institute and the German Hohenstein Research Institute jointly developed the Öko-tex-100 standard and eco-label (www. zinc and copper salts and residues may still be present in cotton-based textiles (Suojanen 1995. For products with the Öko-Tex Standard 100 label.1 Adverse effects of chemicals caused by the production of cellulosic ﬁbres Many chemicals are used in the cultivation of the cotton plant. but potential adverse effects of textile products are not widely appreciated. Marrs 1993. 1987. 1992). Synthetic pyrethroids cause irritation effects (Priha 1988). limiting values have been set for the amounts of recognized harmful chemicals allowed to be present (Öko-Tex Standard 100 1997). 1994. James 1985. 241:1-67. for instance chlorinated phenols. 1999. How- Kuopio Univ.and skin cancer (Lund 1991. Piipari and Keskinen 2003).4 Adverse effects of textile substances Asthma. Some fertilizers contain high concentrations of cadmium (about 138 mg/kg) in phosphoric fertilizers (Malm & Louekari 2000) and some insecticides e. There is less large-scale use of chemicals in the production of other natural cellulosic ﬁbres.2-bis(p-chlorophenyl)ethane (DDT) was found in England and Sweden not only in raw fabrics but also in clothes (Ahonen 1994. Although most of the harmful chemicals are washed away during industrial processing. Lopez-Carillo and Lopez-Cervantes 1993. 1993). Stevens et al. Hayes et al. information about the chemicals of textile products can be important. and Environ. It has also been claimed to be a risk factor in the development of nose. Docker et al. However. 2. This results in the weakening of the lungs and chronic pulmonary inﬂammation (Duffell 1985. 1994. methoxyethylmercurium (Komulainen et al. It has been recognized for over 200 years that prolonged exposure to cotton dust can produce byssinosis. C.g. Luce et al. Sigsgaard 1992. Suojanen 1995). some may still remain in the textile end products. Publ. 1. Textile manufacture is a global activity and it is difﬁcult for consumers to obtain information about the production and chemicals of textile products. Much work has been done in Western Europe and Scandinavia to ensure that textile products do not contain chemicals that might be harmful to the consumer and as consequence many textile chemicals have been banned. 1997). Nat. 1987. However. rhinitis and dermatitis are three of the most common adverse effects evoked by textile processing (Eskelson and Goodman 1963. Serious effects on the central nervous system have been detected following the use of organophosphate insecticides (Minton et al. Christiani & Wang 2003). 1995). 2008 23 . 1988.oeko-Tex. the overall toxic effects of end products are not included and information about overall toxicity is not available.1-trichloro-2. Jahkola et al.Review of the literature 2. Beckett et al.1. Jolanki et al.
1993. Sci. utilises harmful chemicals and these can cause problems. especially when in their powdered form. the register of 24 Kuopio Univ. and Environ. Nat. Manzini et al. Wang et al. the health hazard resulting from exposure to such substances is signiﬁcant (Keneklis 1981). Enzymes. For example. although Hatch et al.2 Adverse effects of reactive dyes Symptoms of asthma. Park et al. 241:1-67. 2007). 1991). 1987). Dyes containing anthraquinone or azo structures are known to cause contact dermatitis (Estlander 1988. 1994). Virtanen and Hannuksela 1999).Kaisa Klemola: Textile Toxicity ever. TMp 838/1993. 1978. The cause of skin reactions is difﬁcult to trace because the dye usually acts as a delayed sensitizer and as such does not cause an immediate response (Hatch and Maibach 1995. 2002). However. Nilsson et al. and chlorinecontaining organic solvents are known irritants. 2008 . in Finland. The international register of cancer-causing chemicals includes many textile dyes (including those based on benzidine and o-toluidine) and their raw chemicals for synthesis (IARC 1987. However. it should be noted that dermatological problems associated with dyes in textiles are relatively rare (Maurer et al. (2003) have noted such effects and have given the name ‘colored clothing allergic contact dermatitis (ACD)’ to these symptoms. 2. rhinitis and dermatitis have been frequently detected in workers exposed to reactive dyes (Hatch 1984. C. 1989. including strong acids. bleaches and brighteners can evoke respiratory allergic reactions. However. Wilkinson and McGechaen 1996). Vanhoorne et al. Aromatic. the actual end products remain to be more thoroughly investigated. 1991). It was noticed as early as 1981 that some sulphonyl ethyl sulphate derivatives were carcinogenic (Keneklis 1981). Topping et al. It has been assumed that since the properties that enable the dyes to react with textile ﬁbres also allow them to bind to body protein. Acidic detergents can cause eczema and contact dermatitis. which may act as irritants. there is minimal information for consumers about these adverse effects. 1996. 1986. The result of a clinical and immunological investigation of respiratory disease indicated that about 15% of 400 workers handling reactive dyes experienced work-related respiratory and nasal symptoms (Docker et al. Although cellulosic ﬁbre processing has been extensively studied. Some of these harmful substances may remain in the textile end products. Other auxiliaries. In conclusion. Publ. The most widely used chemicals after ﬁbre formation are surfactants and auxiliaries. Park et al. even their presence in clothes. 2006. Since reactive dyes are chemically very active. Moreau & Goossens (2005) reported similar effects and stated that reactive dyes should be classiﬁed as potential allergens. 1995). Aalto et al.4. Thoren et al. Many studies have also found statistically signiﬁcant relationships between reactive dyes and increasing immunoglobulin blood values in workers who have been contact with these dyes (Alanko et al. they can cause harmful effects. adverse effects on the eyes and respiration have been detected in persons who have been exposed to carbon disulﬁde which is a known neurotoxic compound (Aaserud et al. mineral oils and salts can cause irritation and allergic reactions (Estlander and Jolanki 1980. 1990. the production of cellulosic ﬁbres utilises chemicals that can evoke adverse effects including allergic reactions and irritation to the skin and the respiratory system. the production of regenerated cellulosics such as viscose.
Polybromide diphenylethers (PBDE) may form polychloride dioxins and furans in a ﬁre and their use is now greatly restricted. 1996. it has been noted that PBDE disrupts spontaneous behaviour. 1995. polybromide biphenyls have been found to cause adverse effects and have been removed from sale (WHO 1997). Sci. in the USA. 2002/61/EY and 2003/3/EY). In addition. Due to the problems associated with textile dyes. ﬂame prooﬁng and antimicrobial treatments. Suomen säädöskokoelma 210/88).4. Carlson et al. although not all the dyes tested were reactive dyes. Mathur et al. 1998). Mathur et al.4`. The UK Health and Safety Executive warns industrial workers who have contact with reactive dyes that they may become sensitized. Schneider et al. then it is important to conduct tests for mutagenicity and genotoxicity (Przybojewska et al. 2005b. the use of textile dyes releasing certain aromatic amines at concentrations above 30 ppm is forbidden (VNa 694/2003. eyes and the respiratory tract (James 1995) and it may also cause asthma (Piipari and Keskinen 2003). and Environ. Priha et al. 2004).3 Adverse effects of ﬁnishing chemicals used for cellulosic textile materials Many ﬁnished fabrics may release formaldehyde. 2003). An increase in the amounts of PBDE has been found in the environment and this chemical is even present in mother`s milk. All these studies have revealed adverse effects of dyes. 1988. Scheman et al. 2008 25 . Publ. However. With regard to ﬂame retarding agents. IARC 2004. If one wishes to detect any adverse effects of textile dyes. 1982. the dyeing process is under constant development. 1996. WHO 1989. This problem is associated especially with the permanent press. stressing the potential for respiratory sensitization. limiting values have been set for the amount of formaldehyde allowed in fabrics (Priha 1995. the measured amounts of brominated ﬂame reagents in the environment have been claimed to pose no threat to the health of children (Hays and Pyatt 2006). Priha 1992. 1983. 241:1-67. carcinogenicity (De Roos et al. 2. Vuorela 2003). impairs learning and memory in adult mice (Vikberg et al. Roberts and Rossano 1984. In Finland. 1987. In many European countries. 2002). 1989. According to IARC (1998) it is not certain whether the widely used phosphonium salt as ﬂame retardants are carcinogens. Formaldehyde has been demonstrated to cause harmful effects (James 1985. THPC was not shown to be carcinogenic in Kuopio Univ.1995. C.5-pentabromodiphenyl ether has been shown to cause altered susceptibility in the cholinergic neurotransmitter system in the adult mouse (Vikberg et al. 1999. Priha et al. 1986. Priha et al. Dogan et al. Speciﬁc effects include irritation to skin. Garcia et al. 2006).2´. 2005a. 2005) and teratogenicity (Birhanli and Ozmen 2005).4. Neonatal exposure to brominated ﬂame retardant. 2. Jolanki et al. the international register of cancer causing chemicals classiﬁes formaldehyde to The Group 1: carcinogenic to humans (IARC 2004). Since high concentrations of formaldehyde may cause cancer. 2004. Nat. with increasing attention being paid to the ecological effects of these chemicals. 2005). The effects of formaldehyde in textile work places have been widely studied (Nousiainen 1979. PBDE and polychlorinated PCBs can interact and enhance developmental neurobehavioral effects when the exposure occurs during a critical stage of neonatal brain development (Eriksson et al.Review of the literature individuals exposed to carcinogens contains little information about textile industry workers (Kauppinen 1999. Tetrakis-hydroxymethyl-phosphoniumchloride (THPC) was tested and treated fabrics did not cause skin irritation to humans. Jahkola et al.1984:a and b.
von Essen 1996. cellular or organ dysfunction and leads to deleterious effects.5. Publ. 2008 . 1988). Indigo-dyed denim fabrics were shown to be mutagenic. The causes of toxicity of a fabric can be difﬁcult to trace since they may be due to the combined effects of several of the chemicals present in the textile. Sci. Kalimo and Lahti1999. these reagents are also available in aerosol forms where they are combined with carbohydrates and ﬂuorochemicals. after which the ultimate toxicant interacts with endogenous target molecules. dermal studies with rabbits have shown that THP salts are promoters but not initiators of skin cancer (WHO 2000). The textile industry utilises different washing. as with the production of cellulosic ﬁbres. 2006). 2006). Nat. However. and Environ. Other ﬁnishing agents: water repellents. C.Kaisa Klemola: Textile Toxicity rats and mice in a 2-year bioassay. These aerosol products are available for home use by consumers. Yazdankhah et al. 16 of them only after metabolic activation. Vernez et al. Although the problems caused by many textile chemicals are recognized. Many antimicrobial agents are relatively toxic and have been found to sensitize humans (Kanerva 1998. cel- 26 Kuopio Univ.5 Toxicity tests 2. potential problems in fabrics have not been widely studied and. Sometimes a xenobiotic does not react with a speciﬁc target molecule but rather adversely inﬂuences the biological (micro) environment causing molecular. showing the importance of analysing the ﬁnished fabrics in addition to the pure chemicals. These chemicals and their metabolites are strongly corrosive and have been found to be toxic to the aqueous environment and to humans. almost 20% of the silk fabrics and over 10% of the cotton fabrics gave positive results in this test for mutagenesis (Pﬁtzenmeier 1990). VNa 596/2004). when azo-dyed fabrics for clothing were tested with the Ames bacterial assay. 2. (Gregus and Klaassen 2001) The most complex path to toxicity involves four steps. Knasmuller et al. These chemicals have been claimed to cause hormonal changes and therefore the European Union has recommended that their use should be limited (2001/838/EY. Nonetheless. there is limited information available to consumers. and in these situations they have caused pulmonary inﬂammation (Wright and Lee 1986. 1992). organellar. (1993) showed that 18 of 196 fabric samples examined were mutagenic. First. dyeing and ﬁnishing chemicals which can contain nonylphenol and nonylphenolethoxylates.1 Mechanisms of toxicity The toxicity is of a compound becomes apparent when a toxicant is delivered to its target and reacts with it and the resultant cellular dysfunction manifests itself in toxicity. Since the mutagenicity of indigo was low. 1993). Though many azo dyes have been shown to be mutagenic (Chung and Cerniglia 1992. it seems that azodyed fabrics are not toxic (Kaur et al. However. dirt repellents and antistatic agents have seldom caused skin or other health problems (Priha et al. 241:1-67. triggering perturbations in cell function and/or structure. which initiate repair mechanisms at the molecular. the toxicant is delivered to its target or targets. Antimicrobial treatments are not widely used on clothing: their use is limited mainly to tent fabrics and woollen carpets. Kaur 1993). the genotoxicity of denim extracts must have been due either to some unknown chemicals or to some unknown reactions (Rannung et al. stain repellents.
241:1-67.ymparisto. strong acids and bases. Biotransformation of a compound to a harmful compound is called metabolic activation. benzene can be oxidized to a variety of quinines and semiquinones that can cause hematopoietic toxicities and leukaemia. nicotine. This conversion is called metabolic inactivation.ﬁ-REACH). (Bend and James 1978.5. some regulations have been introduced. Some of these competing pathways are considered as bioactivation. These guidelines are expected to be followed when toxicity tests are conducted in support of the introduction of a chemical Kuopio Univ. reduction and hydrolysis. Phase II reactions in the second phase are conjugation reactions with compounds having hydroxyl -OH. The functional groups may be present in the parent compound or may have been formed during phase I reactions that lead to toxicity. whereas the toxicity of other compounds is largely due to metabolites. amine –NH2 or carboxylic –COOH groups. (Gregus and Klaassen 2001) A number of xenobiotics such as heavy-metal ions. (Gregus and Klaassen 2001) One chemical may yield several ultimate toxicants. When different cell signalling pathways become disrupted. Gregus and Klaassen 2001). and ethylene oxide are directly toxic. for instance. Thus. Benzene and many other volatile organic compounds (VOCs) are converted via multiple metabolic pathways to products with varying toxicities. When the perturbations induced by the toxicant exceed the repair capacity or when repair becomes malfunctional. During the past years. the toxicity of a chemical may involve several mechanisms which can interact with and inﬂuence each other in an intricate manner. cancer and ﬁbroses. similar to other aromatic compounds. and the reaction with one type of target molecule may have a number of different consequences. functions of enzymes) can inﬂuence the prominence of the different pathways and hence alter toxicity outcomes. However. 2008 27 . the conversion of a bioactive parent compound to a less bioactive or inactive metabolite(s) that is/are efﬁciently eliminated is mostly usual. then toxicity occurs. Publ. For instance. Sijm and Opperhuizen 1989. A variety of factors (for example differences between species. the cell has typically become exposed to some toxic substance (Alberts et al. C. Sci. the Food and Drug Administration (FDA).2 Testing for toxicity Toxicity tests are not designed to demonstrate that a chemical is safe but to characterize the toxic effects it can produce. or detoxiﬁcation. (Parkinson 2001) Non-polar xenobiotics accumulate into lipid-containing tissues or are metabolised to a more water soluble compounds. is oxidised into a variety of reactive metabolites which are normally more toxic than the original compounds. Parkinson 2001) Benzene. The ﬁrst step is xenobiotic metabolism. one ultimate toxicant may react with several types of target molecules. others as detoxiﬁcation pathways. and Environ. 2002. Environmental Protection Agency (EPA) and Organization for Economic Cooperation and Development (OECD) have issued good laboratory practise standards (GLP). the so-called phase I reactions that consisting of non-synthetic reactions like oxidation. Currently there is a new set of regulations and toxicity tests that have to be performed on chemicals intended for commercial use (www.Review of the literature lular and/or tissue level. The examples of chemically induced toxicities followed by this four-step course are tissue necroses. Nat. 2.
each with its own particular advantage in the area of toxicological research. In addition to ethical beneﬁts. Long-term or chronic exposure studies are performed similarly to subchronic studies except that the period of exposure is longer than three months. ion transport and cell division. and Environ. However. 1992). 241:1-67. Subacute toxicity tests are performed to obtain information on the toxicity of a chemical after repeated administration and as an aid in deciding how to conduct subchronic studies. (Eaton and Klaassen 2001. IC50 values are used to describe the inhibition of the viability of the cells in culture and the inhibition of the movements of spermatozoa. energy metabolism and synthesis and degradation of cellular constituents.Kaisa Klemola: Textile Toxicity to the market. when the inhibition of enzyme activity is measured as an endpoint to indicate the viability of the cells in culture. it is often possible to calculate relatively safe doses for humans. The toxic value from an in vitro test is typically stated as the effective concentration EC50 which represents the dose that causes 50% of cells to die. which use much less animals and in which the lethal dose is not determined any more (Worth and Balls 2002). At present. In the area of toxicology. cellular metabolism. In addition. The test for mutagens that has been most widely used in toxicology is the Salmonella/microsome test developed by Ames et al.g. many in vitro tests based on cell cultures have been developed. Clinical chemistry and histopathology are performed after 14 days of exposure. jrc/it). but they have not yet been accepted for regulatory purposes (http://ecvam. It has also become increasingly evident that 28 Kuopio Univ. Nonetheless. Publ. in all chemical safety testing animal experiments must be applied. and replaced by three ethically more acceptable reﬁnement tests. LD50 is no longer in use. skin corrosion. It is known that in vivo animal tests do not always predict toxicity in humans. in vitro tests can provide mechanistic data and also these kinds of tests are useful for screening of chemicals. oral communication). (1975). Another value often used in in vitro studies is the IC50 value. The most important results obtained from toxicity tests are the values of LOAEL (the lowest dose that causes adverse effects) and NOAEL (the dose that does not cause any adverse effect) which set the limiting values of toxicity. 2008 . Nat. several tests have been validated by the European Centre for the Validation of Alternative Methods ECVAM. However. (Eaton and Klaassen 2001) The typical in vivo acute toxicity test (LD50) is estimated by determining the number of animals that die in a 14-day period after treatment of a single dosage of the chemical. NOAEL is the most important value and is helpful when the limiting values for chemicals need to be set. usually from six months to two years. Liesivuori. The safety data sheets still include results of LD50 assays. Some genetic alterations can be visualized with the light microscope. In addition. C. In the present study. this test has been removed from the guidelines (in 2002). In vitro tests for mutagens are in widespread use. severe eye irritation and mutagenicity. (Eaton and Klaassen 2001) Numerous in vivo and in vitro procedures have been devised to test chemicals for their ability to cause mutations. phototoxicity. Sci. these tests are cheaper and quicker to carry out and generate smaller quantities of toxic waste than other tests (Baksi and Frazier 1990). compounds can be selectively cytotoxic or cause cytotoxicity by interference with cell-speciﬁc functions (Seibert et al. e. In the OECD and EU guidelines there are in vitro replacement tests only for skin absorption. Cytotoxicity can include changes in the integrity of membranes and cytoskeleton. In addition.
1993.c. Hengstler et al. when studying the effects of various anticancer drugs during the early phase of the drug development. 1995.g. Seibert and Gosch 1990. 241:1-67. 2004). The spermatozoa in vitro test has been found to be useful when detecting the presence of hazardous substances in indoor building materials subjected to moisture damage and containing complex microbial communities of bacteria and fungi (Andersson 1999). both hyperactivation and inhibition. 1994a. They are completely dependent on their surrounding environment for nutrients and they are also not able to detoxify toxic end products due to the low concentrations of detoxifying enzymes in the cell cytosol. tumour lines. paper materials have been studied (Severin et al. The motility of semen can be measured in several ways e. 1995). Dybing and Sanner 1999. 1996). Cells can be useful for analysing different kinds of materials to reveal acute toxicity. These cell lines have many uses e. Kopponen et al. normal lines and transformed lines. 1991.Review of the literature chemicals causing carcinogenicity in animals are not necessarily carcinogenic to humans (Grisham 1997. 2005). In different studies. Bovine spermatozoa have been used in studying cytotoxicity (Seibert et al. 1994. Törrönen et al. Retinal pigment epithelial cell lines have been exposed to anti-oestrogenic drugs in studies of eye toxicity (Toimela et al. but the combined effects are difﬁcult to study and are not often available in the literature.3 The use of cells in vitro A large range of cell lines is available for studying toxicity: human and animal cells. Sci. Hyperactivation of motility is associated with membrane hyperpolarisation (Zeng et al. 2004). Information about the toxicity of individual chemicals may be available. it is possible to study the overall toxicity of materials with unknown chemical compositions. 1989. 1992. 1999). lines from pathological cases. 1989. Severin et al. 1997). ﬂy ash samples and paper products (Kopponen et al. positive carcinogenicity tests in animals are usually interpreted as being indicative of potential human carcinogenicity (Eaton and Klaassen 2001). Boar spermatozoa cells have a simple metabolism compared to somatic cells. INVITTOX Protocol 21 1991). Bacterial toxins have been found to affect the cell membrane and the mitochondrial membrane potential in the sperm cells at very low concentrations (Hoonstra et al. C. 1991. 2005). 2. 1994. However. boar spermatozoa cells.g. 1992a. Many physiological processes in spermatozoa are controlled by membrane potentials and ion ﬂuxes (Mann and Lutwak-Mann 1982).b. In addition. and Environ. 2008 29 . 1990. 2004) and the effects of radiation with malignant pleural mesothelioma cell lines (Häkkinen et al. for regulatory and risk assessment purposes. Mäenpää et al.5. In addition. Since 1989. (1997) conducted some preliminary studies with textile dyes and fabrics using the hepa-1 cytotoxicity test. Hepa-1 mouse hepatoma cells (Hepa-1c1c7) are a commonly used cell line to assess the potential toxicity of dioxin and dioxin-like compounds. the Hepa-1 cytotoxicity test has been included as a part of the Multicenter Evaluation Kuopio Univ. materials such as laboratory animal beddings and feeds.b. The effects of hyperoxia have been studied with cervical cancer cells (Campian et al. Publ. Inhibition of motility is a consequence of membrane depolarisation (Gao et al. Nat. Kärenlampi and Törrönen. hepa-1 mouse hepatoma cells and human keratinocyte HaCaT cells have been found to be useful.
Nat. In this test. 30 Kuopio Univ. 2. Mitochondrial viability and apoptosis induced by aluminium. some dead cells may also be measured and may cause variation in the viability results. 2007). The cells have also been reported as being useful in clarifying cell signalling pathways (Assefa et al. HaCaT cells are human skin cells and for this reason they have been considered as relevant when human toxicity has been studied. A water-soluble tetrazolium assay. which is similar to the MTT test.5-dimethylthiazol-2-yl]-2. For example. The Hepa-1 cytotoxicity test has given satisfactory values for practically all chemicals tested and these mouse cells are. O`Reilly and Mothersill 1997). 2005). In that study. In living cells. the MTT-test has been used (Kidd et al. HaCaT cells have been used for investigating adverse effects of UV-radiation (Isoherranen et al. INVITTOX Protocol 112 1995). However. 1994). 2008). 241:1-67. 1999). 2008). C. the WST-1 test. the energy of the exposed cells was also investigated by measuring their ATP content. HaCaT cells have been widely used for instance in evaluating skin irritation (Wilhelm et al.5-diphenyl tetrazolium bromide) measures the activity of mitochondrial succinate-tetrazolium reductase system of cells (Kwang-Mahn et al. Shimizu et al. but are not as versatile as hepa-1 cells. the test has been used when the proliferation rate of human osteoblast-like cells has been studied (Weibrich et al. 2008 . the content of the total protein provides valuable information about viability. 1999. Apoptosis and cell viability have also been measured with the MTT-test when anticancer drugs have been evaluated (Giovagnini et al.Kaisa Klemola: Textile Toxicity of In Vitro Cytotoxicity (MEIC) programme. Protocol 64). Human keratinocyte HaCaT cells possess some metabolic activity. Publ. 1996).5. 1999). The MTT-test (3-[4. 1997. 2005) Immunotoxicological activity has been tested by exposing mouse macrophages and measuring the endpoints by the MTT-test when indoor air of moisture-damaged buildings has been studied ( Huttunen et al. The programme revealed that the various cell lines and growth end point measurements gave similar cytotoxicity results in most cases. and Environ. has been performed to measure the mitochondrial synthesis and cellular proliferation rate (Kwang-Mahn et al. better indicators of human toxicity than rat cells. 2004). neutral red penetrates cell membranes and accumulates in lysosomes and can be measured photometrically (INVITTOX. in which the overall toxic potencies of a wide range of chemicals relevant to human toxicity have been studied (Clemedson et al. desipramine has been evaluated in human PC3 prostate cancer cells (Chang et al. skin cancer (Merryman et al. genotoxicity and mutagenicity of textile dyes (Wollin et al. in the studies where the skin models have been validated. 2002).4 Endpoints used in evaluation of toxicity in in vitro cell tests The total protein content of the cells is a widely measured and validated endpoint (INVITTOX Protocol 112 1995). The neutral red (NR) cytotoxicity test determines the number of living cells in the culture. 2004) and the cytotoxicity caused by potential contact with nickel and chromium (Little et al. in general. mercuric mercury and methylmercury have been assessed with the WST-1 test (Toimela and Tähti 2004). 1996. In particular. In addition. pyrethroid compounds in neural cell cultures have been examined with the WST-1 test (Kakko et al. 2008). The rate of apoptosis has been measured when the antidepressant drug. Sci. In addition.
C. 241:1-67. Kuopio Univ. 2008 31 . Sci. methylmercury and aluminium on gial ﬁbrillary acidic protein expression in rat cerebellar astrocyte cultures have been studied (Toimela and Tähti 1995).Review of the literature Lactate dehydrogenase leakage is coupled to energy production of the cells. The enzyme catalyzes the ﬁnal reaction of anaerobic glycolysis. LDH has been used as an indicator of cellular damage of membrane when the effects of mercury. several types of cells are available for use and several endpoints are available for measurement. Publ. the reduction of pyruvate to lactate (Campbell and Farrell 2006). Nat. and Environ. If one wishes to understand the mechanisms of toxic effects.
OBJECTIVES There is limited information about the toxicity of textile substances when they are present in ﬁnished textile articles. The aim of this study was to evaluate the toxicity of textile reactive dyes and fabric extracts with cell tests in vitro. and Environ. The aim was to study the overall toxicity of the dyes in fabrics (I-III). 3. Publ. To examine whether dyed materials differ in their toxicity from the pure reactive dyes. C. To evaluate the toxicity of common commercially available fabrics. 2. 241:1-67. To assess the usefulness of two cell lines and bovine spermatozoa for the determination of possible acute toxicity of textile reactive dyes (I-III). The aim was to determine if the materials after ﬁnishing and dyeing differ in their toxicity compared to the raw materials (IV). the purpose was to study whether data from cell tests could provide useful information about potential toxicity or safety of textile products. Nat. The detailed aims were as follows: 1.Kaisa Klemola: Textile Toxicity 3. In addition. Sci. 4. To deﬁne which tests are useful in providing information about the toxicity of reactive dyes. It was also of interest to examine if these tests could be further developed into routine tests for use in the textile industry. 2008 . 32 Kuopio Univ.
W. those fabrics dyed in industrial processes are called commercial fabrics. All commercial fabrics were available to study in the form of raw fabric material. University of Kuopio. 4.1.2 Origin of the cells Boar semen for testing was available from the Insemination Center of Pieksämäki. The fabrics were ﬁnished in textile factories in Finland. Finland. Hepa-1 mouse hepatoma cells (the wild type Hepa-1c1c7) were obtained from The Department of Physiology. The other fabric was reactive dyed and press shrunk: brown and blue.Materials and methods 4. there were two different types of knitted fabrics: 100% cotton 160g/m2 and 50% /50% cotton/modal 145g/m2. Sci. and Environ.1. MATERIALS AND METHODS 4. Colour Index numbers for the dyes were: Reactive Red 241.1. obtained from a commercial fabric shop. 4. University of Kuopio. One fabric was vat dyed and treated with ﬂame retardant: blue. In addition. They are also the basic three dyes used when mixing different colour combinations. Ohio.2 Fabric A typical sheeting fabric was used in this study: plain weave bleached cotton (white).1 Reactive dyes The three reactive dyes studied were monchlorotriazinyl dyes commonly used in cloth-making and by workers in the crafts industry. The samples of the dye powder were dissolved in the appropriate cell medium solution. D. 4. It was observed that the material contained some brightener. Reactive Yellow 176 and Reactive Blue 221 (Drimarene red CL-5B. Two qualities of 100% cotton fabrics for working clothes were investigated.1 Dyes and fabric samples 4. red and blue dyes respectively. One sample was self dyed with an unknown reactive brilliant yellow dye. twill woven (175g/m2). Finland. the chemical content of the raw fabrics was not known. USA in 1982. Kuopio Univ. Publ. More detailed information about these fabrics was not available. twill woven (260g/m2). Drimarene yellow CL-2R and Drimarene blue CL-2RL respectively).3 Commercial fabrics In this study. University of Cincinnati. The cells were originally obtained from Dr. Knitted fabrics were reactive dyed with yellow. The dyes were obtained from Clariant Ltd. 2008 33 . 241:1-67. Department of Environmental Health. However. Human keratinocyte HaCaT cells were obtained as a gift from the Department of Anatomy. C. Nat. Finland. Nebert.
The samples were added to semen. Before analysis. C. The fabric extracts were sterile ﬁltered through polyester and cell growth medium compounds were added to the extracts before exposure to the cells. the fabrics were spooled in cool and warm water baths and kept in pure boiling water for 10 minutes. using 2ml of semen with 40µl of the dye samples or fabric extracts.3 The spermatozoa motility inhibition test The spermatozoa test based on that used by Andersson (1999) and it was modiﬁed for use with textile samples. A total of 3% dye represents a strong colour on fabrics. Exposure continued for 24 hours and/or 72 hours at room temperature.3 Procedures for sample preparation and toxicity testing 4. The dye bath contained 400 ml water with 50g Na2SO4/l H2O and 20g Na2CO3/l H2O. The samples were shaken thoroughly before centrifugation for 5min at 4500 rpm. The fabric extracts contained the same concentration of cell growth compounds as in the controls of pure growth medium. Valinomycin dissolved in dimethylsulphoxide (DMSO) in several concentrations was used as the positive control: 2ng. The samples were gently mixed and the amount of living spermatozoa cells was qualitatively observed by light microscopy (100 – 400 x magniﬁcation).3. After 24 hours and/or 72 hours of exposure.3. sperm motility was measured and compared to the motility of sperm in the plain semen controls.Kaisa Klemola: Textile Toxicity 4. However. 4ng. there was only one extract sample for each commercial fabric extract. Publ. 4. A solution of valinomycin in DMSO with no semen was also tested. After gently mixing the tubes. Plain semen and water were used as negative controls to determine the level of normal values. 8ng and 16ng/2ml respectively. All processing was carried out under sterile conditions.2 Preparation of fabric extracts The fabric pieces (1cm x 1cm) were extracted with sterilized water (1g fabric/20ml H2O) in laboratory test tubes. 241:1-67. 200µl samples were taken to small plastic tubes for incubation at 37ºC for ﬁve minutes before microscopic analysis. The effects of water and DMSO were also tested. The temperature of the pre-warmed objective glasses used in the microscopic analyses was also 37ºC. All samples were compared to the control sample of plain semen. and Environ. The results reﬂected the motility capacity percentages of living cells. The dyeing process in the present study was based on the common procedures used in industry and by crafts workers.1 Procedure for dyeing the fabrics Fabric samples (10g) were washed gently without soap. There were 3-4 parallel samples. Nat. After dyeing. Na2CO3 was added to the dye bath ten minutes after the beginning of the dyeing process to adjust the pH.3. the tubes were gently mixed manually. 4. The tubes were inverted once a day. Sci. 2008 . The tubes were shaken at room temperature for two hours and incubated at 37ºC for 18 hours. The optimal value of the plain 34 Kuopio Univ. The amount of dye used was 3% on each fabric. Dyeing continued for one hour at 55ºC.
Nat. Before the addition of sodium phosphate buffer it was possible to observe the viability of the cells by light microscopy to obtain preliminary information.2). ability to move forward.4dinitrophenol was used as a positive control at three concentrations: 0. The limiting value of 25% therefore represents the IC50 (inhibitory concentration) which means the concentration where 50% of the original living spermatozoa (optimal value of control was set 50%) were dead.cells move forward. The plates were allowed to stand at room temperature for 15 min before being stirred in a microtitration plate shaker for one minute. the plates were thawed for 15 min and cell growth was detected by assaying the total protein content in the cultures (Kennedy et al 1993. 241:1-67. have strong vibration but less activity than controls. 50µl of sodium phosphate buffer (0.08mM in acetonitrile). All results were compared to these controls.most of the cells are dead. most of the cells are rotating. The total protein content in each well was measured using a plate reading ﬂuorometer at a wavelength of 405/460nm. hepa-1 cells were grown in -MEM (Minimum Essential Medium) and HaCaT-cells were grown in DMEM (Dulbecco’s Modiﬁed Eagle’s Medium). many dead cells. The protein (bovine serum albumin BSA) standard curves were measured in each bioassay.0) was added to each well before freezing the plates for at least 15 min at -70ºC.cells move forward.5 mg 2. only some are moving forward very slowly.45% . the hepa-1 cell cytotoxicity test was used (modiﬁed from INVITTOX [data bank on the use of in vitro techniques in toxicology and toxicity testing] protocol number 112).isolated cells are vibrating slowly. 2. pH 8.05mM. the culture was about 60% conﬂuent. except for the Kuopio Univ. Sci. the cells were washed twice with PBS-buffer (phosphate buffered saline. All processing. The results were recorded within these 5% boundaries. The crude categories were: 50% . After 72 hours of exposure.05mg/ml and 0. 40 . The toxicity limit was considered to be 25% of unexposed cells: levels of 25% and lower represent toxicity.4-dinitrophenol/ml DMSO was used as one control and diluted to concentrations of 0. 30 . damage that can be detected by microscopy.005mg/ml medium respectively. The test was carried out in 96-well plastic microplates seeded with a 200µl cell suspension per well (5x104 cells/ml). the following observations of sperm activity were conducted: speed of movement. vibration and dead cells. 20 – 25% . In the evaluation. 10% foetal calf serum and 1% penicillin/streptomycin solution. 4. Publ. some are rotating. have strong vibration and high activity. After breaking the frozen cells. Subsequently.2µm pore size) fabric extracts. 2008 35 .3. The same modiﬁed method was used for human keratinocyte HaCaT cells.15% . weakly moving or rotating at the end of the exposure time.Materials and methods control sample was set at 50% because the semen always contains dead and damaged cells. and Environ. The cells were grown as a monolayer at 37ºC in an atmosphere of 5% CO2.only some cells are moving slowly. 150µl of sodium phosphate-buffer was added to the wells followed by 50µl of cold ﬂuorescamine (1. Non-exposed cells with medium were used as a negative control. The toxic effects on spermatozoa were qualitatively evaluated in the ﬁeld of vision. 1995). The cells were exposed to the dye samples or to the samples of the sterilized ﬁltered (0. After growing for 24 hours. most of the cells dead and 5%. Both medium solutions were supplemented with 1% glutamine. pH 7.4 Cytotoxicity test with hepa-1 mouse hepatoma cells and human keratinocyte HaCaT cells In order to assess the potential toxicity of dyes and fabric extracts.some vibration remaining. rotation. 10 . nonmotile.35% . C.
4. The sample was considered to have low toxicity if the protein content was less than 80%. In the hepa-1 and HaCaT cell tests. A reduction of more than 50% in the protein content would represent a clearly toxic effect. The inhibitory concentration IC20 denotes the sample concentration where the protein content is 80% compared to the protein content of non-exposed cells. Sci. The number of the samples represented the number of independent measurements and the number of plates. was carried out under sterile conditions. C.5 Statistical methods The coefﬁcients of variation (C of V % were used as evidence of reliability with lower values than 10%) and standard deviations (SDs) were calculated for all measurements to evaluate the reliability. ANOVA. 36 Kuopio Univ. Every plate contained 3-4 parallel dye or fabric extract samples. 241:1-67. the number of the parallel samples was 3-4. and Environ. Statistical signiﬁcance of differences between three reactive dyes was performed for each cell test by variance analyse. 2008 .05 were considered as statistically signiﬁcant. The inhibitory concentration value. The IC20 value was considered to represent the value of LOAEL (lowest observed adverse effect level) which is the lowest concentration value of the sample showing adverse effects. Values of p < 0. The number of parallel samples was in the range of 3-11 when the spermatozoa cells were used as the test cells.3. Nat.Kaisa Klemola: Textile Toxicity protein assays. the IC50 value is the concentration of the sample in which the wells with exposed cells have only 50% of the protein content compared to that of the non-exposed cells (100%): the level of the protein content is a reﬂection of the viability of the cells. In such a case. but more than 50% compared to that of non-exposed cells. the studied dye or fabric extract has inhibited the cell proliferation by 20 %. Publ.
The dyes were assayed with boar spermatozoa cells. hepa-1 mouse hepatoma cell line and human keratinocyte cell line. 2008 37 . Cell growth was assessed as the amount of cellular protein after the exposure (Figures 8-10). and Environ. *n=3-10. Figure 9.2).1-9). RESULTS 5. Sci. Reactive Yellow 176 and Reactive Blue 221.1 The IC50 and IC20 values for three reactive dyes The three reactive dyes studied were Reactive Red 241. C. n = 2-7. Figure 8. Nat. 241:1-67. Kuopio Univ. SD = ±(2. exposed for 24 hours (I). *The number of the samples (n) represents the number of the plots at the various concentrations of the dye on the curve. HaCaT-cells. The inhibitory concentrations measured as IC50 and IC20 values were mathematically calculated from the functions which were drawn from the mean values of different dye concentrations.SD = ±( 2-5. exposed for 72 hours (II). IC50 and IC20 values for the yellow dye when hepa-1 cells were used. Publ.Results 5. IC50 value for the yellow dye when boar spermatozoa cells were used.
HaCaT cells were of intermediate sensitivity also showing somewhat different responses to the different dyes. 241:1-67. Publ. and Environ. Summary of the mean concentration values of IC50 and IC20 (µg/ml) for the dyes studied. after 72 hours of exposure. n = 2-6. exposed for 72 hours (III). 2008 . Table 6). The IC20 mean values showed that with the reactive dyes studied. However. 38 Kuopio Univ. the relative toxicity of the dye studied depended on which cells were used. The spermatozoa cells were most sensitive and displayed toxicity with the lowest dye concentrations. hepa-1 cells tolerated the highest dye concentrations. the blue dye evoked such high toxicity to the spermatozoa cells that it was not possible to determine the IC50 value (I. Nat.(3-9) According to the IC50 mean values. SD = ±(3. The results for all three dyes studied showed the same trends (I-III: Table 6). IC50 and IC20 values for the yellow dye when HaCaT-cells were used. in HaCaT cells the blue dye was less toxic than the yellow dye (I-III: Table 6). However. Table 6. Sci. The red dye was the most toxic to HaCaT cells and this value was even lower than the IC50 value for spermatozoa cells after the same 72 hours of exposure (Table 6).Kaisa Klemola: Textile Toxicity Figure 10.5-9. the hepa-1 cells had the highest tolerance (II-III: Table 6).8). HaCat 72 h µg/ml (n) IC50 IC50 IC50 IC20 IC20 IC20 Reactive Red 241 Reactive Yellow 176 Reactive Blue 221 Reactive Red 241 Reactive Yellow 176 Reactive Blue 221 155 (5) 237 (6) 278 (2-4) 28 (5) 78 (6) 112 (2-4) Hepa-1 72 h µg/ml (n) 370 (4-6) 392 (4-7) 361 (2-4) 108 (4-6) 176 (4-7) 158 (2-4) spermatozoa 24 h µg/ml (n) 124 (4-9) 135 (4-9) 127 (4-10) 72 h µg/ml (n) 46 (3-9) 60 (4-9) . Of the three cells used. C. Thus.
the dyed fabrics were not toxic.Results 5. In addition. The same fabrics after industrial dyeing and ﬁnishing were then analyzed. C.50% of the non-exposed cells (100%) when studied with hepa-1 and HaCaT cells. none of the untreated fabrics studied were considered toxic. The spermatozoa cells did not show toxicity with the fabric extracts apart from the ﬂame retardant fabric. (IV). The plain weave bleached cotton fabric samples which were used as controls were toxic when studied with hepa-1 and HaCaT cells. the plain cotton fabric was dyed with the same reactive dyes as above: Reactive Red 241. Over 50% of the spermatozoa cells retained motility (n=3-6). 2008 39 . However. The studied knitted fabrics did not display any toxicity in the spermatozoa inhibition test after commercial dyeing and ﬁnishing (n= 3-11. The protein contents of the samples measured as an indication of cell growth were found to be 35 . The extracts from the two types of untreated knitted fabrics were not toxic when assayed with the three different cell tests. Thus. Figure 11. Sci. The protein contents in the extract samples indicated cell growth levels of over 80% compared to the non-exposed cells (100%) when hepa-1 and HaCaT cells were used (n=3: IV). In addition to the dyed fabrics. Publ. according to the spermatozoa motility inhibition test and the cytotoxicity tests with hepa1 and HaCaT cells. Figure 12: IV). The yellow cotton was not toxic Kuopio Univ. and Environ. after industrial dyeing and ﬁnishing. fabric extracts were toxic to the hepa-1 and HaCaT cells (n=3: Figure 12) though the yellow and red cotton fabrics were exceptions (IV). However.2 The toxicity of the fabric extracts In the present study. The cytotoxicity of the dyed plain weave and commercial ﬂame retardant fabrics. which displayed low toxicity (n=3-11: IV). a commercial ﬂame retarded fabric was studied. The fabric extracts from the commercial woven fabrics dyed with reactive dyes resulted in decrease in measured protein contents to less than 20% compared to the nonexposed cells when hepa-1 and HaCaT cells were used (IV). Nat. 241:1-67. It was not toxic to these cell lines (IV: Figure 11). Reactive Yellow 176 and Reactive Blue 221. a commercially obtained fabric dyed with an unknown brilliant yellow reactive dye was studied.
and the red cotton was not toxic in the hepa-1-cytotoxicity tests (IV). Reactive Yellow 176 and Reactive Blue 221 (I-III).Kaisa Klemola: Textile Toxicity in the HaCaT. Coefﬁcient of variation values (C of V %) for dye samples and fabric extracts. and Environ. Before and after dyeing and ﬁnishing. most knitted fabrics were toxic to hepa-1 mouse cells and on HaCaT cells (n=3-4). Table 7. The spermatozoa motility test showed only the ﬂame retardant fabric to be toxic. toxic results spermatozoa (dye) spermatozoa (dye) hepa-1 (dye) HaCat (dye) spermatozoa (fabric) spermatozoa (fabric) hepa-1 (fabric) HaCat (fabric) after 24h exposure after 72h exposure after 24h exposure after 72h exposure 31-69 0-58 9-46 7-21 non-toxic results 0-18 9-37 <10 2-14 10-16 8-35 10-17 9-13 40 Kuopio Univ. the blue knitted fabrics were most toxic to the hepa-1 cells (IV). The percentage of protein in cell exposure by knitted fabric extracts compared to the nonexposed cells when hepa-1 and HaCaT cells were used (n=3: IV). The C of V values ranged from 2 –24% (IV). Publ. The fabrics were dyed with Reactive Red 241. 241:1-67. In addition. Figure 12.3 Reliability of the results The commercial fabrics in the study were industrially dyed and ﬁnished. * CO=cotton. the coefﬁcients of variation (C of V) were 3 – 13% (n=3) (IV). C. These woven fabrics were not toxic when tested with hepa-1 and HaCaT cells. The industrial fabrics had C of V values of 0 – 18 % (n=3-11) (IV). When the materials were industrially dyed and ﬁnished. CMD=modal 5. When the blue knitted fabrics were studied in hepa-1 cells the extracts evoked strong toxic effects compared to the situation with the same extracts in HaCaT cells. Sci. Nat. The industrial untreated knitted fabrics showed C of V values of 4-13%. 2008 .
p = 0. The low control concentration of DNF (0. 5.122. the differences between the three studied dyes were statistically signiﬁcant. 2. As a positive control.05mg/ml) resulted in 60% protein content (C of V 11-18%). Publ. for the high concentration of the dyes (196µg/ml). 2008 41 . DNF was used as a positive control and the mean value of the protein content measured from cell suspensions after exposure at low concentrations was 80% of the protein content of the non-exposed cells (II-IV).763. p = 0.225. p = 0. In the HaCaT and hepa-1 assays. non-exposed cells with medium were used as negative controls and all results were compared to them. p = 0. for the concentration of 100µg/ml. Sci. p = 0.4-dinitrophenol (DNF) was used giving C of V values of 2 – 25%. but not at the high concentration of the dyes (600µg/ml). Two concentrations.000. High C of V values were noted in the toxic range. IV). Kuopio Univ. (II-IV). Hepa-1 cell test showed statistical signiﬁcant differences compared to control at a concentration of 150µg/ml. C. for the low concentration (39µg/ml). The values covered the toxic and non-toxic areas.Results For assays using spermatozoa cells. p = 0. valinomycin was used as a positive control.4 Statistical signiﬁcance In the spermatozoa 24-h test. 241:1-67.002.003. and Environ. Nat. With HaCaT cells. while the highest control concentration of DNF caused severe inhibition of cell growth. for 380 µg/ml. for 190 µg/ml.022. DMSO was not toxic at the concentrations used in the tests (I. the difference in the results between the three studied reactive dyes was not statistically signiﬁcant. only 10 % remaining of protein content of the control (C of V 2-25%: II-IV). p = 0. 2ng/2ml and 4ng/2ml of valinomycin had no adverse effects after 24 hours exposure but 8 ng/2ml and 16 ng/2ml concentrations did cause toxicity.
Spermatozoa are dependent on their surrounding environment for nutrients and also for removal of toxic end products since they have low levels of detoxifying enzymes in their cytosol. Reactive Yellow 176 and Reactive Blue 221).Kaisa Klemola: Textile Toxicity 6. This can be explained by the ability of hepa-1 hepatoma mouse cells to metabolise xenobiotics (INVITTOX Protocol 112 1995). Publ.it). The cytotoxicity tests with hepa-1 and HaCaT cells provided information about the toxicity of the substances studied. 241:1-67.1 Reactive dyes in the cell tests When the reactive dyes were studied in vitro (Reactive Red 241. This may be attributable to differences in the metabolic abilities of the cell lines. and Environ. Hoonstra 2004). it seemed that hepa-1 cells tolerated higher dye concentrations than HaCaT cells. In addition. Kennedy et al. When the dyes were studied. not information about their exact metabolic effects. The IC50 and IC20 values for the above mentioned reactive dyes were higher with hepa-1 cells than with HaCaT cells. The enzyme cytochrome P4501A1 is inducible in the epidermis by agents that are inducers also in other tissues. Induction of CYP1A1 (detected as arylhydrocarbon hydroxylase and 7-ethoxyresoruﬁn o-deethylase. Stegeman and Lech 1991). Kopponen et al. 1994a). no enzyme activities were measured as the aim was simply to assess cell viability to reveal acute toxicity of the studied substances. the aim was to obtain information about acute toxicity and therefore only the viability of the human keratinocyte cells was evaluated. 2008 . 1987). toxicity was detected with the spermatozoa cells at low dye concentrations. Since the various cells used in the present study possess different capabilities it is not unexpected 42 Kuopio Univ. Hepa-1 cells have been widely used as a model for studying the induction mechanism of CYP1A1 (Kärenlampi and Törrönen 1990. the phase I metabolism in the skin is usually only a small fraction of that found in the liver. 1995). Sci. The physiological processes in spermatozoa are controlled by membrane potentials and ion ﬂuxes (Mann and LutwakMann 1982) and therefore the spermatozoa cells may be useful for indicating acute toxicity: they can be assumed to give relevant information about toxicity (Seibert 1992. as reﬂected by the higher IC50 and IC20 values with hepa-1 cells than with HaCaT cells. In this thesis. carcinogenicity and other adverse effects. this activity occurs at a much lower rate than that observed in the liver. DISCUSSION 6. In this study. Human keratinocytes have some ability to metabolise xenobiotics. Nat. a phenomenon observable in cell culture (Walsh et al. the enzymes participating in phase II metabolism are expressed in the skin. Though metabolism of xenobiotics usually facilitates the excretion of chemicals. but exceptions are evident. certain steps in the biotransformation pathway can be responsible for the activation of foreign chemicals to reactive intermediates that ultimately result in toxicity. EROD activity) has been shown to be useful for the assessment of the biological potencies of pure chemicals like PCBs and for the estimation of dioxin-like compounds in extracts of environmental samples (Kennedy et al. 1987. The cell lines have been widely used and different tests have been developed as an alternative for animal tests (http://www.jrc. 1995).ecvam. However. In general. 1993. Thus exposure to this kind of agent could inﬂuence skin biotransformation and even sensitize epidermal cells to other agents that are not good inducers themselves. as in the case of quinone reductase (Khan et al. This has been clearly detected in aquatic species (Varanasi et al. C.
Sci. but it is not possible to obtain information from the manufacturers about what other chemicals are present. In the Safety Data Sheet. chlorine. the growth medium was buffered (pH 7. In the present study. all IC50 values with hepa-1 cells were in the range 158 . containing a sulphonyl-group. 2008 43 . In the present study. It is possible to determine which concentration of the particular dye evokes the ﬁrst level of adverse effects and when the material is clearly toxic to the cells. exposed for 24 hours). but also the other chemicals may evoke adverse effects. The sample concentrations during the tests were low (less than 400µg/ml). According to toxicity tests conducted with activated sludge.Discussion that they exhibit some differences in the results obtained. The Chemical Safety Data Sheets for the reactive dyes in this study indicate the blue and red dyes were more toxic than the yellow dye. When the spermatozoa cells were exposed to the blue dye for 72 hours. the red and yellow dyes had IC50 values higher than 1000µg/ml. all cells displayed no motility. ﬂuorine and nitrogen. for instance. When hepa-1 and HaCaT cells were used. the concentrations of the samples were lower Kuopio Univ. the IC50 mean values from the spermatozoa test and hepa-1 test for the yellow dye pointed to higher values than those obtained for the red and the blue dyes. but these dyes were not the same as those used in the present study. and Environ. (1997) studied reactive textile dyes using hepa-1 cells. They contain. Publ. In addition. In the present study. The discussion of the results of this study relates to the toxicity of a mixture of different chemicals and not to the pure molecules. When the spermatozoa cells were used. carboxymethylcellulose CMC. The dyestuffs used in the present study are. the toxicity of the blue dye measured as EC50 (the molar concentration of an agonist. The IC50 values were in the range 53 –825µg/ml. Nat. orally) for the yellow dye is 5000mg/kg. these dyes had LD50 values of 2000 – 9000mg/kg. Kopponen et al. In the present study. when the dyes were studied similarities were noted in the end points and the results supported each other. The dyes are monochlorotriazinyl dyes e. However. exposed for 72 hours). the IC20 mean values from the HaCaT test for the red and the yellow dyes indicated that these compounds may be more toxic than the corresponding values for toxicity published in the Chemical Safety Data Sheets.5 and it is evident that these pH values do not cause toxic effects to the cells. the IC50 values for the blue dye were 127µg/ml (spermatozoa cells. exposed for 72 hours). in fact.1-7. which produces 50% of the maximum possible response for that agonist) was greater than 100µg/ml. This was evaluated via the determination of the IC20 and IC50 values. mixtures of different chemicals (I-IV). 241:1-67. The IC20 values in the present study for the blue dye were between 112158µg/ml with HaCaT and hepa-1 cells. This information would be useful in assessing the toxicity of dye molecules. According to the OECD 209 method (an acute toxicity test for testing sludge). It is possible that not only the dye molecules. The pH of the dyes is in the range 4.392µg/ml (I-III). the LD50 value (rats.g. and for the red and blue dyes 2000mg/kg. This study was not designed to evaluate the effects of a pure dye. it is possible that the dye formulations which were studied are not consistent. The IC20 mean values with HaCaT cells were lower than 100 µg/ml for the red and the yellow dyes (exposed for 72 hours).5 – 6. 361µg/ml (hepa-1 cells.2) and the samples diluted. In the Safety Data Sheets. C. According to the OECD 203 method (an acute ﬁsh toxicity test). but the precise chemical structures of the dye molecules are not available. and the blue dye contains copper. 278µg/ml (HaCaT cells. the LC50 values were claimed to be in excess of 100µg/ml. calcium stearate and other chemicals.
The commercial untreated industrial woven and knitted fabrics were not toxic (the protein content of the cells did not decline to any signiﬁcant degree. after reacting with ﬁbre molecules. It can be assumed that washing and dyeing must have removed harmful chemicals (I-IV). in the present study there was no information available about what kind of different chemicals were used when the knitted fabric was manufactured. The protein contents of the samples taken as indications of cell growth were in the range 35-50% of control. washing of newly dyed products is recommended (Moreau and Goossens 2005). 1988. The knitted fabrics were coloured with reactive dyes. It is also well known that free formaldehyde in textiles can cause toxicity (Priha 1992. 1993. This indicates that for these reactive dyes studied. before it was dyed. i. the ﬂame retardant fabric displayed low toxicity when incubated with the spermatozoa cells. the material was no longer toxic with cells demonstrating over 80% protein content of control. Manzini et al. 1996. It has been observed that some fabric softeners may cause adverse effects in ﬁsh (Wester and Roghain 1992). It can therefore be assumed that the dyes may be more toxic during the dyeing process than during the conditions of the present series of studies. Priha et al. The other commercial woven fabrics were not toxic. these agents should not produce adverse effects in the fabrics. Sci. Hatch and Maibach 1995. However. the plain cotton fabric (bleached) became less toxic after the dyeing process. 1996). 6. Although reactive dyes themselves cause adverse effects like asthma.Kaisa Klemola: Textile Toxicity than those incubated with the hepa-1 and HaCaT cells.e. Publ. the fabric became non-toxic. all data obtained in the spermatozoa motility tests conﬁrmed these non-toxic responses (IV). Therefore. evidence that some harmful chemicals were present. The fabrics to be dyed in the present study were more toxic in the HaCaT cell tests than they were in the hepa-1 cells. However. The fabric itself. In addition. after industrial dyeing and ﬁnishing. Nat. Although the extracts of some fabric samples contained colour. after dyeing. Nilsson et al. less than 20 %). This is because dye molecules are easily hydrolysed in water. any unbounded dye remaining in the fabric could cause allergic reactions. However.2 The fabric extracts in the cell tests Reactive dyes bind to the ﬁbre covalently (Trotman 1984). the extracts were not toxic. Since reactive dyes and their hydrolysis products are water-soluble. According to the present study. the dyes are stable and should not be toxic within the fabric. 2008 . In addition. the knitted fabrics evoked adverse effects in hepa-1 and HaCaT cells (protein contents of cells were considerably below 80% of control with the exception of the yellow cotton in HaCat cells and the red cotton in hepa cells). It is usual to treat knitted fabrics with softeners. unbounded dye can be washed off. C. the dye bath is alkaline because reactive dyes demand a high pH for good reactivity. Wilkinson and McGechaen 1996). more information is needed about the overall toxicity of reactive dyes and dyed fabrics. 1995. In the textile dyeing process. the dye molecule loses its reactivity. rhinitis and dermatitis (Estlander 1988. After the industrial dyeing and ﬁnishing processes. so it can be assumed that the softeners may have caused some toxic effects. 241:1-67. and Environ. However. it is possible that the 44 Kuopio Univ. However. In the present study. was toxic. the end products of the dyeing process do not cause toxic effects in cell tests: i. The binding is very stable and during the process.e.
while in others. since the protein content of some dead cells may be also measured even though the washing procedure should remove the dead (unattached) cells. In addition. However. 6. and variations may occur in the results. The hepa1 cell test has been found to be sensitive and reliable (INVITTOX Protocol 112 1995. there were differences in the quality of the spermatozoa cells used in the present assays and this affected their motility. protein measurement is a widely used procedure and it does provide some information about the toxicity of the studied material. The knitted fabrics produced a particularly clear toxic effect on hepa-1 cells. However. In some assays. despite these limitations. Törrönen et al. Nat. However. 1994. 1992. Kopponen et al. these cell cultures were sensitive to the toxic effects of the compounds in the textiles under study. Since the evaluation of the spermatozoa motility inhibition is subjective. the test can be used reliably for screening samples and when combined with other tests: the inhibition of boar spermatozoa motility has proved valuable when evaluating the toxicity of food constituents (Salkinoja-Salonen et al. it was noted that the cell growth at the edges of the plates was often less impressive than in the middle. Publ. several researchers studied the same samples and the spread of their results was within 5%. The NR test is useful if one wishes more exact information about the cells (INVITTOX Protocol 64 Kuopio Univ. However. 2001/838/EY) have been found. In the cell viability experiments. in the present study with the knitted fabrics. 241:1-67. In this study. In addition. C. However. the total protein contents were measured to indicate the viability and proliferation capacity of the cells. it was interesting to note that hepa-1 mouse cells were more sensitive to toxic knitted fabrics than human keratinocyte HaCaT cells. this test is a qualitative test.Discussion studied material contains chemicals for binding the extra dye to the ﬁbre to improve the wet-fastness. However. 2004). and Environ. a signiﬁcant proportion had good motility. When hepa-1 mouse cell line and human keratinocyte HaCaT cell line were used.3 In vitro cell tests for assaying textile substances The cells in culture usually grow well. The present study indicated that the spermatozoa test is also valuable for studying the potential toxicity of textile substances. In the present study. avoiding possible errors due to differences in the quality of the cells. this is not a very exact method for assessing the viability of the cells. This was opposite to the anticipated results. 2008 45 . However. further development will be necessary when fabric extracts are going to be studied. in the present study. 1997). Kopponen et al. However. and in the present tests the quality of the cells was standardized. There is also the possibility that some living cells are lost before protein measurements during the washing procedures of the plates. residues of harmful nonylphenols (surfactants. The results of the spermatozoa test are read within 5% bands and this can have an extra effect on the variation inherent in the results. the exposure results in every cell culture assay as in the spermatozoa motility assay were compared to those of the non-exposed cells. Sci. although these cells would have been expected to be resistant since they possess better metabolic capabilities than HaCaT cells. 1999). a substantial number of cells were broken. the test has demonstrated its value as a cell toxicity assay in detecting hazardous substances in products without the need for whole-animal exposure or foetal calf serum for cell cultures (Hoornstra et al. it remained unclear which chemical or combinations of chemicals produced the adverse effects on the cells.
However. When the three reactive dyes were compared to each other. Statistical signiﬁcance in hepa-1 cells was observed when the concentrations of the dyes were low. Kwang-Mahn et al. Kakko et al. further development may lead to better reproducibility. cell energy and membrane effects have been studied by ATP and LDH measurements (Toimela and Tähti 1995. e. The extraction method for the spermatozoa motility test needs further development. However. C. The physical property that commonly enables xenobiotics to be absorbed through the cell membrane is the lipophilicity (Gregus and Klaassen 2001). The concentrations of the samples were the same for the hepa-1 and HaCaT cell assays. However. the present study shows that these tests are promising novel methods for studying the toxicity of components on textiles. the quality of the spermatozoa cells can lead to variations in the ﬁnal results. for instance by studying a fuller range of fabric extracts. The fabrics may contain compounds that are lipid soluble. in the future. In addition. the range of substances should be extended and the validation of the test should be made in co-operation with other laboratories. the results of the spermatozoa test (exposed for 24 h) did not reach statistical signiﬁcance (p > 0. it may be useful to expose cells in the medium that contains a lower concentration of serum. the results did not show any adverse effects. However. In the hepa-1 and HaCaT tests. It is clear that there is a need to develop further the spermatozoa test.05). in the present study. all concentrations of the tested dyes resulted in statistically signiﬁcant changes. it seems that the optimal exposure time is 24 hours since this results in the lowest C of V values. The dye molecules are hydrolysable and hence lose their reactivity in aqueous solutions (Trotman 1984). Mitochondrial activity can be studied by MTT and WST-1 assays (Toimela and Tähti 2004. The fabric extracts were not concentrated because that process may have produced chemical reactions during the procedure. 6. For the standardization of the tests. Sci. Nat. 2004). and Environ. the limiting values for toxicity and non-toxicity were clearly identiﬁed. but the spermatozoa cell concentration was lower. It has to be emphasised that the spermatozoa motility test is a qualitative test. In addition. it will be important also to use lipophilic solvents when extracting fabric materials.Kaisa Klemola: Textile Toxicity 1992). the methods to study overall toxicity of textile substances were the focus of interest and more detailed information about mechanism of toxicity in the cells was not the target of the study.4 The reliability of the tests The coefﬁcient of variation was typically high when cell toxicity was present. Polyester cannot bind covalently to dye molecules. since they contained free dye molecules that had passed through the ﬁlter. Publ. According to the C of V values in this study (0-18%). possibly affecting their toxicity. 241:1-67. 2005). Therefore. This is understandable when living material is being analysed. All three methods must therefore be developed and validated further if they are to be used routinely. In HaCaT cells. 46 Kuopio Univ. as this was a requirement of the assay method. Some extracts were coloured. The high concentrations of the serum used may cause mistakes since it may be able to bind certain molecules. 2008 .g. The fabric extracts were not concentrated. The fabric sample extracts were ﬁltered through polyester. The spermatozoa motility inhibition test is only suitable for crude screening of chemical samples.
241:1-67. ECVAM. In vitro cytotoxicity tests provide preliminary information about acute toxicity and other parameters. The evaluations of 20000 of these chemicals have been planned to be performed without animal experiments by using all information available and also by increasing the use of validated alternative tests. Evaluation and Authorisation of Chemicals) demands that the safety of 30000 industrial chemicals should be evaluated by the year 2018. In addition. 2008 47 . In the European Centre for the Validation of Alternative Methods. differentiated. the surface with a type of human collagen (Valerie et al. Thus. 2005). and Environ. more advanced studies in the area of toxicology are needed. especially using a range of cell tests. although subchronic and chronic effects with animals may still needed in the foreseeable future. nor have the combined effects of different chemicals been studied. 2005). Some information about the impacts of waste waters from textile processing is available. The EpiDerm model consists of a multi-layered. Nat. One of the most sensitive biochemical responses is the induction of speciﬁc cytochrome P450 (CYP) enzymes. Two skin models have been validated: the EPISKIN model and the EpiDerm model (EU. fabrics may contain chemicals that are not assessed in the Öko-Tex-100 standard. Another approach would be to use macrophages to evaluate the effects of the compounds on immunological activity. Sci. in vitro model of the human epidermis (Valerie et al. Publ. It would be interesting to study further in more detail the speciﬁc effects of textile dyes and fabric extracts. so the cell tests represented here are useful in conﬁrming the safety of the product. Cell proliferation studied by MTT and WST-1 assays may be useful when more detailed information about the effects of textile substances needs to be obtained. the ATP balance in cells exposed to different textile chemicals. However.Discussion 6. ECVAM 2007. 2005) and it would be useful to use HaCaT cells in toxicity tests with different textile chemicals. However. skin models have been validated and endorsed as suitable for testing skin corrosion and irritation in vitro. The effects of organochlorine pesticides have been studied using HaCaT cells (Ledirac et al. formed from a bovine collagen matrix. El Ghalbzouri et al. Safety Data Sheets show information about the toxicity of individual chemicals. for instance their effects on mitogen activated protein kinase pathways and other cell signalling pathways in human keratinocytes. 2008).g. since there is a scarcity of research providing information for the consumer about the potential toxicity of textile end-products. The cell tests can provide extra information to the standard tests. it would be of interest to measure the energy metabolism of the cells. 2007. Cell tests can be used not only for pure chemicals but also for testing materials containing different chemical components. this standard does not require that common biological tests have to be carried out to detect any adverse effects of the products. for instance heavy metals (Öko-Tex Standard 100 1997). Textile materials are often in contact with the skin. the ecology of laundry services (Kalliala 1997) has been studied. In addition.4 The possibilities for utilizing cell-based tests for studying textile substances Improvements have occurred not only in the working conditions in the textile industry but also in the environmental-friendliness of industrial dyeing-ﬁnishing processes (Nousiainen 1997). The EU new chemical legislation (REACH. C. It is also recommended that more tests that do not involve the use of animals should be devised. The Registration. in the fu- Kuopio Univ. The Öko-Tex-100 textile standard assesses the amounts of certain harmful compounds in textile products related to prescribed limits. The EPISKIN model is a three-dimensional human skin model. Grindon et al. e. In addition.
for instance. 2007). The data is also difﬁcult to obtain since in most cases this information is regarded as a trade secret. 2002). cell tests could be useful in developing ecologically sound textile processes. C.g. 2001. Ciardelli et al. For consumers. 48 Kuopio Univ. such labelling could provide information about the safety of the product. Khan and Husain 2007. However. 2006). 1997. and Environ. ion exchange methods (Lin and Chen 1997) and coalabsorption (Santhy and Selvapathy 2005. Publ. aerobic/anaerobic treatments (Liakou et al. The cell tests used in the present study represent assays that could also provide information about the toxicity of wastewater. In vitro cytotoxicity tests can also be used if wastewaters need to be studied. In vitro cell tests are useful when one wishes to study overall cell toxicity. it could be possible to develop a new labelling scheme for textile materials. Asgher et al. biological cell tests can provide simple and rapid methods to evaluate the potential toxicity of textile products. related to the Öko-Tex standard 100. The manufacturing of textiles is a global activity and often virtually nothing is known about the chemicals used. Sci. the ability of microorganisms to carry out dye decolorization (Banat et al. with the luminescent bacterium Vibrio Fischeri (Wang et al. Dincer et al. In quality control laboratories. Marquez and Costa 1996. The toxicity of wastewaters has been evaluated with different techniques. Nat. some residues of the chemicals used may remain in the textile products causing health problems for some consumers. 2007). 241:1-67. In the future. based on measured biological effects. Cell tests are effective if materials with unknown chemical contents need to be studied and thus they are suitable for studying textile materials.Kaisa Klemola: Textile Toxicity ture studies of textile substances it is recommended that these skin models should be used. Frijters et al. There have been many investigations for developing cleaning techniques in wastewaters emerging from the textile industry: e. 1996. In addition. 2008 . The toxicity of the textile material which was found in this study may be attributable to some individual chemical or to the combined effects of several chemicals. resulting in a labelling scheme analogous to the Öko-Tex standard 100 used today by some textile manufacturers.
CONCLUSIONS In the present series of studies. All cell tests showed clearly that the three reactive dyes (Reactive Red 241. C. After dyeing and ﬁnishing. Sci. In the hepa-1 and HaCaT cell tests. the commercial knitted fabrics studied did not evoke any adverse effects in the cells. After dyeing with reactive dyes. the same sample concentrations were used. However. but extracts from reactive dyed fabrics were not toxic even when they had been coloured with a dye which on its own was toxic. all tested concentrations of these dyes evoked statistically signiﬁcant effects on the viability of HaCaT cells. These facts support the need for using a combination of different cell lines in parallel to provide comprehensive information about toxicity. Reactive Yellow 176. Reactive Blue 221) were toxic. rapid test and may be used for screening of samples. The in vitro cell tests can clearly reveal acute toxicity. for instance by the chemicals losing their reactivity. However. These tests were found to be suitable when materials such as textiles with unknown components need to be analysed and this study shows that they may be valuable in forming the basis for developing routine tests for use by the textile industry. Nat. whereas the concentration was lower in the spermatozoa test. Human keratinocyte HaCaT cells were more sensitive than hepatoma hepa-1 liver cells when incubated with the dyes and reactive dyed fabric extracts were. The spermatozoa inhibition test is still a qualitative. Kuopio Univ. caution is required not to overestimate the capabilities of these cells to display all responses which can be extrapolated to humans. three in vitro cell tests were used to assess the acute toxicity or potential adverse effects of textile reactive dyes and reactive dyed fabrics. This might be attributable to the fact that liver cells have a good capability to metabolise different substances. Publ. 241:1-67. The dye in the extracts had been apparently hydrolysed and therefore had not the same level of reactivity as the intact dye. a previously toxic textile material was no longer toxic apparently because during the dyeing process. Since these fabrics had been dyed with reactive dyes (which are not toxic in the dyed fabric) it is hypothesized that the toxicity is caused by some unknown chemical(s) used in ﬁnishing. There were no major differences in the toxicities of the three studied reactive dyes when the dye concentration was low with hepa-1 cell line. 2008 49 .Conclusions 7. some commercial fabrics did cause toxicity. This latter test needs further reﬁnement to be of practical use for analysing fabrics. harmful chemicals must have been removed or the material had become inactivated in some other way. Before industrial ﬁnishing. and Environ.
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