CHRONIC

HEALTH

EFFECTS TESTING

FOR HYDROQUINONE

Nonprescription Drug Manufacturers Association+ 1150 Connecticut Ave. NW Washington, DC 20036 (202) 429-9260

May 12, 1992

’ The Nonprescriprion Drug Manufacturers Associarion (NDMA) is a illyear-old U. S. trade organization representing the manufacturers and distributors of nonprescription (or over-the-counter)medicines.

CHRONIC HEALTH EFFECTS TESTING FOR HYDRQQUINONE I. Executive Summarv From an in-depth review of the entire databaseon hydroquinone, the Nonprescription Drug Manufacturers Association (NDMA) Hydroquinone (HQ) Task Group concludes that hydroquinone in OTC skin lightening preparationsdoes not present a human carcinogenic risk when used according to label directions. This conclusion is basedon differences in speciesand strain-specific mechanismsof toxicity which contribute to the animal bioassay results and an evaluation of the genotoxicity database. For several years, hydroquinone @ IQ) has been the subject of a series of chronic health effects studies under the testing programs of the U.S. Environmental Protection Agency (see the Toxic SubstanceControl Act Section 4) and the U.S. National Toxicology Program. These studies have included endpoints for neurotoxicity, reproductive toxicity, developmental toxicity, genotoxicity, pharmacokinetics,chronic toxicity, and carcinogenesis. The results of these studies and many more in the published literature have created a data base for HQ that is much more extensive than is available for many other chemicals. Much of these data are derived from studies designedto maximize the likelihood of producing a toxic responseby administering HQ in a high-dose bolus by the gavage route. While on the one hand such studies may be well designedfor hazard identification, they have the potential for producing misleading results for risk assessment purposes. This is particularly the case for chemicals such as HQ where the primary route of exposure is dermal (not oral, as in the case of the animal bioassays)and where dermal absorption does not result in significant toxicity. In such circumstances, the results of oral or parenteral studies should be considered crude estimatesof effects. Such studies are adequatefor preliminary hazard identification, which can often -- and should, where possible -- be refined by additional research, particularly involving studies by the dermal route. Furthermore, the situation with HQ is more complex than simply trying to extrapolate data from studies using an inappropriate route (oral) to a relevant route (dermal), since toxicity related to specific speciesor strains can affect interpretation of the observations. Thus, the results of the recent animal bioassayson HQ should not be extrapolateddirectly for human risk assessment purposesbecausethe tumor endpoint of concern (kidney adenomas)appears to be related to a species-specifictoxicity particular to the male F-344 rat.

(continued . . .)

Page 2

The other genotoxicity assaysconductedwith HQ should not be interpreted to indicate that HQ is likely to present a genotoxic risk to humansor be usedJo support the idea that HQ exposure results in tumorigenicity. This is becausenone of the assaysare designedto be directly extrapolated for human risk assessment purposes. These in vitro studies on HQ greatly exaggeratethe risks associatedwith HQ exposure, and HQ has produced negative results in Ames/Salmonellaand Drospophila assaysconducted by routine procedures. Chromosomal effects, particularly those relating to micronuclei, do not predict bioassay results in the target tissue (bone marrow) or the target species(mouse)and therefore can not be used to predict effects in humans or other animal species. Finally, a substantial amount of researchis now underway including additional studies designedto better understandthe mechanisticissues related to the speciesand strain specific toxicity that HQ possesses. Notwithstanding the ongoing researchprogram, a review of the entire HQ database,with appropriate weight given to mechanistic data on speciesand strain-specificity of the carcinogenic response, yields a conclusion that HQ in nonprescription skin lightening creams does not present a human carcinogenic risk when used according to label directions. The sections of this report are organized as follows: II. Index Section I. II. III. Page .

Executive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Results Following Dermal Application , , . . . . . . . . . . . . . . . . . . . . . . . . . 4 A. B.
C.

HQ PercutaneousAbsorption is Vehicle Dependent . . . . . . . . . . . . . . . Toxicity Following Dermal Application of HQ is Unremarkable . . . . . . .

4 5

HQ Has Been Reported to Have Inhibitory Activity on B[a]Pinduced Carcinogenesisand Promotes Survival of Animals With Transplanted Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

(continued . . .)

Page 3

IV.

Oral Bioassay Results: An Issue of Species-and Strain-Specificity . . . . . . . . . A. B.

7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Qualitative Assessmentof the Oral HQ Bioassay Results . . . . . . . . . . . 7

c.

HQ Exposure is Associated with Significant Renal Toxicity Only in the F-344 Rat . . . . . . . . . . . . . . . . . . . . . . ._. . . . . . . . . . . . . 9 1. Renal Tumors in F-344 Male Rats Exposed to HQ are Associated with Significant Renal Toxicity and Chronic Progressive Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . Renal Adenomas in Male Mice Are Associated With Large Oral Exposures to HQ Which Are Likely to Overwhelm Normal Metabolic Pathways Resulting in Nephrotoxicity . . . . . . . . . . . HQ Does Not Act Like a Classical Renal Carcinogen . . . . . . . .

16

2.

20 21 22 23 23 23

3.

D. E. V.

Mononuclear Cell Leukemia in Female F-344 Rats Is Not a Reproducible Effect Indicative of Carcinogenicity . . . . . . . . . . . . . . . The Incidence of Hepatocellular Adenomas in B6C3F, Mice is QuestionableEvidence of Carcinogenicity . . . . . . . . . . . . . . . . . . .

Genotoxicity, Carcinogenicity and HQ Exposure . . . . . . . . . . . . . . , . . . . . A. B. Introduction . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . The In Vitro Studies for HQ Greatly Exaggeratethe Risks Associated With HQ Exposure and Can Not Be Used For Risk AssessmentPurposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomal Effects, Particularly Micronuclei, Did Not Predict the Bioassay Results with HQ . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary....................................

34 34

C. D.

. . . . 35

(continued . . .)

Page 4

VI.

ResearchWork Underway or Planned ............... Conclusion ........................................... Reference. ............... :. ...........................

; ..........

35 42 43

VII. VIII.

III.
A.

Results FollowinP Dermal Am&cation

HQ Percutaneous Absorption is Vehicle Dependent Percutaneousabsorption of HQ in aqueoussolutions is poor. Alcoholic solutions may increase the penetration of HQ, but even under these conditions, HQ elimination from the body is rapid and, therefore, HQ body burdens remain low. Commercial OTC preparations contain only aqueousvehicles -- not alcoholic vehicles, which are known to enhancethe absorption of HQ. As such, HQ absorption from straight aqueous vehicles would be expectedto be less than that from experimental alcoholic vehicles. The percutaneouspenetration of HQ has been reported in three studies: an in vivo human assessment; evaluation using excised human skin and skin from hairless an mice in vitro; and an in vivo study in rats. Bucks et al.2 (1988) applied 14C-labeledHQ in an alcoholic vehicle’ to the forehead skin of male human volunteers and measuredHQ excretion in the urine. Using different formulations containing 2% HQ in alcoholic vehicles -- including other ingredients known to enhanceabsorption -- varying amounts of the applied dose were absorbed in 24 hours. Curiously, the skin penetrantenhancer, l2dodecylazacycloheptan2-one,did not affect the penetration of HQ, while the addition of 2-ethylhexyl ester of 4-(dimethylamino) benzoic acid significantly decreasedabsorption.

’ Note:

Commercial OTC preparationscontain only aqueousvehicles -- not alcoholic vehicles. As such, HQ absorption from OTC aqueousvehicles would be expectedto be less than that from experimental alcoholic vehicles. See also Marty et al. (1981) and English et al. (1988). (continued . . .)

Page 5

In contradistinction to the results of Bucks et al.3, Marty et al.2 (1981) studied the in vitro and in vivo penetration of HQ in an oil/water smulsion through the skin of hairless rats and in vitro through human skin. Under these conditions, HQ was poorly absorbedthrough rat skin and absorption was seven times slower through human skin than through rat skin. Similarly, English et al.2 (1988) found that 14C-labelledHQ was poorly absorbed through the skin of F-344 rats in vivo from an aqueoussolution applied to the skin for 24 hours. The foregoing indicate that the percutaneousabsorption of HQ in ordinary aqueous solutions is low, and that alcohol -- but not azone or the sunscreen2-ethylhexyl ester of 4-(dimethylamino)benzoicacid -- can increasethe penetration of HQ. But, even under these conditions, HQ elimination from the body is rapid and, therefore, HQ body burdens remain low. B. Toxicity Following Dermal Application of HQ is Unremarkable The majority of studies of HQ following dermal application are unremarkable in terms of the nature and extent of potential toxicity from HQ. Burnett et al.3 (1976) studied the percutaneoustoxicity (i.e., twice weekly dermal application to rabbits for 13 weeks) and teratogenicity (i.e., dermal application to pregnant rats on 1, 4, 7, 10, 13, 16, and 19 days of gestation) of a hair dye containing 0.2% HQ and found no evidence of toxicity. Similarly CTFA commissioned dermal teratology studies (i.e., dermal application from day 6 through day 19 of gestation in pregnant rats) of HQ and a skin lightener containing HQ which did not show evidence of developmentaltoxicity (see CTFA,E 1980). NTPE (1989) conducteda 14-day dermal toxicity study using groups of five rats or mice per sex. Rats were given up to 3.8 g/kg of HQ in 95 % ethanol and mice received up to 4.8 g/kg HQ five days per week for 12 doses over 14 days. Even though HQ was qualitatively detected in the urine of male rats exposed to HQ, no evidence of toxic effects was seen. An exception is the report of Sivchev et al.a (1966) in which guinea pigs were covered (90% of body surface) with a Vaselineointment containing up to 20% HQ daily for 50 days. The 20% HQ ointment provided approximately 500 mg of applied

(continued . . .)

Page 6

HQ per day. The application areas were not covered, and it therefore is noteworthy that the guinea pigs apparently were free to ingest the ointment. Under these conditions, histologic changeswere reported in the liver, kidneys, heart, spleen, lungs, and adrenal glands and hematologic changeswere found. In retrospect, a number of the lesions observedmay have been due to intercurrent infectious disease,as evidenced histopathologically by signs of pneumoniain three animals and catarrhal inflammation of the bronchi and bronchioles in the remaining animals.. C. HQ Has Been Reported to Have Inhibitory Activity on B&]P-induced Carcinogenesis and Promotes Survival of Animals With Transplanted Melanoma In a screening study for co-carcinogenesis tumor promoting agents in tobacco, and Van Duuren and Goldschmidt (1976; Table 1) found that HQ did not induce tumors after skin application and was a weak inhibitor of carcinogenesisinitiated by benzo[aJpyrene(B[a]P). These studies were designedwith groups of 50 female CR/HA Swiss mice given acetone, 5 mg HQ in acetone, or 5 mg HQ in acetone preceded by 5 ug B[a]P three times per week or 150 ug B[a]P once two weeks prior to HQ administration. HQ was administered three times per week for 52-58 weeks. Animals given HQ in acetonedevelopedno tumors, while those given BfaJP and HQ developed fewer neoplasms(7 of 1I mice) than mice given only B[a]P (14 of 16 mice). HQ has also shown a moderateprotectjve effect against a common skin tumor (melanoma) transp!antedinto mice. hlice with tumor transplantsgiven 80 mg/kg HQ subcutaneouslyfor nine days showed survival times which were equivalent to those mice not receiving transplants (Chavin et al.,66 1980). Table 1
HQ Dermal
Rinawny in CRlIlA
Swirc

Mice wlwn

Initiator

Result<:

No tumnn

1IQ Weak lnhihitnr 3 SQ Ceil-lIQ!fhP

IIQ

Wenklnhihttnr

In SQ cdmr

(continued , . .)

Page 7

IV. Oral Bioassay Results: An Issue of Ss A. Introduction

ecificit

There are three HQ bioassays. which have been conductedby oral administration. The protocols and results are outlined in Tables 2a and b on page 8. In the earliest of these studies (Carlson and Brewer,2 1953), HQ was incorporated into diets which were fed to Sprague-Dawleyrats seven days per week for 104 weeks. Carcinogenicity was not observed in any organ system (Table 2a and b). NTPE (1989; Tables 2a and b) conducteda chronic bioassay in F-344 rats and B6C3F mice given HQ by gavage (25 or 50 mg/kg rats and 50 or 100 mg/kg mice). In this study, renal toxicity was observedin male and female rats; renal adenomas were observed in male rats; a trend towards a higher incidence in mononuclear cell leukemia was present in female rats; and an increasein liver tumors (adenomasand carcinomas combined) was seen in female mice. The liver tumor increase in female mice was due to an increase in adenomas. In male mice, a higher incidence.of liver adenomaswas also seen, but becausethere was a decreasedincidence of malignant liver tumors, the combined benign and malignant tumor incidence was not considered significant. NTP classified these responsesas “some evidence”of carcinogenicity. Shibata et a1.z (1991; Tables 2a and b) gave F-344 rats and B6C3F mice diets containing 0.8 % HQ. This diet provided dose levels of approximately 360 mg/kg (rats) and approximately 1300 mg/kg (mice). In this study (Table 2), renal toxicity in male and female rats and kidney tumors in male rats were observed. No mononuclear cell leukemia was observed in female rats. In mice, there was an increase in benign liver tumors only in male mice. Renal toxicity in the form of renal hyperplasia was observed in male mice, and there was a statistically hsignificant increase in benign kidney tumors in male mice as well. The full range of renal pathology changeswas not observed in this study as there were no interim sacrifices to collect renal tissue for histopathology. B. Qualitative Assessment of the Oral HQ Bioassay Results Extrapolation of animal bioassay data should involve a broad ranging assessmentof are all of the data available on a chemical. Risk assessments sometimesbased on data derived from experiments conductedby irrelevant routes of exposure (e.g., use of the

(continued . . .)

r I

Page 8

Table 2a

s
shthnta e1 at

lwY19R9~
,q p&W: Nnf.SexfGrp: F M 4 R~tsl IMCW, Mice

rmr,
F.344 Ratcl
IWCW, Mice

5s
mwl~c Rats Mice 8hfKlo 7 Ratr Mice tfM 2s (c sn SO& Inn 5 103 IO.1

.M
8.8% HQ Diet - ,360 ..

Route:
Dow Levels: (mglkgldq)

- 1.3-m
7 164 96

b-m Frq.:
(day/week)

Stud! Length:
(weeks)

’ The 0.25% Group rat 162.1 rakbxlprp. Snme talk were ttrd for Interim xtttdiw. Expo-wm inrlt&d IIQ (IO rat~‘ srx/~rp); IIQ + 0.1% ritrk acid (lb23 mtsk~xkrp); and t4Q mixed in lard m d hcalcd to IWC fnr -Xl min (I623 ralckxICrp).

Table 2b

HQ Two-Year

Rionrrny

(Oral Rmtles) - 2

No No NO

Kidnrp Toxicity - M T Kidney Adrnomnq - M Mnnrmuclenr Cell Leukemia - F

Kidney Toxicity - M P Kidney Adenomas - M No

Mice Ntl Nn Kidney Hyperplssia - M Kidney Adenomns - M fh’ .S) Liver Adennmas - M NC’

Uver Adenmnm - M W I’
Liver Adenomnq - I;

’ Nn( sitnifiinf

whrn cnmhinrd with rarrinnmnr.

(continued . . .)

Page 9

oral route to assessrisk of human dermal exposure without any regard to routespecific pharmacokinetics)and using inappropriate animal model systems. Both of these errors could potentially be made in the’ assessment the HQ bioassays. of On a qualitative basis, the following statementscan be made about the HQ oral bioassay results. These points are also discussedfurther in Sections C, D, and E below.
q

Renal adenomasin male F-344 rats are associatedwith-an’ interaction between nephrotoxicity and chronic nephropathywhich may be unique to the male F344 rat. Renal adenomasin male mice are associatedwith large oral exposuresto HQ which are likely to overwhelm normal metabolic pathways resulting in nephrotoxicity. Mononuclear cell leukemia in female rats is not a reproducible effect indicative of carcinogenicity. The incidence of hepatocellularadenomasin B6C3F mice is questionable-and certainly not “some” or “definite“ -- evidence of carcinogenicity.

n

m

D

c.

HQ Exposure is Associated with Significant Renal Toxicity Only in the F-344 Rat In reviewing the I-IQ data base, there are several studies which have assessed the effects of HQ on renal structure or function. The endpoints that were used for this review included alterations in kidney weight, renal histopathology, and urinalysis. For the F-344 rat, there are four studies which assessed renal effects associatedwith subchronic or chronic HQ exposure to male or female rats (NTP,E 1989 and Shibata et al 3 1991). The protocols and results of these studies are outlined in Table 3. The yesults of these studies consistently show that HQ alters renal structure in the F344 rat and that subchronic and chronic HQ exposureaffects the male F-344 rat more severely than the female (Table 3).

(continued . . .)

Page LO

Table 3

I-IQ Renal Toxicity - F-344 Rats
NTpB1

I .,
Shihata el al. (1991) 30
0.8% HQ in diet - 360 7 104 Inc. Abs. & %BWT-M Inc. % BWT - F ND Inc. Severity M: 14130 F: 7/30 (minimal)

NoJsexlgrp.: Route: Dose Levels: (mg&Wv) Dose Freq.: (days/week) Study Length: (weeks) Kidney weight:

10 Gavagc in corn oil 25, so, loo, 200,400 5 I3 ND

NW d98_91 lVTP (1989) 10 55
Gavage in waler 25, 50 5 65 Inc. % BWT SO mg/kg M ND Inc. Severity 50 mglkg: 600 M 25 ma/kg: 95 M Gavage in water 25, so 5 103 Inc. % BWT SO mglkg M ND Inc. Severity SO Kg/kg: 2055 M

Urinalysis: Histopathology: (nephropathy)

ND 200 mglkg: 7/10 M 6110 F 100 mg/kg: l/10 F

(continued . . .)

Page 11

.

Table 4 outlines the protocols and results of four studies with HQ exposure to Sprague-Dawley (Topping et al., 541988; Carlson and Brewer,2 1953) or Carworth rats (Christian et al .,2 1976; two studies). The most significant effect on the kidney was slight increasesin relative kidney weight (%BWT). In none of these studies was there evidence of structural damageto the kidney. Tables 5 and 6 outline the protocols and results used to study HQ effects in dogs and humans (Carlson and Brewer,.j 1953). While both of these studies involved relatively small number of subjects, the dose levels, exposureperiods, and detection methods should be consideredadequateto detect nephrotoxicity. None was detected. The 300 mg/day dose level given to humans equatesto approximately 4 mg/kg/day on a body weight basis and is equivalent to a rodent dose of approximately 25 mg/kg/day on a body surface area basis. Based on a comparison of the results of these studies, it is clear that F-344 rats develop nephrotoxicity following HQ exposure and Sprague-Dawleyand Carworth rats do not (Tables 3 and 4). The results of the dog studies, in which no toxicity was found, support the conclusion that the F-344 rat has a unique susceptibility to HQ. For humans, it is clear that significant subchronic exposure does not result in nephrotoxicity (Carlson and Brewer,,z 1953). Carlson and Brewer! (1953) concluded: “Man. Analyses of the urine and blood of 19 human subjects that ingested 300 to 500 mg of hydroquinone daily for 3 to 5 months revealed no abnormal findinaq.” (Carlson and Brewer,? 1953 at page 686; emphasis supplied) This conclusion is supportedby an epidemiology study conductedby Friedlander et al.‘ (1982) on employeesof a photographic processor where HQ exposureswere low 6 (< 0.01 mg per cubic meter) and “no significant excess mortality, sickness absence or cancer incidence” were noted. Additional epidemiologic support is provided by a case report study of ocular toxicity among employeesof a hydroquinone manufacturing plant (Sterner et al. ,E 1947 and Anderson,68 1947) whereby no systemic toxicity was observed under conditions which led to comeal pigmentation. Sterner et al.53 observed: “Repeatedphysical examinationsand laboratorv studies did not reveal anv evidence of svstemic iniury and the degree of dis-

(continued . . .)

- -

Page 12

Table 4
..

1

HQ Renal Toxicity - S-D and Carworth Rats
Topping ei al. A.!WtLCarlson and Ih2!Ydj.(135~~
S-D 46-53’ 0.1, 0.25, 0.5,
1.0% IIQ Diets - 80-800 7 104 ND

I
Christian gt d. (1976.)
Carwort h 20 1, 2, 4 gfL in water 110- 430 7 15 Inc. Rel. W t. 1, 2, 4 g/L (1 g/L F NS) ND No effect

Christian f$ al. -.(I 976)
Carwort h 6 2.5, 5, IO g/L in water 230 - 810 7 8 Inc. @ ,Sand 10 g/L ND No effect

Strain: Na./sex/grp.: Route: Dose Levels: (mglkglday) Dose Freq. : (days/week) Study Length: (weeks) Kidney weight:

S-D 10
Gnvagc in water

20, 64, 200

s
13
No effect

Urinalysis: Histopathology:

ND

No elect

hT) No effect

’ The 0.25% Group was 16-23 rats/sex/grp. Some rats were used for interim studies. Exposures included HQ (10 ratslsexfgrp); IIQ + 0.1% citric acid (16-23 rats/sex/grp); and IrQ mixed in lard and heated to 190°C for 30 min (16-23 rats/sex/grp).

(continued . . .)

Page 13

Table 5
HQ Rcnnl T&city
rWDogF/C.rp.: 1

- Miucd-Reed Dogs
2

I
5 mntcv

Dnqe LQveIs: (mglkgld:ly) Dope Freq.: (days/week) Study Length: (weeks) Kidney Weight: Urinalysis: Histopathology:

I6 7 RO ND No cffw-t No effect

lAand40 7 1.6 mg!kg for 31 wks +40 mg/kg for 49 wks

1fM-l 7 26 Nn No effecf

No effect

No eNect

Dacn from Carbon and f!rc*uc-r, 19.23

Table 6

_.____ ----.-_

Dote Levels: (mgld:lr) Dme Freq.: (days/week) Siud! Length: (weeks) Urinalysis;: 7 20 No effttrt 7 12-20 No eflect

Data from Carlson and lhrucv, I953

(continued . . .)

Page 14

comfort sustainedwas almost entirely the limiting factor in controlling the quinone vapors until recogniticlil of the above local eye injury.” . “Following the identification of the eye effects with the quinonehydroquinone exposures,examination of the exposedworkers for evidence of systemic absorption and intoxication was intensified, but no iniurv other than the 1ocaIeve effects has been found. Comnlete physical examinationsand comprehensive laboratorv studies. including urine inoreanic-total sulfate ratios have been made seriallv. and have been uniformlv nepative.” (Sterner et al., 5f 1947 at page 67; emphasissupplied) “The results of all of these characteristics studied [i.e., values for hemoglobin, leukocyte count, erythrocyte count, cell volume, sedimentationrate, polymorphonuclearcount, icterus index, ‘ stab’cell count, lymphocyte count, eosinophile count, monocyte count, basophile count] show no significant deviations from those of the control groun.” (see Tables 1-12 of Sterner et al ‘ 531947 at page 67-70; emphasissupplied) , Anderson g (1947) described the medical condition of this population as well: “Two of our patients (cases8 and 41) have been hospitalized and subjectedto exhaustive tests. Urinalysis and studies of the blood have been performed repeatedly on all personsemployed in the plant. No sueeestionof svstemic intoxication with the &g, which after all is relatively harmless, has been observed.” (Anderson g, 1947 at page 11; emphasissupplied) In summary, the studies by NTP, Shibata et al., Topping et al., Carlson and Brewer, Christian et al., Sterner et al., and Anderson demonstratedefferencesin sex, strain, and species specificity. The mechanistic reason(s)for the differences in sex, strain, and species susceptibility are not completely known, but are being vigorously investigated. Boatman et al.@ (1992) have developedan acute model for HQ toxicity that should allow for mechanistic studies on the role of various enzyme systems in the production of HQ toxicity. Boatman et al.2 (1992) found that administration of 400 mg/kg HQ to either male or female F344 rats resulted in significant increased excretion of alanine aminopeptidase,n-acetyl glucosaminidase,alkaline phosphatase,

(continued . . .)

Page 15 . gamma-glutamyl transpeptidase,and glucose which are markers of proximd renal tubular damage. Also significantly, Sprague-Dawleyrats given the same dose levels of HQ did not increasetheir excretion of these marker substances. However, Boatman et al.69(1992) were able to induce tiephroto&city in both F344 and SpragueDawley rats by intravenous inj,ection,of a hydroquinone conjugate. Hill et al.70(1992) and Kleiner et al.2 (1992) recently reported on the in vivo and in vitro formation of several conjugatesof HQ in the Sprague-Dawleyrat. They were also able to induce nephrotoxicity in Sprague-Dawleyrats by intraperitoneial or intraarterial (renal artery) injection of HQ conjugates. These data suggestthat the strain difference in susceptibility to HQ may not reside in the kidney since both F343 and Sprague-Dawleymale rat kidneys respond to the HQ conjugate (Table 7). The most likely explanation for the difference in strain responsesis that there is a significant difference in hepatic P450 metabolism that results in differences in the amount of glutathione conjugate formed, in the rate of glutathione conjugate formed, or the type of glutathione conjugate formed. Table 7

F-44 Rats

Mates Females

++ +

ND ND

S-D Rats Car-worth Rats Mixed-Breed Dogs
Human

N-D

w
ND

ND
No Effect

(continued . . .)

Page 16

Similarly, the sex differencein HQ nephrotoxicity be dependent the differmay on encesin hepaticmetabolism HQ or the rate of deliveryof HQ’ of metabolites the to kidneys.
1. Renal Tumors F-344 Male Rats Exposed to HQ are Associated with Significant Renal Toxicity atid Chronic Progressive Nephropathy

In both the studyby Shibata al.2 (1991)and that by NTE@(1989))chronic et HQ exposure associated an increased was with severityof chronicnephropathy is in maleF-344rats. This relationship shownin Table 8 which is modified from a tablein the NTP report to also showthe strongcorrelationbetween the Nine of the twelve occurrence adenomas the severityof nephropathy. of and tumorswere observed rats with end stagerenaIdisease.This strong in association chronicnephropathy, with which is recognized a life limiting as disease malerats, may explainwhy femaleF-344rats did not showan in * increased incidence renal tumor, eventhroughthey sharea susceptibilityto of nephrotoxicity with the malerats.
Table 8 Number of Male Rats with Indicated Severity of Nephropathy (Adenomas) in the NTP Two-Year Gavage Study of Hydroquinone

Severity -_-._. _
No. of rats examined 5s ss 55

No nephropathy Minimal

2

3

0

3
12 26 12 (0)

I
12 (1) 31 (1) 8 (2) (4)

3
5 15 (1) 32 (7) @I

Mild
Moderate ’Marked Total No. Adenomas

HistoricalControt Incidence Range O-6%

(continued . .) .

Page 17

Recently collected data on repetitive dosing of male and female F344 rats provides insight into this strain difference (English, unpublished data). English gave male and female F344 rats 25 or 50. mg/kg/day (5 d/wk) of HQ by gavage for 1, 3, or 6 weeks and found that at six weeks there was an .. increasein the excretion of urinary enzymes and glucose only in male rats (50 mg/kg) and that the increasedexcretion of these materials was associatedwith an increasein the rate of proximal renal tubular cell proliferation. During the NTP HQ bioassay, there was a concern that sex difference in the kidney adenomaresponsemay have been due to a mechanisminvolving the renal accumulation of a-2u-globulin in the male rats. Therefore, the report specifically made mention that hyalin droplets were not observed in male rats during the HQ bioassay. Yet in many ways the pathogenesisof the tumors following HQ administration and in the case of a-2u-globulin may be very similar in that both casesthe test materials induce cell toxicity and cell proliferation very early (between3-6 weeks with HQ) after the initiation of dosing and in both casesthe cell proliferation associatedwith chronic progressive nephropathymay contribute to the latter appearanceof cell hyperplasia and benign tumors (Short and Swenberg,E 1991). Figures 1A and 1B outline the potential interaction between these simultaneousstimulators of cell proliferation.

(Figures 1A and 1B follow on pages 18 and 19; Text continues on page 20.)

(continued . . .)

Page18

Figure IA ..

Proposed Interaction Between’ CPN-induced and HQ-induced . Cell Hyperplasia in the Male F-344 Rat

1Male Rat F-344 1 / Kidney Cell 1
h’ o / / ’
\

; HQ Metabolite

H Q Exposure

“Normal” Aging I Chronic Progressive h’ ephropathy I Regeneration I Cell Proliferation Repair / Hyperplasia I j .

Chronic Toxic Effects I Cell Toxicity and 1 Degeneration I . ’ i

Similar interactions are not expected to occur in female F-344 rats, SD. rats, or other species.

(continued . . .)

Page 19

Figure IB

Hydroquinone Exposure Does Not Result In Kidney Toxicity or Adenomas In Male Sprague-Dawley Rats

+I HQ Metabolite Exposure I No Toxic Effects I No Cell Degeneration Regeneration Cell Proliferation Repair Hyperplasia
1

-

1

1

I No Cell Proliferation

(continued . . .)

Page 20

2.

Renal Adenomas in Male Mice Are Associated With Large Oral Exposures to HQ Which Are Likely to OverwheLm Normal MetaboIic Pathways 1, Resulting in Nephrotoxicity ‘ . Table 9 outlines the protocols and the results of the NT@ (1989) and the Shibata et al.s (1991) studies using B6C3F mice. In-the NTP study, a transitory increasein relative kidney weight (% BWT) was observed only in female mice at 65 weeks of exposure. In contrast, Shibata et al.2 (1991) reported finding renal tubular hyperplasia in 9130 mice and adenomasin 3/30 mice. The latter finding was not considered statistically signjfkwt. Table 9

HQ Renal Toxicity - BK3F,
Pll’ (1989) Tl’ Ndsexlgrp.: Route: 10 Gavage in corn oil 25 so, IO, 20, 400 s 13 ND
Inc. so, 100

Mice

-..
Shihata et al. (1991) 30 0.8% HQ in diet - 1300 .7
I

Dose Levels: (mg:/kg/day) Dose Freq . : (days/week) Study Length: (weeks) Kidney weight:

s

(is

103
Rwr

96 Inc. % BWT Females only ND Tubular Hyperplasia in 9/30 males

%

No efT’ ect

Urinalysis: Histopathology:

ND No effect

SO and 100 mg/kg F ND No ened

ND No effect

(continued . . .)

=

Page 21

The likely explanation for the difference in results between the two studies is that the dose level in the Shibata et al.2 (1991) study (approximately 1300 mg/kg/day) overwhelmed the normal .detoxification process in the liver resulting in non-linearity in HQ pharmacokineticsand nephrotoxicity. This likeli. hood seemshighly probable considering size of the daily dose. Although Shibata et al.% stated that no nephrotoxicity was observed in mice, their study did not include interim sacrifices of animals to determine if nephrotoxicity had = occurred. 3. HQ Does Not Act Like a Classical Renal Carcinogen The USEPAS (1991) has recently profiled the biological characteristics of classical renal carcinogens. In Table 10, the biological profile for HQ is matched against these characteristics. The only match between the two profiles is that HQ is positive in non-bacterial in vitro mutagenicity assays. _ Classical renal carcinogensare typically also positive in bacterial systems where HQ is not active. Thus there is very little basis for considering that HQ is acting biologically like recognized renal carcinogens. Table 10 HQ Dots Not Act Like A Classical 1 Renal Cnrcinycn

I
systems

l l l l l l

Positive in short-term Induces malignant

mutagenicity

assays

l l l l

Negative in hacteriat
No No No

tumors

Induces high incidence of tumors Requires minimal exposure durations

Assnciated with a decreased latency period Little sex difference in susceptibility

* Nrl 0 No

I

USEPA Risk Assessment Forum Report, Alpha-2p-Glnhelin: Association with Chemically-induced Renal Toxicity and Neopl:6~ in the Male Rat, EPA/625/3-91/0t9F, September 1991, Washington, DC.

(continued . . .)

Page22

D.

Mononuclear Cell Leukemia in Female F-344 Rats Is Not a Reproducible Effect Indicative of Carcinogenicity Table 11 includes the mononuclearcell leukemia incidence data available on HQ for female F-344 rats. The only. statistically significant effect was noted at the high dose level in the NTP study. Even at the high dose level, the incidence of mononuclear cell leukemia was within the historical control limits. The incidence data for mononuclear cell leukemia in male F-344 rats and for leukemia in male and female mice in the NTP study did not differ from the respective control groups and thus do not support the premise that HQ is a leukemogenin female F-344 rats. In any case, of particular recent concern is the increasing incidence of mononuclearcell leukemia in F-344 control rats used in the NTP studies. Table 11

Monoryclear Cell Leukemia in Female F344 Rats - 1

hTP Historical Control’ So. Tumors 75/299’

NTP Bioassay (1989) 0 mglkg 9/5s 25 mglkg 15/s* 50 mg/kg 22/55*

Shibata el al. (1991) 0 mglkg 6130 360 mgW 8130

/ 25 f 15’ /

16

/

27

40

/

20

/

26

’ 2 3 ’

hTp Water Gavaged Female F344 Rats No. Tumors/No. Animals Mean f SD Statistically Significant Trend

(continued . . .)

Page 23

..

The instability in the incidence of leukemia can lead to serious interpretation problems. As an example, in a recent NTPg (1991) bioassayon tris (2-chloroethyl) phosphate,the incidence of mononuclearcell leukemia i? female F-344 rats was 1400 or 28% (control), 1660 or 32% (low dose), and 20150or 40% (high dose), results which are very similar to those seen in the NTP HQ bjoassay. Yet the NTP concluded that the increaseswere marginal, within the historical control range, and not clearly related to administration of the test chemical. Considering the problem that unstableincidences of mononuclearcell leukemia can have in interpretation of NTP studies, it may be significant that, in Japan where the incidence rate for thus tumor is stable, no effect on leukemia incidence was observed following HQ exposure (Shibata et al.,50 1991). E. Tbe Incidence of Hepatocellular Adenomas in BK3F, Mice is Questionable Evidence of Carcinogenicit

--Table 12 includes the incidence data for liver tumors in B56C3F mice. These data should rightfully be questionedbecausethe incidence of adenomasin female mice: (a) did not follow a clear dose response;and (b) was close to the upper historical . control range for untreated female mice. Similarly, the data for the male mice, while consistentbetween the NTPS (1989) and the Shibata et a1.s (1991) studies, are close to the historical incidence for these tumor types and in the NTP study actually show a reduction in malignancies. The NTPE (1989) interpreted the male mouse data as no evidence of carcinogenicity, while Shibata et a1.s (1991) interpreted similar data in the opposite way. V. Genotoxicitv. Carcinogenicitv and HO Exposure

A.

Int reduction HQ has been tested in a large number of assaysfor genotoxic effects. Many of these assayswere tabulated in the NTPZ (1989) bioassay report. Tables 13 (a-h) include data from the NTP report and additional studies which have been reported and reviewed since the publication of the NTP report. In general these reports fall into a number of categories including bacterial mutagenesisassays, Drosophila sex-linked

(continued . . .)

Page 24

Table 12
... -_

1

Hepatocehlar
NTP Historical Control’

Adenomas in BW3F, kIic6

Females
No. Tumors Incidence (a) No. Tumors Incidence ’ 2 ’ ‘ ’ 22134Y 6 * s

w347 16 f 4

hTp Water GavagedB6C3F, Mice No. Tumors/No. Animals Mean f SD Statisticalty Significant for Adenomas Statistically Significant Negative Trend for Carcinomas

(continued . . .)

Page 25

recessive mutation assays;in vitro chromosomal effects assaysin higher plants, fungi, and mammalian cells; in vivo murine chromosomaleffects assaysby parenteral or oral routes of exposure, and assaysfor DNA adducts in vivo. and in vitro, Interpretation of these assaysis not straight forward and none of the assaysare designed to be directly extrapolated‘ risk assessment for purposes, For most of these assays, correlations between the assay results may be more important than the result of any one test. For example, Ishidate et al.2 (1981) have shown that chemicals which are positive in both the Ames/Salmonellatest and in vitro CHO cell chromosomal aberration test are likely to be associatedwith carcinogenic activity and, conversely, chemicals which are Ames/Salmonellatest negative but positive.for chromosomal aberrations, are unlikely to be carcinogens. The pattern of effects associatedwith HQ exposure in these studies is fairly well recognized. HQ has generally produced negative results in Ames/Salmonella and Drosophila assays, and generally positive results in chromosomal assaysin vitro and in vivo when given by parenteral routes of exposure. DNA adduct formation has been positive in vitro but negative in vivo. When given orally, HQ has produced micronuclei at high dose levels but has been negative for dominant lethal effects. Thus the primary concern raised by genotoxicity screening tests have been for chromosomal damageparticularly aneuploidy and related effects. If the results of the screening assayslisted in Tables 13 (a-h) were all that were known about HQ, they might raise serious concerns. Fortunately, there is much more information avfailablewhich needsto be consideredand which should also reduce the level of concern. This information (describedbelow) leads to two conclusions:
B

The in vitro studies for HQ greatly exaggeratethe risks associatedwith HQ exposure and should not be used as a direct assessment human risk. (See of Section B below.) Chromosomal effects, particularly micronuclei, from HQ at 80 mg/kg (Ciranni et al.,? 1988) and 200 mg/kg (Gad-El Karim et al.,2 1986) do not predict the NTP bioassayresults in the target tissue (i.e., bone morrow) or the target species (i.e., mouse) and therefore can not be used to predict carcinogenic effects in humans or, for that matter, other species. (See Section C below.)

l

(continued . . .)

Page 26

_.

Table 13 a

.

Summary

of Results h

Genetic Toxicohgy Studies of Hydr’ ciqninone - 1

Bacteria
Salmonetkr typhimun’ um Epler et al., 1978

Gcnc mutation

Florin ef al., 1980 Rapson et al., 1980 G(Kke et al., 1981
Haworth et al., 1983 (NTP)
I.

Negative Negative Negative
Pokive (a)

Sakai et al., 1985

Negative Negative

Insects
Drosophila melnnognster Gscke ef al., 1981 NW, (a) 1989

.Sex-linked recessive lethal mutat ions Sex-linked recessive
lethal mutations

Negnt ive Negative

Positive result was obtained with genetically uncharacterized strain in nonstandard medium.

(continued . . .)

Page 27

Table 13 b

‘ ,

of Resrrlis of Genetic Toxicology Studies of Hydroqainone - 2
Summary

Test Sytem/Reference %----_-----

Higher Plants .

.

AIlium cepa Krogulevich and Stom, 1969 Chara zeylanica Chatterjee and Sharma, 1972 Callisia fragrans Roy, 1973

Chromosomal ahcrrat ions Chromosomal thickening

Negative Positive

Chromosomnl hreaks Polyploidy

Positive Negative

Fimgi
Aspergillus nidulans Crehelli et al., 1987 and 1991 Kappas, 1990 Strain 06 Parry et al., 1990 Chromosomal aherrntions Mitotic cross-ovcrs lndwed chromosome loss Positive Positive Positive

(continued . . .)

Page 28

Table 13 c

‘ .

Strmmary of Results of Genetic Toxicology Studies of Hy,droqtlinone - 3 ..

Mammalian

celts (in vitro)
DNA strand breaks Inhibition of DNA synthesis Trifhlorothymidine resistance Negative Positive Positive

Mouse lymphoma cells Pellack-Walker and Blumer, 1986 Pellack-Walker et al., 1985 McGregor et al. 1988 (N-P) Mouse bone marrow cells Lee et al., 1989 Cbinese hamster ovary celis Galloway et al., 1987 (NTP) Chinese hamster Don cells Shimada et al., 1988

DNA synthetic activity Sister chromat id exchanges Chromosomal aberrations Sister chromatid exchanges

Positive Positive Positive Positive

(continued . . .)

Table 13 d

.‘ _

Srlmmary of Results of Genetic Toxicology Studies of Ilydhquinone

-4

Mammalian cells (in vitro) cont.
Human HeLa cells Painter and Howard, 1982 Human lymphocytes Morimoto et al., 1983 Knadle, 1985 l&t Zymbal Gland Cells Reddy ef al., 1990 Human Promyelocyte Leukemia (HL-60) Cells Levay et at., 1991 CHO cells Parry ef at., 1990 Inhihif ion of DNA syntfwsis Sister chromat id exchanges Positive rosit ive Po& ive Positive

DNA adducts

DNA adducts (11) Micronuclei Mit ot ic division nberrat ions

Positive Negative Positive

(II)

HL-60 adducts and calf thymus DNA adducts differ.

(continued . . .)

Page 30

Table 13 e

Summary of Results of Genetic Toxicology Stwk of Hydroqhinone - 5
Exposure Rout-e-

Endpoint

Results

ian ceIIs (in vivo)
Mice (DDY) Shimada et

al., 1988

hlicrontfclci DNA strand Micronttclci Micrwttrclci Chromosomztl Micronuclei

hrcnks

Positive Positive

NS NS SC NS NS IP IP TP

Mice (NMRI) Tunek, 1982 Mice (1021El x C3H/El)F’ Xu and Adler, 1990 Mice (NMRI) Cocke et al., 1981 Mice (CUBL) Gocke et al., 1983
Mice (fOI/El x C3H/EI)I;,

Positive PositivC Positive Positive Negative Positive

ohcrrafions

Gene mttfatiort Micronttclei

Adler and Kliesch, 1990 NS - Route not specified.

(continued . . .)

Page 31
-_

Table 13 f

Summary

of Results of

Genetic Toxicology

Studies of Ilydroqninone

- 6

. Exposure ._ -Route

Test System/Reference Mammalian
Mice (Swiss CD-l)
Barale ef nl., 1990

I$rdpair?t.

Retits

ce!Is (h vhw) co,nt.
Positive
Micronwlei TP

Mice (Swiss CD-l) Ciranni et al., 1988 Mice (CBA) Jenssen and Ramel, 1980 Mice (102/El x C3HIEl)F, Ciranni and Adler, 1991 Mice (CS’ 7BlICne x C3H/Ck)F, Pacchierotti et al., 1991

Positive

ir
JP
IP

Micronuclei Chromosnmal slwrrations (male germ cells) JMicronoclci Cell-cyle lengf hcning

Positive Positive

Positive Positive
Positive Negative

Hyperpioidy Sister chromatid exchange

3P IP ‘ TP IP

(continued . . .)

P a g e3 2

-.

Table 1 3 g

S lrm m n r y o f R e st& s o f

G e n e tic Toxicology S tu d ies o f H y d r o q u in o n k - 7

T a t Syste m lRpfe r e n c e M a m m a lia n cells (in viva) c o n t.
M ice ( 1 0 2 /E l x C 3 H /E l)F, X u a n d A d ler, 1 9 9 0 M ice (lO lIE x C 3 H /E l)F, M iller a n d A d ler, 1 9 8 9 M ice Marrazzini e f al., 1 9 9 1

l?ndpoint

Resuits

Cltramowmnl c-Mitotic

n fwrrnf ions

P o s i tive P o s i tive P o s i tive, P o s i tive P o s i tive P o s i tive

IIP IP IP ? IP ? IP ? IP ?

e ffects

M icronuclei C h r o m o s o m e !tihcrrations Aneuploidy P o lyplaidy

M ice (CD-l) Cad-El-Karim e t al., 1 9 8 6 M ice (Swiss CD-l) Ciranni e f al., 1 9 8 8

M icronrlrlci M icronuclei ’

P o s i tive P o s i tive

PO PO

(contin u e d. . .)

=

Page 33

Table 13 h

Summary of Restitfs of Genetic Toxicology Studies of Hydroqttinone - 8
..

Test System/Reference Matimxhn cells (in vivn) cont.

Fjndpoint

-- _ Res$ts

Exposnre Route -_--_ .e

Rat (S-D) Ktssavage, 1984 Rat (S-D) Reddy, 1990

Dominant

Lethal

Negative Negative

PO PO

DNA Adducts (c)

(c)

Bone marrow, Zymhal gland, liver and spleen were studied using PI-enhanced

32Ppostlabeling.’

(continued . . .)

Page 34

B.

The In Vitro Studies for HQ Greatly Exaggerate the Risks &ociated Exposure and Can Not Be Used For Risk Assessment Purposes

With HQ

One major problem with in vitro assaysof HQ is that the relative water solubility of HQ allows exposure of target tissues at much higher concentrationsin vitro than can be achieved during in vivo testing. This is a problem associatedwith in vitro assays of many chemicals and is not unique to HQ studies. A larger problem is that the in vitro studies of HQ are essentially irrelevant for risk assessment purposesbecausethe reduction oxidation potentials in the culture systems were uncontrolled. Greenleeet al.z (1981) have shown that HQ in a phosphatebuffer @H 7.4) exposedto air autoxidizes to p-benzoquinone. At a concentration of 0.4mM, HQ autoxidizes to P-benzoquinone a rate of 4.8 + 0.3 nmol/min. In a at similar fashion, Irons and Neptun? (1980) showed that hydroquinone inhibition of microtubule polymerization, and accelerationof the decay of tubulin-colchicine binding activity did not occur if HQ autoxidation was prevented. Becausecell culture systems are artificial systems lacking the protective mechanisms present in the whole organism, it is not possible to make a direct extrapolation between the cell culture conditions and the in vivo situation. The importance of cellular and whole organism homeostaticmechanismsis most clearly demonstratedby contrasting the DNA adducts measuredby 32P-postlabeling using isolated calf thymus DNA, HL-60 cells and intact rats. In isolated DNA and HL-60 cultures, Levay et al.32(1991) were able to detect HQ-DNA adducts but the type of adduct differed in the two systems suggestingthe cellular mechanisms modified the HQ-DNA interaction. In contrast to these results, Reddy et al.47(1990) found no HQ-DNA adducts in the liver, spleen, bone morrow, and Zymbal glands of intact rats. C. Chromosomal Effects, Particularly Micronuclei, Did Not Predict the Bioassay Results with HQ The largest number of in vitro and in vivo assayswith HQ have studied chromosomal damage, particularly using red blood cell micronuclei as a significant endpoint. For 9 example, doses of 80 mg/kg (Ciranni et al.,- 1988) and 200 mg/kg (Gad-El Karim et al.,c 1986), HQ produced micronuclei in CD-l mice. Based on these findings, it is reasonableto expect that chronic oral HQ exposure should induce hematopoietic tumors in mice. Yet the male and female mice in the study by Shibata et a.l.s (1991)

(continued . . .)

Page 35

and the male mice in the NTP study showed no increasein hematopoietic tumors at approximately 1300 mg/kg/day and 50 and 100 mg/Jg/day, respectively. The,female mice in the NTP study actually showed a statistically significant trend towards a reduction in hematopoietictumors. As a result, in the target species (mouse) and tissue (bone marrow), clastogenicity did not correlate with tufnorigenicity. D. Summarj7 In summary, then, the following points can be made in relationship to a qualitative assessment the in vitro data on HQ: of HQ is not genotoxic in bacterial assays. In vitro cell assay results are not relevant for predicting genotoxicity for HQ. In viva assaysfor genotoxicity are negative except at high dose levels of HQ when exposure is by a relevant route. In viva clastogenicity following HQ exposure is not relevant to predicting animal or human tumorigenicity. . \‘ I. Research \I’ ork Underwav or Planned

An in-depth ResearchProgram to further the understandingof HQ toxicity is underway or planned. The Researchprogram is outlined in Figure 2. These studies are directed totyard developing a nephrotoxicity model system in rats to better understandingof speciesdifferences in HQ metabolism. This work has demonstratedthat nephrotoxicity can be produced in F-344 rats with a single oral dose of HQ and that SpragueDawley rats do not develop nephrotoxicity under similar conditions (i.e., oral dosing). Development of this system has already allowed identification of a nephrotoxic HQ metabolite that can induce nephrotoxicity in F-344 and Sprague-Dawleyrats. Preliminary results of this work were presentedat the 1992 Society of Toxicology meeting. Studies to measurecell proliferation in the kidneys of F-344 rats given HQ are also underway but final results are not available at this time. The aim of this program is to develop an integrated model for HQ toxicity which includes a biological model component and a physiologicallybased pharmacokinetics model component. (Figure 2 and Tables 14 and 15 follow.)

(continued . . .)

Page 36

Figure 2: Timeline for Research Activities on Hydroquinone Jan. 1992 Feb. 1992 Pharmacokineticstudy completed. Kidney cell proliferation study (oral dosing) underway. Presentationto Society of Toxicology on acute-dosemodel system for study of HQ nephrotoxicity.

study in rats (oral dose) underway. March 1992 32P-postlabeling May 1992 Oral-dosecell proliferation study in one-year-old rats started. Meet with FDA to discuss researchplans. Decision on specific vehicle for dermal studies. Initiation of 14-day dose-rangingstudy for 13-week dermal study in rats. Initiation of Dermal Absorption/Distribution/Metabolism/Excretion Study. Oral-dose cell proliferation study in young rats completed. 32P-postlabeling study completed. 14-day dermat study in rats completed. 13-week dermal study, including measurement kidney cell proliferation, of underway. In-life phaseof 13-week study in rats completed. Cell proliferation study in one-year-oldrats completed. Full Toxicity Review Two-year dermal bioassay, pending Toxicity Review

June 1992 June 1992 July 1992 Nov. 1992

Jan. 1993 Feb. 1993 April 1993 June 1993

Page 37 -_ Table 14

Summary of HQ Metabolism in a Human Volunteer,

F344 Rats, SD Rats and Mice*

MEI-AROLITE H+Glucuronatc HQSulfate HQ-Mercap 54.8 38.0 7.1 61.913.7 35.8&2.7 < lq90 1.6iO.8 62.Oi6.2 29.9k2.8 3.6i2.8 3.8i3.6

T P ?RCl:l’ 59.7il.4 3%6&0.5 2.lfO.9

Of TOTAL

RECOVEREl-b 85.6f0.4

IN URINE 75.3kO.6 24.7iO.6 S6.2i4.8 41.3i2.2 0.2fO.l < Inq 2.Okl.3 63.7f9.5 26.9f3.8 0.9io.9

75.0&2.2 2o.oi2.2 S.O&:o.O

14.4kO.4

HQ
*

0.01

< Ioq

I--- <Inq I <ioq I
llese

1

8.5f8.3

The human dose was 275 mg or 4 mglkp and the rodent dmex were 25 mglfkg. surfau Irea.

dmcs are mughly quivrlenf

with re~perl lo kd)

0

<lq

= mean vrlue~ were less than the lower limit al quanti!ation.

(continued . . .)

Page 38

Table 15

: Kinetics of Oral Hydroquinone’ (275 mg) in a Male VoIunteer
HQ Concentration 100.0 -

In t)Iood (rig/ml)

Half life = 16.6 mlnules 1o.cI

1.a

2.0

4.0

6.0

6

8.0

10.0

Time Nler Ingesflon (Hours)

(continued . . .)

’ b

,

x

Page 39

Pharmacokinetics studies with HQ were completed as part of the USEPA TSCA Section 4 _ test rule. Additional data on the comparative metabolism HQ in different species of is presented Table 14 and the blood kinetics of HQ in a human volunteeris presented in in Table 15. A proposed metabolic pathways for HQ .biotransformation in F-344 rats is

presented Figure 3. in
Figure 3 shows that significant conjugation of hydroquinone to its glucuronide and sulfate metabolites occurs in the intestinal tract where large amounts of high capacity conjugation enzymes are located (Cassidy and Houston,22. 1984). Hydroquinone which escapes conjugato tion in the intestinal tract is available for absorption and can be transported the liver where further sulfate, glucuronide, or glutathione conjugation can occur. The sulfate and glucuroni@ metabolites are considered detoxification products and can be readily excreted in the urine. Hydroquinone can also potentially be metabolized through a series of reactions to mono-, and di-, or tri-glutathione conjugates which are excreted into the bile and reabsorbed from the intestine as cysteine conjugates. The cysteine conjugates can be metabolized the in liver or more probably in the proximal renal tubular epithelium to a N-acetyl cysteine conjugate of hydroquinone. The, cysteine and N-acetyl cysteine conjugates may accumulate in the renal epithelium becausethey move into the cells by transport systems, while they leave the epithelium only by diffusion. The metabolic cycling between these two materials may lead to oxidative stress in the renal epithelial cells and tdxicity in the F-344 rat. All of this work to date has been conducted using oral HQ exposures. With the low degree of penetration seen after HQ application to the skin and becauseclearance mechanisms rapidly remove HQ from the blood, the risks associatedwith dermal exposure to HQ should be extremely small even for the F-344 rat. To further characterize this issue, NDMA’ s Hydroquinone Task Group plans to implement a long term dermal bioassay according to the draft protocol provided in the references (see NDMA,g*c 1991). The Task Group is in the end stages of completing this protocol in order to initiate the studies in the very near future (seeFigure 2). NDr\lA would estimate at this time that the completion of the dermal bioassay would take approximately 2.5 to 3 years. In summary, based on the data available, we have generated a hypothesis about how and why HQ exposure results in renal tumors in male F-344 rats. When HQ is given orally to the F344 rat, it is rapidly absorbed and rapidly metabolized in the liver to sulfate, glucuronide, and glutathione conjugates. The glutathione conjugate and/or its metabolites are mildly toxic to the renal tubular epithelium which undergoes regenerative hyperplasia or cell proliferation. The susceptibility to nephrotoxicity can be transferred to Sprague-Dawley rats by intravenous injection of the HQ-glutathione conjugate. Both male and female F-344 rats are susceptible

(continued . . .)

Page 40

Figure 3

..

Proposed Site-Specific Metabolism For Hydroquinone
Per OS

..

oj

Rena1 Epitheiial Cell /, I \\

\1

.

I v

:Lumen JMucosai

‘ Blood

Urine

Intestine

Liver

Kidney

Page 41

Legend for Figure 3

a

--_

I 2 3 4 5 6 7 g 9 i-i B G &
BD

B-GS R-2(GS) H-Glut H-GS
H-2&S)

H-3( GS) H-CyS-NAC
H-C@ H-S O#

Per OS IP

Sulfuryl Transferase Catalyzed .’ Glucuronide Transferase Catalyzed P-450 Catalyzed . GST Catalyzed ‘ -GT Catalyzed y PeptidaseCatalyzed N-acelyltransferase Catalyzed Quinone ReductaseCatalyzed DeacetylaseCatalyzed Hydroquinone Benzoquinone Renal Glomerulus ’ Transport System Bile Duct Benzoquinone Glutathione Conjugate BenzoquinoneDiglutathione Conjugate Hydroquinone Glucuronide Hydroquinone Gtutathione Conjugate Hydroquinone Diglutathione Conjugate Hydroquinone Triglutathione Conjugate Hydroquinone N-Acetyl Cysteine Conjugate Hydroquinone Cysteine Conjugate Hydroquinone Sulfate Conjugate Oral Administration of Hydroquinone Intraperitoneai Administration of Hydroquinone

Page 42

to HQ renal toxicity, but the malesdiffer from the femalesin two ways. The fast is that the response the femalesis less severe of thanthat of the maleswhenthe animalsare exposed to HQ chronicallyor subchronically.The second th;lt, in the.males,spontaneous is chronic are progressive nephropathy much more seriousthanin females. Thesetwo phenomena is likely to lead to higherlevels of chroniccell proliferationin malesthanin females. The consequence this combined of stimulusto cell proliferationwould be expected result in an to increased of tumorigenicityfor maleF-344rats that would not be shared femaleFrisk by 344 rats or other strainsof rats and species animalswhich do not developnephrotoxicity of following HQ exposure.
VII. Conclusion

Therefore,given that foregoingcommentary aboutthe availabledata, NDMA’ s Hydroquinone Task Groupconcludes hydrquinone in OTC skin lighteningpreparations that is not likely to represent humanrisk whenusedaccording label directions. a to Respectfully submitted behalfof the NDMA Hydroquinone on Task Group,

R. William Soller, Ph.D. SeniorVice President Director of Science Technology & Nonprescription Drug Manufacturers Association

(Reference List follows; Tablesfollow references.)

Page 43 -1

VIII. 1.

References

Adler, I.-D. and Kliesch, U. (1990). Comparison of single and multiple treatment regimens in the mouse bone marrow micronucleusassay for hydroquinone (HQ) and cyclophosphamide(CP). Murur. Res. 234, 115-123, Barale, R., Marrazzini, A., Betti, C., Vangelisti, V., Loprieno, N., a,ndBarrai, I. (1990). Genotoxicity of two metabolitesof benzene: phenol and hydroquinone show .I strong synergistic effects in viw. Mutat. Res. 244, 15-21. Bucks: D.A.W’ , McMaster, J.R., Guy, R.H., and Maibach, H.I. (1988). Percu. taneousabsorption of hydroquinone in humans: Effect of l-dodecylazacycloheptane2-one (Azone) and the 2-ethylhexyl ester of 4-(dimethylamino) benzoic acid (Escalol 507). .J. Toxicoi. Em*irm. Hlth. 24, 279-289. Burnett, C., Goldenthal, E.I., Harris, S.G., Wazeter, F.X., Stratusburg, J., Kapp, R., and Voelker, R. (1976). Teratology and percutaneoustoxicity studies on hair dyes. J. Tosid. Eu\irm. Hit111, 1027-1040. Carlson, A.J. and Brewner,N.R. (1953). Toxicity studies on hydroquinone. Proc. Sot. Eup. Bid. 81, 683-688. Chatterjee, P. and Sharma, A.K. (1972). Effect of phenols on nuclear division in Chara zqlmico. nll~lei~ 15, 214-218. Christian, R.T., Clark, C.S. ; Cody, T.E., Witherup, S., Gartside, P.S., Elia, V.J., Eller, P.hl., Lingg, R., and Cooper, G.P. (1976). The developmentof a test for the potability of water treated by a direct reuse system. University of Cincinnati, Cincinnati, Ohio. Ciranni, R. and Adler, I.-D. (1991). Clastogenic effects of hydroquinone: Induction of chromosomal aberrationsin mouse germ cells. Mutat. Res. 263, 223-229. Ciranni, R., Barale, R., Ghelardini, G., and Loprieno, N. (1988). Benzeneand the genotoxicity of its metabolites. II. The effect of the route of administration on the micronuclei and bone marrow depressionin mouse bone marrow cells. Mutat. Res. 209, 23-28.

2.

3.

4.

5. 6. 7.

8. 9.

(continued . . .)

Page 44 VIII. References (continued)

10. 11.

Cosmetics Toiletry and Fragrance Manufacturers (CTFA) (1980). Dermal teratology study in albino female Sprague-DawleyRats. Study Project: TR-01-80. Crebelli, R., Conti, G., Conti, L., and Carere, A. (1991). in vitro studies with nine known or suspectedspindle poisons: Results in tests for chromosomemalsegregation in Aspergillus nidulans. Mutagenesis6, 131-136. Crebelli, R., Conti, G., and Carere, A. (1987). On the mechanism-of mitotic segregationinduction in Aspergillis nidulans by benzenehydroxy metabolites. Mutagenesis2, 235-238. a. English, J.C., Deisinger, P.J., Perry, L.G., Schum, D.B., and Guest, D. (1988). Toxicokinetics studies with hydroquinone in male and female Fischer 344 rats (Part I). Chemical Manufacturers Association, Washington, D.C.English, J.C., Deisinger, P.J., Perry, L.G., Schum, D.B., and Guest, D. (1988). Toxicokinetics studies with hydroquinone in maIe and female Fischer 344 rats (Part II). Chemical Manufacturers Association, Washington, D.C. :

12.

13.

b.

14.

Epler, J.L., Larimer, F. W., Rao, T.K., Nix, C.E., and Ho, T. (1978). Energyrelated pollutants in the environment: Use of short-term tests for mutagenicity in the isoIation and identification of biohazards. Environ. Health Perspect. 27, 11-20. Florin, I., Rutberg, L., Curvall, M., and Enzell, C.R. (1980). Screening of tobacco smoke constituents for mutagenicity using the Ames’test. Toxicology 15, 219-232. Friedlander, B.R., Hearne, T., and Newmann, B.J. (1982). MortaIity, cancer incidence, and sickness-absence photographic processors: an epidemiologic study. in J. Occup. Med. 24, 605-613. Gad-El-Karim, M.M., SadagopaRamanujam, V.M., and Legator, M.S. (1986). Correlation between the induction of micronuclei in bone marrow by benzene exposure and the excretion of metaboiitesin urine of CD-l mice. Toxicol. App. Pharmacol. 85, 464-477.

15. 16.

17.

(continued . . .)

Age 45 VIII. References (continued)

18.

Galloway, S.M., Armstrong, M. J., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A.D., Nakamura, F., Ahmed, M., Duk; S., Rimpo, J., Margolin, B.H., Resnick, M.A., Anderson, B., and Zeiger, E. (1987). Chromosomeaberrations and sister chromatid exchangesin Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ. Molec. Mutagen. lO(Supp1.lo), l-l.75. Gocke, E., Wild, D., Eckhardt, K., and King, M.-D. (1983);’Mutagenicity studies with the mouse spot test. Mutat. Res. 117, 201-212. Gocke, E., King, M-T., Eckardt, K., and Wild, D. (1981). Mutagenicity of cosmetics ingredients licensed by the Europeancommunity. Murat, Res. 90, 91-109. Greenlee, W.F., Sun, J.D., and Bus, J.S. (1981). A proposed mechanismof benzene toxicity: formation of reactive intermediatesfrom polyphenol metabolites. Toxicol. Appl. Plmrnmol. 59, 187-195. Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. (1983). Salmonella mutagenicity test results for 250 chemicals. Environ. Mutagen. Suppl. 1, 3-142. Irons, R.D. and Neptun, D.A. (1980). Effects of the principal hydroxy-metabolites of benzeneon microtubule polymerization. ii&r. Toxicol. 45, 297-305. Ishidate, Jr., M., Sofuni, T., and Yoshikawa, K. (1981). Chromosomal aberration tests in vitro as a primary screeningtool for environmental mutagensand/or carcinogens. GANh7Monographs on Cancer Res. 27, 95-108. Jenssen,D. and Ramel, C. (1980). The micronucleus test as part of a short-term mutagenicity test program for the prediction of carcinogenicity evaluated by 143 agents tested. Mrrtat. Res. 75, 191-202. Kappas, A. (1990). On the validation of the system of aspergillus for testing environmental aneugens. Mutation and the Environment, Part B, Wiley-Liss, New York, 267-274.

19. 20. 21.

22.

23. 24.

25.

26.

(continued . . .)

,

9

Page 46 VIII. References (continued)

_

27.

Knadle, S. (1985). Synergistic interaction between hydroquinone and ace@ldehydein the induction of sister chromatid exchange in human lymphocytes in vitro. Cancer .‘ _ Res. 45, 4853-4857. . Krasavage, W. J. (1985). Hydroquinone: A developmental toxicity study in rats. Chemical Manufacturers Association, Washington, D.C. Krasavage, W.J. (1984). Hydroquinone: A dominant lethal assay in male rats. Eastman Kodak Company, Rochester, NY. Krogulevich, R.E. and Stom, D.S. (1969). Effects of quinoid and phenolic analogs of radiotoxins on the chromosome apparatus. Rudiobioiogy (USSR) 9, 241-247. Cited in the NTP Report, Reference 39. Lee, E.W., Johnson, J.T., and Garner, C.D. (1989). Inhibitory effects of benzene metabolites on nuclear DNA synthesis in bone marrow cells. J. Toxicol. Env. Hlth. 26, 277-29 1, Levay, G., Pongracz, K., and Bodell, W.J. (1991). Detection of DNA adducts in HL-60 cells treated tsith hydroquinone and e-benzoquinone by 32P-postlabeling. Carcinogcwrsis 12, 118I- 1 186. Marrazzini, A., Betti, C., Barale, R., Bernacchi, F., and Loprieno, N. (1991). Cytogenetic effects of possible aneuploidizing agents. Mutat. Res. 252, 195196. epierre, J. (1981). Percutaneous Marty, J-P., TrouLin, J.H., Jacquot, C., and b’ pharmacokinetics of “C-hydroquinone. C. -I?. Congr. Eur. Biopharm. Pharmacociner. 2, 221-228. (translation) McGregor, D.B., Riach, C.G., Brown, A., Edwards, Reynolds, D., West, K., and Willington, S. (1988). Reactivity of Catecholaminesand related substancesin the mouse lymphoma L5178Y cell assay for mutagens. Environmental and Molecular Mutagenesis 11, 523-544, Miller, B.M., and Adler, I.-D. (1989). Suspect spindle poisons: Analysis of cmitotic effects in mouse bone marrow cells. Mutagenesis 4, 208-215.

28. 29. 30.

31. i 32.

33. 34.

35.

36.

(continued . . .)

Page 47 VIII. References (continued)

37.

Morimoto, K., Wolff, S., and Koizumi, A. (1983).* Induction of sister chromatid exchangesin human lymphocytes by microsomal activation of benzenemetabolites. Muy. Res. 119, 355-360.
. .-

38.

National Toxicology Program (1991). Toxicology and carcinogenesisstudies of tris(2-chloroethyl)phosphatein F-344/N rats and B6C3F, mice. NIH Publication No. 91-2846, ResearchTriangle Park, NC, page 53.’ National Toxicology Program (1989). Toxicology and carcinogenesisstudies of hydroquinone in F-344/N rats and B6C3F, mice. NIH Publication No. 90-2821, ResearchTriangle Park, NC. Nonprescription Drug Manufacturers Association: Draft Protocol for Chronic Oncogenicity Evaluation of Hydroquinone in Mice (1991). Nonprescription Drug Manufacturers Association: Draft Protocol for Chronic Oncogenicity Evaluation of Hydroquinone in Rats (1991). Pacchierotti, F., Bassani, B., Leopardi, P., and Zijno, A. (1991). Origin of aneuploidy in relation to disturbancesof celi-cycle progression. II: Cytogenetic analysis of various parametersin mouse bone marrow cells after colchicine or hydroquinone treatment. Murugcwsis 4, 307-311, Painter, R.B. and Hou*ard, R. (1982). The HeLa DNA-synthesis inhibition test as a rapid screen for mutagenic carcinogens. Mutut. Res. 92, 427-437. Parry, J.M., Parry, E.hl., W’ T., Lynch, A., and James, S. (1990). The arr, detection of aneugensusing yeast and cultured mammalian cells. Mutation and the Environment, Part B, Wiley-Liss, New York, 247-266. Pellack-Walker, P. and Blumer, J.L. (1986). DNA damagein L5178YS cells following exposure to benzenemetabolites. Mol. Phmnacol. 30, 42-47. Rapson, W.H., Nazar, M.A., and Butsky, V.V. (1980). Mutagenicity produced by aqueous chlorination of organic compounds. Bull. Environ. Contam. Toxicol. 24, 590-596. -*

39.

40. 41. 42.

43. 44.

45. 46.

(continued . . .)

, I

i

Page48 VIII. References(continued)

47.

Reddy, M.V., Bleicher, W.T., Blackbum, G,R., ancj. Mackerer, CR. (1990). DNA adduction by phenol, hydroquinone, or benzbquinone virro but not in vivo: ‘ in nuclease l-enhanced 32P-postlabeling adductsas labelednucleoside P of biphosphates, dinucleotides and nucleoside monophosphate. Carcinogenesis 11, 1349-1357:

48. 49.

Roy, S. C. (1973). Comparative effects of colchicine, caffeine, and hydroquinone on nodal roots of Callisia fragrans. Biol. Plant. (Praha)15, 383-396.
Sakai, M., Yoshida, D., and Mizusaki, S. (1985). Mutagenicity of polycyclic aromatic hydrocarbons and quinones on Salmonella typhimurium TA97. Murat. Res.

156, 61-67.
50. Shibata, hl.A., Hirose, M., Tanaka, H., Asakawa, E., Shirai, T., and Nobuyuki, I. of (1991). Induction of renal cell tumors in rats and mice, and enhancement hepatocellular tumor development in mice after long-term hydroquinone treatment. Jap. J. Cancer Research 82, 1211-1219. Shimada, H., Sato, T., hattori, C., Sat&e, S., and Itoh, S. (1988). Induction of micronuclei by benzeneand its metabolites. Tokyo: Research Institute of Daiichi Seiyaku Co., Ltd. Sivchev, S, Bazhdekov, B., and Durmishev, A. (1966). On the influenceof hydroquinone in percutaneousapplication on guinea-pigs, Nauchni Trudove Visshiya Med. Inst. Sofia 45, 35-42. Sterner, J.H., Oglesby, F.L., and Anderson, B. (1947). Quinone vapors and their harmful effect. I. J. Indrrst. Hyg. Toxicol. 29, 60-73. Topping, D., Bernard, L., Blacker, A., Murphy, S., and O’ Donoghue, J. (1988). Subchronic functional observational battery (FOB) and neuropathology studieson hydroquinone. Chemical Manufacturers Association, Washington, D.C. Tunek, A., Hogstedt, B., and Olofsson, T. (1982). Mechanism of benzene toxicity. Effects of benzeneand benzene metabolites on bonemarrow cellularity, numberof granulopoietic stem cells, and frequency of micronuclei in mice. Chem. Biol. Interact. 39, 129-138.

51.

52.

53. 54.

55.

(continued . . .)

Page 49 VIII. References (continued)

56.

USEPA Risk AssessmentForum Report (1991). Alpha-2p-globulin: Association with chemically-induced renal toxicity and neoplasiain the male rat (Draft). EPA/625/3-91/019A, Washington, D.C. VanDuuren, B.L.and Goldschmidt, B.M. (1976). Carcinogenic and tumor-promoting agents in tobacco carcinogenesis. J. Narl. Cancer Inst. 56, 1237-1242. Xu, W. and Adler, L-D. (1990). Clastogenic effects of known and suspect.:spindle poisons studied by chromosomeanalysis in mouse bone marrow cells. Mutagenesis 5, 371-374. CIR Report: Final Report on the Safety Assessmentof Hydroquinone and Pyrocatechol (1986). J. Am. College Toxicol. 5, 123-165. Schroeder, R.E. and Daly, I. W. (1989). A developmentaltoxicity study in rabbits with hydroquinone. Final Report. Chemical Manufacturers Association, Washington, D.C. Schroeder, R.E. and Daly, l.W’ (1989). A two-generation reproduction study in rats . wiht hydroquinone. Final Report: Volume I of V. Chemical Manufacturers Association, \i’ ashington, D.C. Schroeder, R.E. and Daly, 1.W’ (1989). A two-generation reproduction study in rats . with hydroquinone. Final Report: Volume II of V. Chemical Manufacturers Association, Washington, D.C. Schroeder, R.E. and Daly, 1.W:. (1989). A two-generation reproduction study in rats with hydroquinone. Final Report: Volume III of V. Chemical Manufacturers Association, Washington, D.C. Schroeder, R.E. and Daly, I.W. (1989). A two-generation reproduction study in rats with hydroquinone. Final Report: Volume IV of V. Chemical Manufacturers Association, Washington, D.C. Schroeder, R.E. and Daly, I.W. (1989). A two-generation reproduction study in rats with hydroquinone. Final Report: Volume V of V. Chemical Manufacturers Association, Washington, D .C.

57. 58.

59. 60.

61.

62.

63.

64.

65.

(continued . . .)

Page 50 VIII, References (continued)

66. 67. 68. 69.

Chavin, W., Jelonek, E.J., Reed, A.H., and Binder,%.R. (1980). Survival of mice receiving melanomatransplantsis promoted by hydroquinone. Science208, 408-410. Hunter, C., Blair, D. (1972). Benzene: Pharmacokineticstudies in man. Ann. Occup. Hyg. 15, 193-199. Anderson, B. (1947). Cornea1and conjunctival pigmentation among workers engaged in manufacture of hydroquinone. Arch. Opthalmol. 3, 812-826. . .. Boatman, R.S., English, J.C., Perry, L.G., and Bialecki, V.E. (1992). Sex and strain related differences in the nephrotoxicity of hydroquinone in rats. The Toxicologist 2, 427. Hill, B.A., Monks, T.J., and Lau, S.S. (1992). Metabolism and toxicity of 2- (glutathion-s-y-l) hydroquinone in the in situ perfused rat kidney. The Toxicologist 12, 345. Kleiner, H.E., Hill, B.A., Monks, T.J., and Lau, S.S. (1992). In vivo and in vitro formation of several s-conjugatesof hydroquinone. The Toxicologist 2, 345. Short, B.G., and Swenberg, J.A. (1991). Chronically induced cell proliferation in carcinogenesisin the male rat kidney. In: Chemicallv Induced Cell Proliferation: Implications for Risk Assessment,Butterworth, B.E., Slaga, T-J., Farland, W., and McClain, Al. (eds.), Wiley-Liss, New York, pp. 357-367. Cassidy, M.K., and Houston, J.B. (1984). In vivo capacity of hepatic and extrahep,aticenzymes to conjugate phenol. Drug Metab. Disposit. 12, 619-624.

70.

71. 72.

73.

WSlams 5-l 1-92