INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 157 HYDROQUINONE This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. First draft prepared by Dr M. Gillner, Dr G.S. Moore, Dr H. Cederberg and Dr K. Gustafsson, National Chemicals Inspectorate, Solna, Sweden Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Orgnization Geneva, 1994 The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals. WHO Library Cataloguing in Publication Data Hydroquinone. Environmental health criteria: 157) 1. Environmental exposure 2. Hydroquinones - analysis 3. Hydroquinones - toxicity I.Series ISBN 92 4 157127 8 (NLM Classification QD 341.P5) ISSN 0250-863X The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, Which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1994 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the impression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of every country, territory, city, or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR HYDROQUINONE 1. SUMMARY 1.1. Identity, physical and chemical properties, analytical methods 1.2. Sources of human and environmental exposure 1.3. Environmental transport, distribution and transformation 1.4. Environmental levels and human exposure 1.5. Kinetics and metabolism 1.6. Effects on laboratory mammals, and in vitro systems 1.7. Effects on humans 1.8. Effects on other organisms in the laboratory and field 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.2.1. Reduction-oxidation equilibria 2.2.2. Oxidation of hydroquinone 2.3. Conversion factors 2.4. Analytical methods 2.4.1. Sampling 2.4.2. Methods of analysis 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Anthropogenic sources 3.2.1. Production levels and processes 3.2.2. Uses 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Transport and distribution between media 4.2. Transformation 4.2.1. Biodegradation 4.2.2. Abiotic degradation 4.2.3. Bioaccumulation 4.3. Interaction with other physical, chemical or biological factors 4.4. Ultimate fate following use 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air, soil and water 5.1.2. Food 5.2. General population exposure 5.3. Occupational exposure 6. KINETICS AND METABOLISM 6.1. Absorption 6.2. Distribution 6.3. Metabolic transformation 6.4. Elimination and excretion 6.5. Reaction with body components 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO SYSTEMS 7.1. Single exposure 7.2. Skin and eye irritation; sensitization 7.2.1. Skin irritation 7.2.2. Eye irritation 7.2.3. Sensitization 7.3. Short-term exposure 7.4. Long-term exposure 7.5. Reproduction, embryotoxicity and teratogenicity 7.5.1. Effects on male reproduction 7.5.2. Effects on female reproduction 7.5.3. Embryotoxicity and teratogenicity 7.6. Mutagenicity and related end-points 7.7. Carcinogenicity 7.7.1. Long-term bioassays 7.7.2. Carcinogenicity-related studies 188.8.131.52 Skin 184.108.40.206 Bladder 220.127.116.11 Stomach 18.104.22.168 Liver 7.8. Special studies 7.8.1. Effects on spleen and bone marrow cells; immunotoxicity 7.8.2. Effects on tumour cells 7.8.3. Neurotoxicity 7.8.4. Nephrotoxicity 7.8.5. Interaction with phenols 8. EFFECTS ON HUMANS 8.1. General population exposure 8.1.1. Acute toxicity - poisoning incidents 8.1.2. Short-term controlled human studies 8.1.3. Dermal effects; sensitization 8.2. Occupational exposure 8.2.1. Dermal effects 8.2.2. Ocular effects 8.2.3. Systemic effects 8.2.4. Epidemiological studies 22.214.171.124 Respiratory effects 126.96.36.199 Carcinogenicity studies 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Toxicokinetics 10.2. Animal and in vitro studies 10.3. Evaluation of human health risks 10.3.1. Exposure 10.3.2. Human health effects 10.4. Evaluation of effects on the environment 11. RECOMMENDATIONS 12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES REFERENCES APPENDIX RESUME RESUMEN WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR HYDROQUINONE Members Dr L. Albert, Program of Health and Environment, Centre for Ecology and Development, Xalapa, Veracruz, Mexico (Chairman) Dr H. Cederberg, National Chemicals Inspectorate, Solna, Sweden Dr J. Devillers, Centre de Traitement de l'Information Scientifique (CTIS), Lyon, France Dr D.A. Eastmond, Environmental Toxicology Graduate Program, Department of Entomology, University of California, Riverside, California, USA Dr M. Gillner, Scientific Documentation and Research, National Chemicals Inspectorate, Solna, Sweden (Rapporteur) Dr S. Humphreys, Contaminants, Standards, and Monitoring Branch, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Washington, DC, USA Dr G.A. Moore, Scientific Documentation and Research, National Chemicals Inspectorate, Solna, Sweden Professor H. Naito, Institute of Clinical Medicine, University of Tsukuba, Tsukuba City, Ibaraki, Japan Dr C.O. Nwokike, Medical Division, Lever Brothers (Nigeria) PLC, Apapa, Lagos, Nigeria Dr J. O'Donoghue, Corporate Health and Environment Laboratories, Eastman Kodak Company, Rochester, New York, USA Professor P.N. Viswanathan, Ecotoxicology Group, Industrial Toxicology Research Centre, Lucknow, India Observer Mr P-G. Pontal, Rhône Poulenc Agro, Lyon, France Secretariat Dr M. Gilbert, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary) Dr J. Wilbourn, Unit of Carcinogen Identification and Evaluation, International Agency for Research on Cancer, Lyon, France NOTE TO READERS OF THE CRITERIA MONOGRAPHS Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are kindly requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda. * * * A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Case postale 356, 1219 Châtelaine, Geneva, Switzerland (Telephone No. 9799111). * * * This publication was made possible by grant number 5 U01 ES02617-14 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA. ENVIRONMENTAL HEALTH CRITERIA FOR HYDROQUINONE A WHO Task Group meeting on Environmental Health Criteria for Hydroquinone was held at the British Industrial Biological Research Association (BIBRA), Carshalton, United Kingdom, from 24 to 28 May 1993. Dr D. Anderson welcomed the participants on behalf of the host institution and Dr M. Gilbert opened the meeting on behalf of the three cooperating organizations of the IPCS (ILO/UNEP/WHO). The Task Group reviewed and revised the draft criteria monograph and made an evaluation of the risks for human health and the environment from exposure to hydroquinone. The first draft of this monograph was prepared by Dr M. Gillner, Dr G.A. Moore, Dr H. Cederberg and Dr K. Gustafsson, National Chemicals Inspectorate, Solna, Sweden. Dr M. Gilbert and Dr P.G. Jenkins, both members of the IPCS Central Unit, were responsible for the overall scientific content and editing, respectively. The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged. ABBREVIATIONS AUC area under the curve BP benzo [a]pyrene BHA butylated hydroxyanisole cAMP adenosine 3',5'-phosphate cGMP guanine 3',5'-phosphate CLV ceiling value HPLC high-performance liquid chromatography HQ hydroquinone IL-1 interleukin-1 IL-4 interleukin-4 i.p. intraperitoneal i.v. intravenous MDA malondialdehyde MCL melanotic cell lines NADPH reduced nicotinamide adenine dinucleotide NMCL nonmelanolic cell lines MNNG N-methyl- N'-nitro- N-nitrosoguanidine NOAEL no-observable-adverse-effect level NOEL no-observed-effect level ODC ornithine decarboxylase QSAR quantitative structure-activity relationship s.c. subcutaneous STEL short-term exposure limit TLC thin-layer chromatography TLV threshold limit value TWA time-weighted average 1. SUMMARY 1.1 Identity, physical and chemical properties, analytical methods Hydroquinone (1,4-benzenediol; C6H4(OH)2) is a white crystalline substance when pure, with a melting point of 173-174 °C. The specific gravity is 1.332 at 15 °C, and the vapour pressure is 2.4 x 10-3 Pa (1.8 x 10-5 mmHg) at 25 °C. It is highly soluble in water (70 g/litre at 25 °C) and the log n-octanol/water partition coefficient is 0.59. With respect to organic solvents, the solubility varies from 57% in ethanol to less than 0.1% in benzene. Hydroquinone is combustible when preheated. It is a reducing agent which is reversibly oxidized to its semiquinone and quinone. Hydroquinone in the air is sampled either by trapping in solvent or on a mixed cellulose ester membrane filter. Analysis of hydroquinone is carried out by titrimetric, spectrophotometric or, most commonly, chromatographic techniques. 1.2 Sources of human and environmental exposure Hydroquinone occurs both in free and conjugated forms in bacteria, plants and some animals. Industrially, it is produced in several countries. In 1979, the total world capacity for production exceeded 40 000 tonnes, while in 1992 it was approximately 35 000 tonnes. It is extensively used as a reducing agent, as a photographic developer, as an antioxidant or stabilizer for certain materials that polymerize in the presence of free radicals, and as a chemical intermediate for the production of antioxidants, antiozonants, agrochemicals and polymers. Hydroquinone is also used in cosmetics and medical preparations. 1.3 Environmental transport, distribution and transformation Hydroquinone occurs in the environment as a result of man-made processes as well as in natural products from plants and animals. Due to its physicochemical properties, hydroquinone will be distributed mainly to the water compartment when released into the environment. It degrades both as a result of photochemical and biological processes; consequently, it does not persist in the environment. No bioaccumulation is observed. 1.4 Environmental levels and human exposure No data on hydroquinone concentrations in air, soil or water have been found. However, hydroquinone has been measured in mainstream smoke from non-filter cigarettes in amounts varying from 110 to 300 µg per cigarette, and also in sidestream smoke. Hydroquinone has been found in plant-derived food products (e.g., wheat germ), in brewed coffee, and in teas prepared from the leaves of some berries where the concentration sometimes exceeds 1%. Photohobbyists can be exposed to hydroquinone dermally or by inhalation. However no data on exposure levels are available. Dermal exposure may also result from the use of cosmetic and medical products containing hydroquinone, such as skin lighteners. The European Community (EC) countries have restricted its use in cosmetics to 2% or less. In the USA, the Food and Drug Administration has proposed concentrations between 1.5 and 2% in skin lighteners. Concentrations up to 4% may be found in prescription drugs. In some countries even higher concentrations may be found in skin lighteners. Few industrial hygiene monitoring data are available for hydroquinone. Average concentrations in air during manufacturing and processing of hydroquinone have been reported to be in the range of 0.13 to 0.79 mg/m3. Occupational air exposure limits (time-weighted average) in different countries range from 0.5 to 2 mg/m3. 1.5 Kinetics and metabolism Hydroquinone is rapidly and extensively absorbed from the gut and trachea of animals. Absorption via the skin is slower but may be more rapid with vehicles such as alcohols. Hydroquinone distributes rapidly and widely among tissues. It is metabolized to p-benzoquinone and other oxidized products, and is detoxified by conjugation to monoglucuronide, monosulfate, and mercapturic derivatives. The excretion of hydroquinone and its metabolites is rapid, and occurs primarily via the urine. Hydroquinone and/or its derivatives react with different biological components such as macromolecules and low molecular weight molecules, and they have effects on cellular metabolism. 1.6 Effects on laboratory mammals, and in vitro systems Oral LD50 values for several animal species range between 300 and 1300 mg/kg body weight. However, for the cat LD50 values range from 42 to 86 mg/kg body weight. Acute high-level exposure to hydroquinone causes severe effects on the central nervous system (CNS) including hyperexcitability, tremor, convulsions, coma and death. At sublethal doses these effects are reversible. The dermal LD50 value has been estimated to be > 3800 mg/kg in rodents. Inhalation LC50 values are not available. A formulation containing 2% hydroquinone in a single-insult patch test in rabbits resulted in an irritation score of 1.22 (on a scale of 0 to 4). Daily topical applications for three weeks of 2 or 5% hydroquinone in an oil-water emulsion on the depilated skin of black guinea-pigs caused depigmentation, inflammatory changes and thickening of the epidermis. The depigmentation was more marked at higher concentrations, and female guinea-pigs were more sensitive than males. Sensitization tests in guinea-pigs have shown weak to strong reactions depending on the methods or vehicles used. The strongest reactions were obtained with the guinea-pig maximization test. A cross-sensitization of almost 100% between hydroquinone and p-methoxyphenol was also seen in guinea-pigs, but only restricted evidence of cross-reactions to p-phenylenediamine, sulfanilic acid and p-benzoquinone was obtained. A 6-week oral toxicity study in male F-344 rats resulted in nephropathy and renal cell proliferation. Thirteen-week oral gavage studies in F-344 rats and in B6C3F1 mice resulted in nephrotoxicity in rats at 100 and 200 mg/kg, and tremors and convulsions in rats at 200 mg/kg; reduced body weight gain was seen in both rats and mice. Dosing at 400 mg/kg was lethal in rats. In mice dosed for 13 weeks at 400 mg/kg, tremors, convulsions and lesions in the gastric epithelium were reported. Thirteen-week hydroquinone exposure of Sprague Dawley rats resulted in decreased body weight gain and CNS signs at 200 mg/kg. CNS signs were also observed at a dose level of 64 mg/kg body weight but not at 20 mg/kg. Hydroquinone injected subcutaneously reduced fertility in male rats, and prolonged the estrus cycle in female rats. However, this was not found in oral studies (a dominant lethality study and a two-generation study). In a developmental study in rats, oral doses of 300 mg/kg body weight caused slight maternal toxicity and reduced fetal body weight. In rabbits, the no-observed-effect level (NOEL) for maternal toxicity was 25 mg/kg per day, and it was 75 mg/kg per day for developmental toxicity. In a two-generation reproduction study in rats hydroquinone caused no reproductive effects at oral doses of up to 150 mg/kg body weight per day. The no-observed- adverse-effect level (NOAEL) for parental toxicity was determined to be 15 mg/kg per day, and for reproductive effects through two generations it was 150 mg/kg per day. Hydroquinone induces micronuclei in vivo and in vitro. Structural and numerical chromosome aberrations have been observed in vitro and after intraperitoneal administration in vivo. Furthermore, the induction of gene mutations, sister-chromatid exchange and DNA damage has been demonstrated in vitro. Hydroquinone caused chromosomal aberrations in male mouse germ cells at the same order of magnitude as in mouse bone marrow cells after intraperitoneal injection. Induction of germ-cell mutations could not be established in a dominant lethal test in male rats dosed orally. In a two-year study, oral administration of hydroquinone caused a dose-related incidence of renal tubular cell adenomas in male F-344/N rats. The incidence was statistically significant in the high-dose group. In the high-dose males, renal tubular cell hyperplasia was also found. In female rats a dose-related increased incidence of mononuclear cell leukaemia occurred. Female B6C3F1 mice developed a significantly increased incidence of hepatocellular adenomas. In another study, hydroquinone (at a dietary level of 0.8%) produced a significantly increased incidence of epithelial hyperplasia of the renal papilla and a significant increase of renal tubular hyperplasia and adenomas in male rats. No increased incidence of mononuclear cell leukaemia in female rats was observed. In mice, the incidence of squamous cell hyperplasia of the forestomach epithelium was significantly increased in both sexes. In male mice, there was a significantly increased incidence of hepatocellular adenomas and also of renal tubular hyperplasia. A few renal cell adenomas were observed. In vivo (intraperitoneal injection) and in vitro studies in mice have demonstrated that hydroquinone has a cytotoxic effect by reducing the bone marrow and spleen cellularity and also an immunosuppressive potential by inhibiting the maturation of B-lymphocytes and the natural killer cell activity. Results also indicate that bone marrow macrophages may be the primary target for hydroquinone myelotoxicity. Myelotoxic effects were not observed in a long-term bioassay in rodents. In a 90-day study in rats using a functional-observational battery, dose levels of 64 and 200 mg hydroquinone/kg produced tremors, and 200 mg/kg produced depression of general activity. Neuropathological examinations were negative. 1.7 Effects on humans Cases of intoxication have been reported after oral ingestion of hydroquinone alone or of photographic developing agents containing hydroquinone. The major signs of poisoning included dark urine, vomiting, abdominal pain, tachycardia, tremors, convulsions and coma. Deaths have been reported after ingestion of photographic developing agents containing hydroquinone. In a controlled oral study on human volunteers, ingestion of 300-500 mg hydroquinone daily for 3-5 months did not produce any observable pathological changes in the blood and urine. Dermal applications of hydroquinone at concentration levels below 3% in different bases caused negligible effects in male volunteers from different human races. However, there are case reports suggesting that skin lightening creams containing 2% hydroquinone have produced leucoderma, as well as ochronosis. Hydroquinone (1% aqueous solution or 5% cream) has caused irritation (erythema or staining). Allergic contact dermatitis due to hydroquinone has been diagnosed. Combined exposure to hydroquinone and quinone airborne concentrations causes eye irritation, sensitivity to light, injury of the corneal epithelium, corneal ulcers and visual disturbances. There have been cases of appreciable loss of vision. Irritation has occurred at exposure levels of 2.25 mg/m3 or more. Long-term exposure causes staining of the conjunctiva and cornea and also opacity. Slowly developing inflammation and discoloration of the cornea and conjunctiva have resulted after daily hydroquinone exposure for at least two years of 0.05-14.4 mg/m3; serious cases have not occurred until after five or more years. One report described cases of corneal damage occurring several years after the exposure to hydroquinone had stopped. There are no adequate epidemiological data to assess the carcinogenicity of hydroquinone in humans. 1.8 Effects on other organisms in the laboratory and field The ecotoxicological behaviour of hydroquinone has to be related to its physicochemical properties, which induce sensitivity to light, pH and dissolved oxygen. Its ecotoxicity, which is generally high (e.g., < 1 mg/litre for aquatic organisms), varies from species to species. Algae, yeasts, fungi and plants are less sensitive to hydroquinone than the other organisms generally used for toxicity testing. However, within the same taxonomic group, the sensitivity of different species to hydroquinone may vary by a factor of 1000. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1 Identity Primary constituent Chemical formula: C6 H4 (OH)2 Chemical structure: Relative molecular mass: 110.11 Common name: Hydroquinone CAS registry number: 123-31-9 Synonyms: 1,4-benzenediol; p-benzenediol; benzohydroquinone; benzoquinol; 1,4- dihydroxybenzene; p-dihydroxybenzene; p-dioxobenzene; p-dioxybenzene; hydroquinol; hydroquinole; alpha- hydroquinone; p-hydroquinone; p-hydroxyphenol; quinol; ß-quinol Technical product: Trade name: Tecquinol Impurities: none identified Isomeric composition: None Additives: None 2.2 Physical and chemical properties Physical state: Long needles Colour: White (analytical grade) Odour: Odourless Taste: Not documented Melting point: 173-174 °C Boiling point: 287 °C Flash point: 165 °C (closed cup) Flammability: Combustible when preheated Explosion limits: Slight when exposed to heat. Reactive at high temperature or pressure Vapour pressure: 2.4 x 10-3 Pa (1.8 x 10-5 mmHg) at 25 °C 0.133 kPa (1 mmHg) at 132.4 °C 0.533 kPa (4 mmHg) at 150 °C 8.00 kPa (60 mmHg) at 203 °C Specific gravity: 1.332 at 15 °C Vapour density: 3.81 Log n-octanol/water partition coefficient: 0.59 Solubility: Water: 59 g/litre at 15 °C 70 g/litre at 25 °C 94 g/litre at 28 °C Organic solvents: Soluble in most polar organic solvents ethyl alcohol 57 g/100 grams solvent at 25 °C acetone 20 g/100 grams solvent at 25 °C methyl isobutyl 27 g/100 grams solvent at 25 °C ketone 2-ethylhexanol 12 g/100 grams solvent at 25 °C ethyl acetate 22 g/100 grams solvent at 25 °C Virtually insoluble (< 0.1%) in benzene, toluene and carbon tetrachloride Other properties: Reducing agent; pK1 = 9.9, pK2 = 11.6; Redox active (see below) 2.2.1 Reduction-oxidation equilibria Hydroquinone undergoes reversible redox changes which can involve a variety of pathways and redox couples (see Fig. 1). Each redox couple has an electrochemical potential dependent upon the degree of protonation and electron reduction. Hydroquinone is a reducing agent with an electrochemical potential (E°) of +286 mV for the benzoquinone/hydroquinone (Q/H2Q) redox couple at 25 °C and pH 7.0, and under constant conditions. 2.2.2 Oxidation of hydroquinone Hydroquinone is oxidized by a variety of oxidants including nitric acid, halogens, persulfates and metal salts (NIOSH, 1978). It is also oxidized by molecular oxygen in alkaline solutions. Hydroquinone reacts with molecular oxygen (autooxidation). In an aqueous medium the rate of autooxidation is pH dependent, occurring very rapidly at alkaline pH to produce a brown solution, but very slowly in acidic medium. This reaction is strongly catalysed by copper ions. Some of the possible reactions during autooxidation of hydroquinone in alkaline medium are outlined in Fig. 2. In alkaline solution, p-benzoquinone can further react to form 2-hydroxyhydroquinone. In a similar manner to hydroquinone, 2-hydroxyhydroquinone can be oxidized to 2-hydroxy- p-benzoquinone by electron transfer and disproportionation reactions (4a and b). In addition, 2-hydroxy- p-benzoquinone (QI) is formed from 2-hydroxy-hydroquinone (HQI) by sequential mixed-redox reactions with p-benzoquinone involving comproportionation [Eq. 1] and a redox equilibrium reaction [Eq. 2]. Formation of p-benzoquinone from hydroquinone also occurs in a reverse manner by these mixed-redox reactions once 2-hydroxy- p-benzoquinone is formed. Hydrogen peroxide may be generated by the reaction of hydroquinone and oxygen, and can then react with p-benzoquinone forming 2,3-epoxy-hydroquinone. This latter product, if reduced, forms 2-hydroxy-hydroquinone. Owing to the large number of redox reactions possible between mono-benzo products, the possible dimeric combinations, including formation of charge transfer complexes between equal molar equivalents of hydroquinones and benzoquinones (Q + HQ <-> Q ... HQ), oligomers and polymers with various physical chemical properties are numerous and, hence, their specific chemical formulae are not shown in Fig. 2. Autooxidation of hydroquinone is not synonymous with semiquinone autooxidation, which is also termed quinone redox cycling. The latter phenomenon entails redox cycling between a semiquinone and quinone in the presence of molecular oxygen, generating the superoxide anion radical [Eq. 3]. With p-benzosemiquinone and 2-hydroxy- p-benzoquinone, this reaction is not marked because the equilibrium constant for the disproportionation reaction (Ks) of p-benzosemiquinone to hydroquinone and p-benzoquinone [Eq. 4] is around two orders of magnitude higher than the equilibrium constant (Kc) for autooxidation of benzosemiquinone [Eq. 3]. Thus autooxidation of the semibenzoquinone does not significantly contribute to oxygen depletion as for other hydroquinone/quinone couples. In contrast, superoxide anion radical serves to reduce p-benzoquinone to p-benzosemiquinone. Confusion over the significance of redox cycling [Eq. 3] has arisen from experiments performed in the presence of superoxide dismutase (SOD) which catalyses the dismutation of superoxide anion radical to H2O2 and O2 [Eq. 5]. Experiments in which addition of SOD has been shown to modulate quinone toxicity have often been interpreted as indicating that active oxygen species are involved in hydroquinone/quinone mechanism of action (oxidative stress). In fact, SOD "drives" the autooxidation of p-benzosemiquinone to p-benzoquinone [Eq. 3] by removal of superoxide anion radical [Eq. 5] (Winterbourn, 1981; Rossi et al., 1986). Dry pure hydroquinone is very stable to oxidation by oxygen, darkening slowly upon prolonged exposure to air. 2.3 Conversion factors 1 ppm = 4.5 mg/m3 at 25 °C (1 atmosphere pressure) 1 mg/m3 = 0.222 ppm at 25 °C (1 atmosphere pressure) 2.4 Analytical methods Information about analytical methods for hydroquinone are contained in Devillers et al. (1990) and NIOSH (1978). The procedures reported include colorimetry, column-, paper, thin-layer and gas chromatography, and HPLC. It should be noted that difficulties occur when hydroquinone is analysed by HPLC (Devillers et al., 1990). Trace metal impurities, concentration of dissolved oxygen in the mobile phase, pH of the solution, age of the water sample, and age and history of the guard column may each influence the analysis. 2.4.1 Sampling Sampling techniques for air are outlined in Table 1. 2.4.2 Methods of analysis Analytical methods are summarized in Table 2. Table 1. Sampling techniques for hydroquinone in air in the occupational setting Method Sample type Comments Technique Reference Midget hydroquinone hydroquinone sample time = Oglesby impinger dust absorbed in 5-10 min; sample et al. (1947) isopropyl alcohol rate = 2.82 litres/min in an all-glass impinger Midget hydroquinone hydroquinone air volume= 409-504 litres Chrostek (1975) impinger mist collected in for about 430 min distilled water; disadvantage: sample loss can occur from spillage Mixed cellulose hydroquinone filter with 0.8-µm sample time = NIOSH (1976) ester aerosol pore size and 37-mm 60 min; sample membrane diameter rate = 1.5 litres/min filter recommended; collection is >96% Table 2. Analytical methods Method Sample type Comments Detection limit Reference Potentiometric aqueous hydroquinone extracted twice with ethyl- not stated Stott (1942), titration acetate (<99.4% extraction) followed Levenson (1947), by titration; requires little equipment Stevens (1945) but is difficult and time consuming Oxidiometric aqueous ceric sulfate with o-phenanthrolineferrous not stated; Kolthoff & Lee (1946), titration sulfate complex (ferroin) used as indicator; accuracy <99.98% Brunner et al. (1949) simple and fast with easily discernible colour change Iodometric aqueous single methyl acetate extraction involving not stated; Baumbach (1946), titration potentiometric titration of metol (methyl- p- reproducibility Shaner & Sparks amino-phenol sulfate) followed by oxidation (95.4-97.8%) (1946) of both metol and hydroquinone with iodine Iodometric urine urine hydrolysed at 100 °C for 2 h with conc. not stated Baernstein (1945) titration H2SO4(pH 1.0); pH adjusted to 7.0 with sodium sulfite followed by extraction of phenols for 4 h in a continuous liquid-liquid extractor; hydroquinone precipitated with lead acetate pH 6.5 plus pyridine-acetate buffer; filtrates acidified, reacted with bromine, and excess bromine back titrated with 0.2 mol/litre sodium sulfite after addition of potassium iodide; alternatively an iodine sensitive electrode can be used as indicator; disadvantage: ketones react in a similar manner to hydroquinone Colorimetry aqueous hydroquinone reacted with phloroglucinol 1-35 mg/m3 Oglesby et al. (1947) in NaOH; measured at 520 nm Colorimetry aqueous hydroquinone in styrene reacted with sodium lower limit Whettem (1949) tungstate and sodium carbonate; detected < 0.01 mg/ml by visual comparison with standards Table 2. (contd). Method Sample type Comments Detection limit Reference Colorimetry aqueous reaction with 4-aminoantipyrine; 0.05 ppm Jacquemain et al. disadvantage; reacts with phenols (1975) Spectrophotometry aqueous absorption wavelength not stated not stated Chrostek (1975) Paper aqueous uses various solvent systems; separation qualitative Borecky (1963) chromatography of mixtures with hydroquinone is indistinct Paper aqueous three different solvent systems used; qualitative Stom (1975) chromatography stable derivative formed by reaction with benzene sulfinic acid Paper aqueous developed with potassium meta periodate microgram quantities Clifford & Wight (1973) chromatography Chromatography cigarette methylether hydroquinone derivative formed qualitative Commins & Lindsey, and smoke by reactions of dimethyl sulfate and (1956) spectrophotometry hydroquinone Gas aqueous phenols extracted into methyl isobutyl 0.1 mg/litre Cooper & Wheatstone, chromatography ketone; trimethylsilyl ethers prepared, (1973) separated on a Chromosorb W (AW-DCMS) column coated with 5% tri-2,4-xylenyl phosphate; detected by flame ionization TLC aqueous reaction with feric chloride and qualitative Umpelev et al. (1974) K3 [Fe (CN)6] HPLC aqueous hydroquinone absorbed on mixed cellulose 0.84-4.05 mg/m3 NIOSH (1978) ester filter membrane; filters are extracted with 1% acetate; samples are injected onto a Partisil TM 10-ODS column with 1% ethanoic acid as mobile phase; detected at 290 nm Table 2. (contd). Method Sample type Comments Detection limit Reference HPLC aqueous separated on Merckogel PGM 2000 column not stated Seki (1975) with 0.05 mol/litre Pi (pH 6) followed by 0.05 mol/litre Pi plus 0.66 mol/litre borate pH 6; detected at 280 nm HPLC aqueous separated on µBondapak C18 column with > 2 µM Raghavan (1979) 0.01 mol/litre Pi (pH 7); detected at 280 nm HPLC air hydroquinone oxidized to p-benzoquinone 0.005 mg/m3 Levin (1988) by permanganate impregnated glassfibre in a 5-litre air filter; p-benzoquinone formed is trapped on sample XAD-2 adsorbent and desorbed with acetonitrile; detection at 290 nm 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence Hydroquinone occurs in a variety of forms as a natural product from plants and animals. It has been found in non-volatile extracts of coffee beans (Högl, 1958) and other foods (see section 5.1.2), and as Arbutin (a glucoside of hydroquinone) in the leaves of blueberry, cranberry, cowberry and bearberry plants (Varagnat, 1981). Hydroquinone formation from Arbutin in Pyrus spp. is involved in fire blight resistance (Smale & Keil, 1966; Hildebrand et al., 1969). Hydroquinone is considered to be the most important component of the allelopathic interaction between the perennial weed leafy spurge (Euphorbia esula) and the small everlasting (Antennaria microphylla). A differential ability to detoxify hydroquinone in the two species was observed in tissue cultures (Hogan & Manners, 1990, 1991). Hydroquinones have been isolated from marine sponges of Dysidea sp. (Iguchi et al., 1990) and from the marine colonial tunicate Aplidium californicum (Howard et al., 1979). Hydroquinone is also found in the bombardier beetle where it is involved in defensive biochemistry: the beetle can shoot a hot cloud of quinone, formed by the action of hydrogen peroxide, hydroquinone and catalase-peroxidase in the explosion chamber of the beetle, towards an oncoming enemy (Eisner et al., 1977). The occurrence of hydroquinone in nature can originate from metabolic processes. Direct hydroxylation of phenol to form hydroquinone has been reported to occur when phenol was used as a substrate by cytochrome P-450-enriched extracts of Streptomyces griseus (Trower et al., 1988). Hydroquinone can also occur as a metabolite in the biodegradation of substituted phenols (e.g. Spain et al., 1979; Nyholm et al., 1984). Hydrolytic p-hydroxylation initiates the degradation of many polychlorinated phenolic compounds by Rhodococcus chlorophenolicus with the formation of substituted hydroquinones (Häggblom et al., 1988). 3.2 Anthropogenic sources 3.2.1 Production levels and processes In 1979, the world capacity for the production of hydroquinone exceeded 40 000 tonnes (Varagnat, 1981). The annual production volume of hydroquinone in the USA was estimated to be about 12 000 tonnes in 1985 (US EPA, 1985). Hydroquinone is manufactured in the USA, Japan, France, Italy, and China (IARC, 1977; Varagnat, 1981). In 1992, the world production was approximately 35 000 tonnes (USA: 16 000; Europe: 11 000; Japan: 6000; Central and South America and Asian countries other than Japan: 2000) (personal communication from H. Naito, University of Tsukuba, to the IPCS in 1993). Hydroquinone can be manufactured commercially by several processes. In the aniline oxidation process aniline is oxidized with manganese dioxide and sulfuric acid to quinone; this is followed by reduction of the latter to hydroquinone by an aqueous solution of iron or by catalytic hydrogenation (Varagnat, 1981). Hydroquinone is also manufactured by hydroxylation of phenol with hydrogen peroxide as a hydroxylation agent. The reaction occurs with strong mineral acids or ferrous or cobaltous salts as catalysts (Varagnat, 1981). A third process to produce hydroquinone is hydroperoxidation of diisopropylbenzene. The para isomer is isolated and oxidized with oxygen to produce the corresponding dihydroperoxide, which is treated with sulfuric acid to produce acetone and hydroquinone (NTP, 1989). Hydroquinone can also be formed, based on Reppe's synthesis, by carbonylation of acetylene under pressure. Finally, hydroquinone is obtained from the reaction of p-isopropenylphenol and 30% aqueous hydrogen peroxide in acidic conditions, but these syntheses are not used for commercial production (Varagnat, 1981). 3.2.2 Uses Hydroquinone has a multitude of used. It is used as a developer in black-and-white photography and related graphic arts such as lithography, rotogravure, and for medical and industrial X-ray films (Varagnat, 1981). It is also widely used in the manufacture of rubber antioxidants and antiozonants, monomer inhibitors, and food antioxidants to prevent deterioration in many oxidizable products, e.g., to stabilize vitamin A in fish oil, vitamins D and E, ß-carotene, and antibiotics in feeds, and as a chemical intermediate for the production of agrochemicals and performance polymers (Varagnat, 1981). Hydroquinone and products containing hydroquinone are used in cosmetics and medical skin preparations as a depigmenting agent to lighten small areas of hyperpigmented skin. It is also used in the treatment of melasma, freckles, senile lentigines, and postinflammatory hyperpigmentation (Varagnat, 1981; CIR, 1986). It is used as a coupler in oxidative hair dyeing (CIR, 1986). In 1977, the use of hydroquinone in the USA was estimated to be as follows: photographic developers, 45%; antioxidants and polymerization inhibitors, 50%; other uses, 5%. Corresponding figures in western Europe were, respectively, 70%, 15% and 15% (Varagnat, 1981), and in Japan 30%, 50% and 20% for 1992 (personal communication from H. Naito, University of Tsukuba, to the IPCS in 1993). In 1981, hydroquinone was an ingredient of 147 hair dyes and colour preparations and 23 skin care products, including products intended for medical use as skin lighteners in the USA (CIR, 1986). Like hydroquinone, many of its derivatives are reducing agents and have a wide variety of applications. Hydroquinone derivatives that are used as rubber antioxidants and antiozonants include dialkylated hydroquinone, N-alkyl-p-aminophenol and diaryl- p-phenylenediamines. The main food antioxidants are butylated hydroxyanisole (BHA) and tert-butylhydroquinone. 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1 Transport and distribution between media A calculation of fugacity, according to Mackay's model level I (Mackay & Paterson, 1981), shows that hydroquinone will be distributed mainly to the water compartment when released in the environment. This was also concluded by Devillers et al. (1990). 4.2 Transformation 4.2.1 Biodegradation Biodegradation of hydroquinone is closely related to many variables such as pH, temperature and whether conditions are aerobic or anaerobic (Devillers et al., 1990). It also depends on the acclimation level of the microorganisms involved (Tabak et al., 1964; Harbison & Belly, 1982). Harbison & Belly (1982) investigated various pure cultures of microorganisms for their ability to utilize hydroquinone as sole carbon source. The pure cultures were isolated from soil, photographic sludge and laboratory sludge. When incubated with 750 mg/litre the isolates gave an average TOC (total organic carbon) removal of 97.5% in 5 days. After various incubation periods, the possible metabolites and end-products were analysed; 1,4-benzoquinone, 2-hydroxy-1,4-benzoquinone and ß-ketoadipic acid were detected as metabolites. None of the compounds persisted in the cultures. Neujahr & Varga (1970) proposed that the first step in the degradation of hydroquinone by Trichosporon cutaneum should be a hydroxylating step to hydroquinol. The ring fission should then probably result in ß-hydroxymuconate. The BOD5 (biological oxygen demand in 5 days)/COD (chemical oxygen demand) ratio, which is an indicator of biodegradability, has been reported to be 0.37 by Dore et al. (1975) and 0.53 by Young et al. (1968). This indicates that under aerobic conditions hydroquinone is readily biodegradable. Devillers et al. (1990) have summarized various metabolic pathways (Fig. 3). Young & Rivera (1985) studied the methanogenic degradation of hydroquinone. When the microbial community from a municipal sewage treatment plant digester was acclimated to hydroquinone, the rate of metabolism and gas formation increased. The rate of substrate metabolism was 23.6 ± 2.0 (n=6) with acclimated microorganisms compared to 5.7 ± 1.4 (n=6) mg/litre per day with non-acclimated organisms. The rate of gas production (CO2 + CH4) was 9.33 ± 1.7 and 5.70 ± 1.1 ml/litre culture fluid per day for acclimated and non acclimated organisms, respectively. Prior to mineralization hydroquinone was metabolized to phenol. The authors have summarized various anaerobic degradation steps and proposed the scheme in Fig. 4. Stoichiometrically the anaerobic bioconversion of hydroquinone is described as follows: C6H6O2 + 3.5 H2O -> 2.75 CO2 + 3.25 CH4 4.2.2 Abiotic degradation The photodegradation of hydroquinone has been discussed by Devillers et al. (1990). Due to its intrinsic properties hydroquinone is relatively readily degraded by means of photodegradation. Phototransformation may occur from direct excitation or from induced or photocatalytic reactions. Freitag et al. (1985) reported that when 62 ng hydroquinone adsorbed on silica gel was exposed to ultraviolet light (290 nm) for 17 h, 57.4% of the hydroquinone was mineralized. Tissot et al. (1985) measured changed toxicity due to phototransformation (Table 3). The phototransformation products were p-benzoquinone after 0.5 h and hydroxy p-benzoquinone after 4 and 22 h. Table 3. Photoirradiation of hydroquinone and toxicity to Daphnia magna measured as inhibition of motility after 24 h (from: Tissot et al., 1985) Initial Irradiation % EC50 (mg/litre HPLC analysis at the concentration time degradation initial end of the irradiation (h) concentration) period 67.1 mg/litre 0 0 0.15 (6.1 x 10-4 M) 0.5 15 0.2 p-benzoquinone 4 49 0.2 10-4 M hydroxy p-benzoquinone 22 80 0.5 1.4 x 10-4 M hydroxy p-benzoquinone 4.2.3 Bioaccumulation With a log n-octanol/water partition coefficient of 0.59 it can be considered that hydroquinone does not bioaccumulate. The bioconcentration factors found in the literature for static tests are listed in Table 4. Table 4. Bioaccumulation factors (BCF)a Species Test Hydroquinone BCF Comment duration concentration (days) (mg/litre) Activated sludge 5 0.05 870 dry weight basis Algae Chlorella fusca 1 0.05 40 wet weight basis Fish Leuciscus idus melanotus 3 0.05 40 wet weight basis a From: Freitag et al. (1985) 4.3 Interaction with other physical, chemical or biological factors Tratnyek & Macalady (1989) report on direct abiotic reductions of nitro groups from nitro aromatic pesticides to amines by hydroquinones. In homogeneous solutions of quinone-hydroquinone redox couples, which were selected to model the redox-labile functional groups in natural organic matter, rapid abiotic reduction of nitro aromatic pesticides occurred. The authors proposed that hydroquinones contribute to the reduction of pollutants in the environment, but their role is likely to be complex. The water hyacinth (Eichhornia crassipes), which is used for water treatment, clears more than 98% hydroquinone (50 mg/litre) after about 48 h (O'Keeffe et al., 1987). This property has been attributed to enzymatic metabolism by polyphenol oxidases. 4.4 Ultimate fate following use Hydroquinone occurs in photo-processing effluents (Dagon, 1973; Harbison & Belly, 1982). However, it is not certain that it reaches the water ecosystem, because reliable monitoring data are not available. 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air, soil and water No monitoring data have been found concerning ambient free hydroquinone concentrations in air, soil or water. However, hydroquinone has been identified in tobacco smoke and measured in mainstream smoke from non-filtered cigarettes at amounts ranging from 110 to 300 µg per cigarette, with a ratio of the sidestream to mainstream concentration of 0.7-0.9 (IARC 1986). 5.1.2 Food Free and conjugated (Arbutin) hydroquinone exist as natural components of a variety of plant-derived beverages and food products. Högl (1958) identified hydroquinone in the non-volatile extract of coffee beans. Hydroquinone concentrations in roasted coffee have been reported to range between 25 and 40 mg/kg (Maier, 1981). Gold et al. (1992) estimated that one cup of coffee would contain approximately 100 µg hydroquinone. Teas prepared from leaves of blueberry, cowberry, cranberry and bearberry have been reported to contain hydroquinone at concentrations sometimes exceeding 1% (Deichmann & Keplinger, 1981). The concentrations of free and total (free hydroquinone and Arbutin) hydroquinone have been measured in a variety of foods and beverages by Hill et al. (1993); results indicate that significant exposure to hydroquinone can occur through dietary sources. In most of the samples (Table 5) derived from plant sources, the levels of Arbutin are considerably higher than those of free hydroquinone. However, Arbutin is hydrolysed readily by dilute acids yielding hydroquinone and glucose. Therefore, both free hydroquinone and Arbutin may contribute to hydroquinone exposure from natural sources as well as to the daily intake of dietary antioxidants. Adhesives containing trace amounts of hydroquinone are permitted as a component of food packaging in the USA (FDA, 1981; 1991). Table 5. Concentrations of free and total hydroquinone in various foods and beverages Food sample Concentrations (mg/kg ± SD)a Free HQ Total HQb Wheat germ, toasted 0.591 8.352 Drip-brewed coffee (pre-ground) 0.293 ± 0.003 0.385 ± 0.016 Whole wheat bread (100% whole wheat) 0.584 ± 0.202 0.893 ± 0.480 Whole wheat cereal (commercially available) 0.205 ± 0.019 0.992 ± 0.161 Processed corn cereal (commercially available) bkgb bkg Pear skin (D'Anjou, fresh) bkg 38.057 Pear flesh (D'Anjou, fresh) bkg 1.301 Milkfat (2%) homogenized milk bkg bkg Yogurt (black cherry) bkg bkg Cantaloupe bkg bkg Diet cola 0.0362 0.0287 a bkg = background levels comparable to that observed in control blanks b Includes free hydroquinone and hydroquinone released following treatment of the samples with ß-glucosidase 5.2 General population exposure Photohobbyists, who develop their own black-and-white films (a process which utilizes hydroquinone) may be exposed dermally. Exposure to dust is also possible when preparing developer solutions. In 1980, the number of photohobbyists was estimated to be about 2.2 million in the USA (US EPA, 1985). There are no data on exposure levels. Dermal exposure to hydroquinone may also occur from products intended for cosmetic and medical use. In the USA, hydroquinone has been used in cosmetics, and in over-the-counter (OTC or non-prescription) and prescription drugs. Both OTC and prescription drugs are used to lighten areas of hyperpigmented skin. In cosmetics, concentrations of < 0.1% to 5% have been reported (CIR, 1986). OTC skin lighteners may contain up to 2% hydroquinone and prescription drugs may contain higher concentrations. In the EC countries, hydroquinone is restricted for use in cosmetics to 2% or less (Boyle & Kennedy, 1985). The US Food and Drug Administration has issued a Notice of Proposed Rule-making for the use of hydroquinone as a skin lightener in OTC drugs at concentrations below 1.5-2.0% (FDA, 1982). Skin-lightening creams containing hydroquinone are frequently inadequately labelled and the concentration often exceeds the limit of 2%; it is likely to be much stronger than 2% (Brauer, 1985; Godlee, 1992) and even up to 7% (Boyle & Kennedy, 1986). A 2% upper limit on hydroquinone concentration, set by the South African government in 1980 and followed by the United Kingdom and USA, was based on tests of cutaneous irritancy (Arndt & Fitzpatrick, 1965) and contact dermatitis (Bentley-Philips & Bayles, 1975). 5.3 Occupational exposure Hydroquinone can be encountered in solid form or in solution during its production and use (NIOSH, 1978). It has a very low vapour pressure, but can be oxidized in the presence of moisture to form quinone, which is more volatile. The saturated concentration in air for hydroquinone vapour under standard conditions is estimated to be 0.108 mg/m3 (approximately 0.024 ppm at 25 °C) (NIOSH, 1978). There are some industrial hygiene monitoring data available for hydroquinone. Oglesby et al. (1947) reported 20-35 mg hydroquinone/m3 in a packaging area without exhaust cabinet and 1-4 mg/m3 in a packaging area with exhaust cabinet in a plant manufacturing hydroquinone. However, the analytical methods did not distinguish between hydroquinone and quinone. Industrial data, provided to the US EPA (1985), indicated worker inhalation exposure due to closed production processes within one manufacturing facility at an arithmetical average concentration of 0.79 mg/m3 (± 0.52 standard deviation) and a highest average concentration of 0.2 mg/m3 in another facility. In the unloading area of a production facility the arithmetic average air concentration was reported to be 0.13 mg/m3 (± 0.15 standard deviation). The concentration of hydroquinone was measured in the workroom air in 12 Finnish plants (altogether 36 samples) during the period 1950-89 (Rantanen et al., personal communication to the IPCS, 1992). Most samples were collected in the printing industry (23 samples from five plants). The occupational exposure limit of 2 mg/m3 was exceeded in only one measurement: 9.5 mg/m3 during charging of hydroquinone in a gas plant in 1962, a short operation carried out once every three weeks. Approximately 470 000 workers in the USA are potentially exposed to hydroquinone in about 137 occupations (US EPA, 1985). Certain occupations in which hydroquinone exposure may occur are listed in Table 6. Some of the national occupational air exposure limits used in various countries are compiled in Table 7. Table 6. Occupations with potential exposure to hydroquinonea Antioxidant makers Drug makers Hair dressers and cosmetologists Hydroquinone manufacturing workers Paint makers Photo processors Organic chemical synthesizers Photographic developer makers Plastic stabilizer workers Rubber coating workers a From: Key et al. (1977); NIOSH (1978); NIOSH (1990) Table 7. National occupational air exposure limits used in various countries (from: IRPTC, 1987; ILO, 1991) Country TWAa STELb CLVc (mg/m3) (mg/m3) (mg/m3) Australia 2 Belgium 2 Denmark 2 Finland 2 4 France 2 Germany 2d The Netherlands 2 Poland 2 2 Romania 1 2 Sweden 0.5 1.5 Switzerland 2 4 United Kingdom 2 4 USA: ACGIH 2 NIOSH/OSHA 2 Yugoslavia 2 a TWA = time-weighted average; a maximum mean exposure limit based generally over the period of a working day b STEL = short-term exposure limit c CLV = ceiling level value d inhalable dust 6. KINETICS AND METABOLISM The main results pertinent to this chapter, with the exception of section 6.5, have been summarized in Table 8 and will be expanded upon only where necessary. Table 8 shows that the majority of studies have been performed in Fischer-344 rats. 6.1 Absorption Absorption of orally administered or intratracheally instilled hydroquinone is rapid and extensive (Garton & Williams, 1949; Divincenzo et al., 1984; English et al., 1988). However, the rate of hydroquinone absorption through the skin is low. Marty et al. (1981) reported that the in vitro permeability constants for rat and human skin were 28 x 10-6 and 4 x 10-6 cm/h, respectively. Based on the data of Bucks et al. (1988), an in vivo human dermal absorption rate of 3 µg/cm2.h and a permeability constant of 2.25 x 10-6 cm/h can be calculated. The actual amount of hydroquinone absorbed following dermal exposure depends on the exposure concentrations, length of exposure and vehicle, as well as other factors. When Bucks et al. (1988) applied 14C-labelled hydroquinone in an alcoholic vehicle to the foreheads of human volunteers for 24 h, 57% of the total 14C label was excreted in the urine after 5 days. Addition of a sun screen to the hydroquinone solution reduced total excretion to 26%. 6.2 Distribution Following the oral administration of radiolabelled hydroquinone to F-344 rats, radioactivity was widely distributed throughout the animal tissues. The highest activity was localized in the kidney and liver (Divincenzo et al., 1984). However, on a quantitative basis, the amount retained within the animal was low, representing < 2% of the total dose 48 h after exposure (Divincenzo et al., 1984; English et al., 1988). Widespread distribution and extensive elimination was also observed following intratracheal administration of hydroquinone to F-344 rats (Lockhart & Fox, 1985b). However, following the intravenous injection of radiolabelled hydroquinone to F-344 rats, radioactivity was shown, using whole body autoradiographic techniques, to concentrate in the bone marrow, thymus and white pulp of the spleen (Greenlee et al., 1981a). Subsequent experiments indicated that significant acid soluble and covalently bound radioactivity could be recovered in the thymus, bone marrow and white blood cells 24 h after intravenous administration (Greenlee et al., 1981b). These results indicate that the route of administration may influence the profile of distribution and elimination observed following hydroquinone administration. Table 8. Summary of toxicokinetic data for hydroquinone (HQ) Species and Absorption Distribution Metabolic Elimination and Reference treatment transformation excretion Oral administration Species: rabbits less than 1% of the dose Garton & Treatment: 3-6 rabbits was excreted unchanged; Williams received 100 or 200 mg/kg about 80% of the dose was (1949) HQ as a single dose; urine recovered as glucuronide metabolites analysed after and monosulfate conjugates 24 h in the urine Species: Sprague-Dawley T1 & T2: rapid and T1 & T2: for 200 mg/kg the major radio- mainly in the urine; Divincenzo rats (m) extensive based 0.28-1.25% and labelled species in elimination for T1 and et al. Treatment: (T1) 2-4 rats upon urinary 0.26-0.56% of the urine were: HQ T2 was similar; after (1984) per group received 5, 30 excretion administered radioactivity monoglucuronide, HQ 48 h around 95% of or 200 mg/kg [14C]-HQ as a recovered in carcass monosulfate and HQ dose had been excreted single dose; rats were and tissues after 48 h (T1: 56%, 42% and in urine (90%), faeces (4%) observed for 48 and 96 h and 96 h, respectively; 1%; T2: 72%, 23% and CO2 (0.4%); no before sacrifice. widely distributedand 1%) difference in elimination (T2) 4 rats pretreated with throughout tissues between single and 200 mg/kg unlabelled HQ with highest concentration repeated doses once a day for 4 days in liver and followed by 200 mg/kg kidney [14C]-HQ on day 5; rats observed for 48 h Table 8. (contd). Species and Absorption Distribution Metabolic Elimination and Reference treatment transformation excretion Species: Fischer-344 T1 & T2: rapid and T1 & T2: < 1% of T1 & T2: the major Excreta English rats (m,f) extensive based administered radiolabelled species T1 & T2: mainly excreted et al. Treatment: (T1) 8 (m,f) upon peak blood radioactivity recovered in the urine were in the urine; after 48 h (1988) rats per group received 25 concentration in carcass and HQ monoglucuronide around 90% of the or 350 mg/kg [14C]-HQ as within 0.8 h of tissues for each dose (44-54%), HQ mono- administered radioactivity a single dose. dosing and urinary after 48 h; twice as sulfate (19-33%), was recovered as urine (T2) 8 rats (m,f) excretion; much radioactivity HQ (0.25-7%), HQ (approx.78%), cage rinse pretreated with 25 mg/kg no sex recovered in the mercapturate (approx.12%) and faeces unlabelled HQ once a day differences liver and kidney (0.16-4.68%) and (approx.2.2%); dose-related for 14 days followed by 25 of females compared p-benzoquinone differences were observed mg/kg [14C]-HQ on day 15; with males (0.24-0.84%); no at 8 h, 54% (m) and 45% (f) after oral administration sex difference of the dose was excreted 4 rats per dose and renally by the high-dose sex were designated group compared with 81% (m) for blood collection and 82% (f) for the low- samples (up to 96 h) dose group and for excreta and radiodistribution Blood kinetics (up to 7 days) AUC values were increased by 17-fold (m) and 26-fold (f) for a 13- and 14-fold higher mean dose; most of radioactivity was excreted by 8 h and was associated mainly with alpha- elimination phase (T´ = 0.23-1.72 h); accurate ß T´ could not be determined because of the appearance of a second peak in the blood concentration versus Table 8. (contd). Species and Absorption Distribution Metabolic Elimination and Reference treatment transformation excretion Species: Fischer-344 rapid and extensive low distribution, by 8 h, major time curve by 24 h, Lockhart rats (m,f) absorption as i.e. less than 1%; metabolites found recovery was more than 92% et al. Treatment: a single dose indicated by marked no significant in urinewere HQ in urine, approx.2% in (1984); of 5, 25 or 50 mg [U-14C]- recovery of [14C] differences between glucuronide faeces and less than 0.2% Lockhart HQ/kg body weight by in urine by 24 h sexes (approx.50%), in CO2 & Fox gavage HQ sulfate (1985a) (approx.30%) and HQ (approx.2%); neither dose- nor sex-dependent Species: Fischer-344 rapid and extensive low distribution: by 8 h, major by 24 h, recovery was Lockhart rats (f) absorption as approx. 0.55% in liver and metabolites found in approx.92% in urine, & Fox Treatment: 5 rats per dose indicated by marked 0.64-0.9% in carcass urine were HQ approx.2.6% in faeces and (1985a) group 5, 25 or 50 mg recovery of [14C] glucuronide (approx. approx.0.3% in CO2 [U-14C]-HQ/kg body weight in urine by 24 h 46%), HQ sulfate (single gavage dose) (approx.29-36%) and HQ (approx.2.5%) Dermal administration In vitro: Rat or human overall absorption Marty et skin biopsy; repeated and permeability al. (1981) dosing with 40 mg/cm2 in constant were low water; observed for 24 h but on average 7-fold greater for rat than human skin In vivo: Mouse or rat absorption by local cutaneous combined urine and faecal mouse was low; distribution was high elimination was low; 1.6% after 6 h in rat approx.10% after 96 h in the rat Table 8. (contd). Species and Absorption Distribution Metabolic Elimination and Reference treatment transformation excretion Species: human average percutaneous Bucks et Treatment: 6 normal adult taneous absorption al. (1988) male volunteers had 2% estimated from (w/w) HQ in ethanol urinary elimination (approx.70%) plus 0.2% data was 57% after ascorbic acid applied to 120 days; sun- their foreheads for 24 h; screens decreased single dose = 125 µg/cm2; absorption but observed for up to 120 h penetration enhancers were without effect Species: Fischer-344 skin irritated but after 1 week, 15-18% HQ English rats (m,f) poorly absorbed; was recovered in urine and et al. Treatment: 8 (m,f) rats large interanimal cage rinsings, 1.7-3.7% in (1988) per group were dermally variation in the faeces, 2.6 to 12.9% exposed for 24 h to 25 disposition; removal in the body and 0.14 to or 150 mg/kg [14C]-HQ of HQ after skin 2.2% in the excised dissolved in distilled washing with soapy skin exposure site water for 24 h water was close to 100% after 10 min of exposure or around 65% after 24 h of exposure Table 8. (contd). Species and Absorption Distribution Metabolic Elimination and Reference treatment transformation excretion Intravenous administration Species: Fischer-344 rat (m) whole body Greenlee Treatment: 1.3 mg/kg autoradiography showed et al. [14C]-HQ in saline that [14C] concentrated (1981a) administered as a single most in the white pulp dose; one group of rats of the spleen, bone marrow was pretreated with and thymus; Aroclor 1254 Aroclor 1254 (250 mg/kg pre-treatment decreased i.p.) the tissue/blood optical density by approx.60% for the thymus and bone marrow Species: Fischer-344 rat (m) acid-insoluble Greenlee Treatment: rats received radioactivity associated et al. 14 mg/kg [14C]-HQ as a with protein increased (1981b) single administration; one with time in the bone group of rats was marrow > thymus > liver; pretreated with Aroclor pretreatment with Aroclor 1254 resulted in a significant decrease in the radioactivity measured in the bone marrow Intratracheal instillation Species: Fischer-344 rapid and extensive < 0.13% in lung, less by 8 h, major by 48 h recovery was Lockhart rats (m) absorption as than 1% to other organs metabolites recovered more than 92% in urine, & Fox Treatment: 5 rats per dose indicated by in the urine were approx.2% in faeces and (1985b) group: 5, 25 or 50 mg recovery in urine HQ-glucuronide less than 0.2% in CO2 [U-14C]-HQ/kg; 2 rats per within 24 h (approx.50%), control group HQ-sulfate (approx.30%) and HQ (approx.2%) Table 8. (contd). Species and Absorption Distribution Metabolic Elimination and Reference treatment transformation excretion Intraperitoneal administration Species: Wistar rat (f) metabolites recovered elimination was rapid with Inoue et Treatment: 9 rats received in urine were 1,2,4- 84% of the metabolites al. a single 50 mg/kg dose benzenetriol (11%), recovered within 4 h after (1989a) catechol (1%) and administration; by 24 h, hydroquinone (87%) recovery of 1,2,4- benzenetriol, catechol and hydroquinone in the acid- hydrolysed urine comprised 38% of the administered dose Species: Japanese white metabolites recovered by 24 h, recovery of Inoue et rabbits in urine were 1,2,4- 1,2,4,-benzenetriol, al. Treatment: 5 rabbits benzenehydrotriol catechol and quinone in (1989b) received a single 50 mg/kg (12%), catechol (1%) the acid-hydrolysed urine dose and hydroquinone comprised 40% of the (86%) administered dose 6.3 Metabolic transformation Hydroquinone is converted mainly by Phase II metabolism to water-soluble conjugates, as shown by the recovery of only little parent compound and p-benzoquinone (0.25-7%) but large amounts of hydroquinone-monoglucuronide and hydroquinone-monosulfate (>90%) in the urine (Divincenzo et al. 1984; English et al. 1988). A small percentage of the dose was recovered as the mercapturic acid conjugate of hydroquinone, suggesting the intermediate formation of a glutathione conjugate of hydroquinone. Divincenzo et al. (1984) demonstrated that repeated dosing with 200 mg hydroquinone/kg did not alter the relative or absolute rat liver weight or induce the hepatic mixed-function oxidase system, nor did hydroquinone undergo Phase I oxidation to other metabolites such as 1,2,4-trihydroxybenzene. In addition, the formation of 1,2,4-trihydroxybenzene was not observed in the urine after oral administration of hydroquinone to rabbits (Garton & Williams, 1949). However, following intraperitoneal injection of hydroquinone (50 mg/kg) in Wistar rats and Japanese white rabbits, 1,2,4-trihydroxybenzene represented a significant proportion (approximately 12%) of the metabolites recovered in the urine (Inoue et al., 1989a,b). This apparent difference in the metabolic profile observed when hydroquinone is administered by the intraperitoneal route rather than the oral route is probably related to the efficient ability of the gastrointestinal system to conjugate phenolic compounds absorbed in the intestine, thus reducing the amount of free hydroquinone available for Phase I metabolism in the liver (Powell et al., 1974; Cassidy & Houston, 1980a,b; Cassidy & Houston, 1984). Fig. 5 shows proposed metabolic pathways for hydroquinone biotransformation in Fischer-334 rats. 6.4 Elimination and excretion Hydroquinone is excreted mainly in the form of water soluble metabolites via the urine (about 90%). Dose-related differences have been observed for rats receiving 25 or 350 mg/kg, which suggests that elimination processes are saturated at high-dose levels (English et al., 1988). The area under the curve (AUC) values for plasma concentration, which provide an index of bioavailability, also showed that saturation of elimination had occurred at high-dose levels, particularly for females. The fact that most of the radioactivity excreted is associated with the alpha-elimination phase suggests that this may be due to conjugation of hydroquinone to readily excreted metabolites. The appearance of a double peak in the blood concentration versus time curve indicates that enterohepatic recycling of hydroquinone may have occurred. 6.5 Reaction with body components The available studies suggest that hydroquinone derivatives are responsible for many of the toxicological effects associated with in vivo and in vitro hydroquinone exposure. Hydroquinone itself may be responsible for the acute CNS signs (tremors and convulsions) that are seen within the first hour following hydroquinone exposure (see section 7.8.3), since the signs appear soon after exposure when significant metabolism has probably not occurred. However, it is possible that derivatives even have a role in inducing CNS effects. The derivatives formed from hydroquinone may differ between in vivo and in vitro studies. Even when the in vivo situation alone is considered, the derivatives may vary qualitatively and quantitatively, and the concentrations of derivatives in the various body compartments may be different depending on the route of exposure. When hydroquinone is administered by expected routes of exposure, the primary derivatives should be largely glucuronide and sulfate conjugates, which are quickly exported, as well as glutathione conjugates, which may represent activated metabolites. When hydroquinone is given by intraperitoneal or intravenous routes, the primary metabolites are expected to be 1,4-benzoquinone and 1,2,4-trihydroxybenzene. In most in vitro systems the primary metabolite is expected to be 1,4-benzoquinone. Hydroquinone-and oxygen-derived radical species are also likely to be formed both in vivo and in vitro. The hydroquinone-derived metabolites and radical species formed in vitro will depend on the oxygen content, the pH, the ionic strength, the autooxidant and the protein content of the culture or reaction medium used in the study, as well as other factors including the metabolic capacity of the test system. The differences in the potential derivatives and the concentrations of the derivatives occurring in the different in vivo and in vitro exposure systems studied indicate that extrapolations from in vitro to in vivo systems and between routes of exposure need to be made with a great deal of care. The main results pertinent to this section have been summarized in Table 9, which shows that many of the interactions of hydroquinone have been identified in vitro but not all have been demonstrated in vivo. Hydroquinone reacts with many different biological components, including macromolecules such as protein, DNA, tubulin, lipids, and low molecular weight molecules such as sulfydryls and nucleotides, is toxic to different cell types, has affects on cellular metabolism, and modulates enzyme activities. Covalent binding and oxidative stress are mechanisms postulated to be associated with hydroquinone-induced toxicity. Both oxidized hydroquinone species ( p-benzosemiquinone radical and p-benzoquinone) and thiol-hydroquinone/quinone conjugates are believed to contribute to hydroquinone toxicity. Oxidized hydroquinone derivatives can covalently bind cellular macromolecules or alkylate low molecular weight nucleophiles, e.g., glutathione (GSH), resulting in enzyme inhibition, alterations in nucleic acids and oxidative stress; however, redox cycling is not likely to contribute significantly to oxidative stress in contrast with other hydroquinones and quinones (see section 2.2; Rossi et al., 1986; O'Brien, 1991). The reaction of benzoquinone with GSH results in the formation of GSyl conjugates which can be processed to cysteine conjugates. These latter thiol conjugates have been speculated to mediate cellular toxicity in the kidney by alkylation and/or oxidative stress, possibly involving redox cycling (Lau et al., 1988). Table 9. Summary of the reactions of hydroquinone (HQ) with biological componentsa Index studied Method Result Reference Reactions with macromolecules Covalent binding to 14 mg/kg [14C]-HQ was administered acid-insoluble radioactivity associated with Greenlee et al. cell protein i.v. as a single dose to Fischer-344 rats protein increased with time in the bone (1981b) (in vivo) (m); after 2 and 24 h, acid-insoluble marrow > thymus > liver; pretreatment with radioactivity associated with proteins Aroclor resulted in a significant decrease in was determined; one group of rats was the radioactivity measured in the bone marrow pretreated with Aroclor 1254 Covalent binding to 25-75 mg/kg [14C]-HQ was incubated radioactivity associated with protein; the Eastmond et al. boiled rat liver in the absence or presence of phenol presence of PhOH enhanced this association (1987) protein (in vitro) (PhOH) (75 mg/kg) with H2O2-horseradish peroxidase or freshly isolated human polymorphonuclear leucocytes in the presence of boiled rat liver protein Covalent binding to 75 mg/kg [14C]-HQ alone or coadministered after 18 h of administration, acid-insoluble Subrahmanyam et cells (in vivo) with phenol (PhOH) (75 mg/kg) radioactivity was found associated with al. (1990) was administered i.p. (probably as a kidney > blood > bone marrow; coadministration single dose) to pathogen-free male with PhOH significantly (statistically) B6C3F1 mice (5-12); after 4 and 18 h, increased binding to blood and bone marrow acid-insoluble radioactivity associated but not kidney or liver with macromolecules was isolated and analysed for covalent binding Covalent binding to isolated liver microsomes from male radioactivity associated with microsomal Wallin et al. microsomal proteins S-D rats, either treated or untreated with proteins; binding was more extensive than (1985) (in vitro) phenobarbital or 3-methyl cholanthrene, that of phenol and independent of electron was incorporated with [14C]-HQ, both with donors and without NADPH Table 9. (contd). Index studied Method Result Reference Covalent binding to peritoneum macrophages isolated from [14C]-HQ was activated by macrophages to Schlosser et cells (in vitro) male C57BL/6 mice were incorporated metabolites that bind irreversibly to protein; al. (1989); with [14C]-HQ activation was inhibited by peroxidase inhibitor Schlosser & aminotriazine and the nucleophile cysteine and Kalf (1989) enhanced by arachidonic-acid-mediated prostagladin synthesis catalysed reaction Covalent binding to bone marrow macrophages and a fibroblastoid radioactivity associated with macrophages was Thomas et al. cells (in vitro) stromal cell (LTF) line obtained 16-fold higher than for LTF cells; DT- (1989); Ross et from male B6C3F1 mice were incorporated diaphorase activity [Q -> HQ] was 4 times al. (1990) with [14C]-HQ higher on LTF cells than in macrophages; slightly decreased (approx.16%) by addition of dicoumarol, an inhibitor of DT-diaphorase, for LTs but not macrophages Chromosomal aberration example: bone marrow cells were isolated micronuclei induced in polychromatic Tunek et al. (in vivo) (see also from male NMRI mice (4 per group) erythrocytes (1982) section 7.6) administered between 20 and 100 mg HQ/kg by subcutaneous injection once a day for 6 days Mitochondrial DNA mitoplasts isolated from rabbit bone covalent adduct formed with guanine Rushmore et (in vitro) marrow cells were prelabelled with al. (1984) [3H]-dGTP incorporated with [14C]-HQ and assayed for guanosine adduct formation DNA damage (in vitro) example: calf thymus DNA was incubated two adducts identified Jowa et al. (see also section 7.6) with [14C]-HQ in the presence of Fe3+ at (1990) pH 7.2 Microtubulin binding T1: isolated brain microtubulin from male T1: HQ inhibited microtubulin polymerization Irons & Neptun (in vitro) Fischer-344 rats was incubated with between and bound to high molecular weight tubulin; (1980) 1 and 1.5 x 10-4 mol/litre [14C]-HQ anaerobic conditions inhibited polymerization Table 9. (contd). Index studied Method Result Reference T2: isolated spleen lymphocytes from rat T2: HQ suppressed lectin-induced blastogenesis Pfeifer & Irons were incubated with HQ (10-6-10-4 mol/litre) and concomitant inhibition of cell (1983) agglutination Lipids (in vivo) SD rats received a single oral dose of 100 urinary MDA increased in HQ-treated rats Ekström et or 200 mg HQ/kg; malondialdehyde (MDA), al. (1988) a lipid peroxidation product, was analysed in the excreted urine for up to 18 h Cytochrome c3+ stop- and continuous-flow experiments HQ reduces cytochrome c3+ via Yamazaki & Ohnishi reduction (in vitro) p-benzosemiquinone; reaction accelerated by (1969); Ohnishi p-benzoquinone et al. (1969) Reactions with low molecular weight molecules Thiol conjugation glutathione thiol conjugates formed by reductive addition; Tunek et al. (in vitro) monothiol HQ conjugate formed by the reaction (1980) of p-benzoquinone with thiol after oxidation of HQ to the semiquinone or quinone glutathione di, tri and tetra (GSyl)-HQ conjugates are Eckert et al. formed by reductive addition of the oxidized (1990) (GSyl)-HQ conjugate, i.e. quinone conjugate with GSH monocysteine-HQ conjugate HQ oxidized by prostaglandin H systhetase Schlosser et al. (1990) Nucleotide adduct [3H]-deoxyguanosine and [14C]-HQ two doubly labelled products isolated; adduct Jowa et al. (in vitro) incubated in the presence of Fe3+ at pH 7.2 2: 3-OH benzethano (1, N2) deoxyguanosine (1990) 2-Thiobarbituric glutamate or deoxyribonucleic acid 2-thiobarbituric acid produced; hydroxyl radical Rao & Pandya acid (in vitro) incorporated with HQ plus Cu2+ at pH 7.4 (OH€) formation thought to be involved (1989) Table 9. (contd). Index studied Method Result Reference Toxicity to cells Erythrocytes (in vivo) polychromatic erythrocytes isolated from 20 mg/kg: no haemotoxic effect; 100 mg/kg: Tunek et al. male NMRI mice (4 per group) haemotoxic effects (suppressed bone marrow (1982) administered between 20 and 100 mg cellularity) HQ/kg s.c. once a day for 6 days Bone marrow (in vivo) i.p. administration of HQ (100 mg/kg, twice transient, mild suppression in bone marrow Eastmond et daily for 12 days to six male B6C3F1 mice) cellularity al. (1987) i.p. co-administration of HQ (25-75 mg/kg) significant decrease in bone marrow cellularity; Eastmond et and phenol (75 mg/kg) twice daily for phenol enhanced HQ-induced myelotoxicity al. (1987) 12 days to groups of six male B6C3F1 mice Isolated rat spleen responses of spleen cells from F-344 rats low concentrations (10-7-10-6) enhanced Irons & Pfeifer and lymphocytes were assayed after addition of mitogen and mitogenesis, higher concentrations (10-5) (1982) (in vitro) phytohaemagglutinin A suppressed mitogen response Pigment cells (in vitro) toxic effects of HQ on melanotic cell lines toxic effects occurred between 0.625 and HU (1966) (MCL) and non-melanotic cell lines (NMCL) 2.5 µg/ml for MCL and NMCL was studied Cell line (in vitro) lymphoma-derived cell line Raji, erythro- percentage survival decreased for all cells Picardo et al. leukaemia cell line K 562 and human (approx. 65%, low dose) (approx. 20%, high dose) (1987) melanotic cell lines IRE 1 and IRE 2 were incubated with 0.01 and 0.1 mmol HQ/litre Bone marrow cells bone marrow cells isolated from the femurs HQ decreased the number of mature surface King et al. (in vitro) and tibias of male B6C3F1 (C57BL/6J x IgM+ B cells and adherent cells; HQ may block (1987) C3h/HeJ) mice were incubated with final maturation stages of B cell between 10-7 and 10-5 mol HQ/litre differentiation Table 9. (contd). Index studied Method Result Reference Cell line (in vitro) bone marrow macrophage and a fibroblastoid HQ (10-4 mol/litre) decreased viability and Thomas et al. stromal cell line isolated from male colonies for macrophages (60% and 70%, (1989b) B6C3F1 mice were incubated with respectively) and stromal cells (30% and between 10-8 and 10-4 mol HQ/litre 0%, respectively) Cell line (in vitro) bone marrow stromal cells isolated from HQ cytotoxicity was greater in stromal cells Twerdok & Trush male DBA/2 mice and C57BL/6 mice derived from DBA/2 than C57BL/6 mice; tert- (1990); Twerdok were incubated with HQ butylhydroquinone (tBHQ) or 1,2-dithiole-3- et al. (1992) thione (DTT) preincubation protected against HQ-induced toxicity; dicoumarol-sensitive quinone reductase activity was increased by tBHQ and DTT, and levels of GSH increased with DTT Cell line (in vitro) human promyelocytic leukaemia cell line, HQ dose-dependently inhibited TPA- and 1,25- Oliveira & Kalf which can be induced to differentiate to dihydroxy vitamin D3-induced (but not (1992) both monocyte and myeloid cells, was interleukin (IL)-1) acquisition of incubated with HQ (0.01 µmol/litre to differentiation characteristics of monocytes 10 µmol/litre) (adherence, nonspecific esterase activity and phagocytosis), but had no effect on cell proliferation; retinoic-acid- or DMSO-induced differentiation to granuloctyes was not inhibited at the same doses Hepatocytes (in vitro) freshly isolated hepatocytes (106/ml) time-dependent cell death; complete by 120 min O'Brien (1991) incubated with 850 µM HQ; % cell viability measured by Trypan blue inclusion Effects on cellular metabolism Haemoglobin (Hb) five cats were treated every second day up 15-30% Hb oxidized to ferric form Jung & Witt (in vivo) to 12 times with HQ (40-160 mg/kg) (1947) Table 9. (contd). Index studied Method Result Reference Reduction of haemoglobin HQ incubated with Hb in presence of O2 very slow formation of ferrihaemoglobin Oettel (1936) (Hb) (in vitro) Iron utilization female Swiss albino mice administered inhibition (70%) of erythroid 59Fe utilization Guy et al. (in vivo) between 25 and 100 mg HQ/kg 3 times at occurred only at the highest dose; (1990, 1991) 64, 48 and 40 h prior to administration coadministration of PhOH or muconaldehyde of 59Fe; coadministration with phenol enhanced the inhibitory effects of HQ (PhOH) (50 mg/kg) Cellular RNA and DNA the effects of HQ on nucleoside HQ selectively inhibited the metabolism of MCL Pennay et al. synthesis (in vitro) incorporation in two melanotic cell lines cf. NMCL; [3H]-uridine incorporation was de- (1984) (MCL) and three non-melanotic cell lines creased approx. 30-fold in MCL cf NMCL; [3H]- (NMCL) was observed uridine incorporation was more sensitive to HQ than [3H]-thymidine; DNA and RNA syntheses were decreased by 80 and 50%, respectively, in MCL; HQ may exert depigmenting effect by selective action on MCL metabolism rather than specific effect on melanin synthesis Mitochondrial synthesis mitoplasts isolated from rabbit bone RNA synthesis inhibited (IC50 = 5.0 x 10-5 mol Rushmore marrow cells were incorporated with HQ HQ/litre) et al. (1984) and assayed for RNA synthesis Cyclic nucleotides three different melanomas were treated cAMP and cGMP were elevated in 3/3 and 2/3 Abramowitz & (in vitro) with HQ and assayed for cAMP and cGMP tumours, respectively Chavin (1980) by radioimmuno assay Cytokine synthesis murine P388D1 macrophages or bone HQ caused a concentration-dependent inhibition Renz et al. marrow stromal macrophages were of the processing of 34-Kd pre-interleukin-1 (1991) incubated with HQ (0.5-10 µmol/litre) alpha (IL-1alpha) to 71-Kd mature cytokine in both types of macrophages; lipopolysaccharide- induced production of the pre-IL-1alpha precursor or cell viability or DNA and protein synthesis were not inhibited Table 9. (contd). Index studied Method Result Reference Effects on enzymes Tyrosine-tyrosinase radiometric assay tyrosinase inhibited by HQ (9 x 10-4 mol/litre); Usmani et al. (tyrosine --> dopa) suggested that HQ is a competitive inhibitor (1980); Palumbo (required for skin et al. (1991) pigmentation) Catalase (in vivo) male Wistar rats received 5 mg HQ/kg per 5 mg HQ/kg per day inhibits catalase activity in Vladescu & day p.o. for 10 days and H 18R tumour- the liver, spleen, blood and H 18R tumour Apetroae (1983) bearing rats received 5 mg HQ/kg per day p.o. for 7 days; [14C]-HQ was administered i.p. Ornithine decarboxylase SD rats received a single oral dose of 100 ODC activity was increased in a dose- Ekström et al. (ODC) or 200 mg HQ/kg; liver ODC activity was dependent manner (1988) measured after 18 h of exposure Horseradish peroxidase [14C]-phenol incubated with H2O2-HRP in radioactivity associated with protein Eastmond et (HRP) the presence of boiled rat liver protein; decreased when coincubated with HQ; al. (1987) coincubated with HQ competitive inhibition presumed Table 9. (contd). Index studied Method Result Reference Reaction of HQ metabolites Benzoquinone (BQ)/ numerous: BQ/SQ formed by autooxidation rate of enzyme-mediated formation of BQ is Yamazaki et al. benzosemiquinone (SQ) and 1e-mediated enzymic oxidation (e.g., much faster than autooxidation; formation of (1960); Sawada horseradish peroxidase (HRP), BQ is enhanced by coincubation of HRP and et al. (1975); myeloperoxidase (MPO) and prostaglandine MPO with phenol and other compounds; O'Brien (1991); H synthase (PHS)) of HQ indomethacin partially inhibits H2O2-dependent Smith et al. HQ oxidation with HRP or MPO or PHS, but (1989); Hsuanyu substantially inhibits arachidonate-dependent & Dunford (1992); oxidation mediated by PHS; this latter finding Eastmond et al. indicates the involvement of cyclooxygenase; (1987); Thomas both BQ and SQ are electrophiles which readily et al. (1989); react with low and high molecular weight Subrahmanyam et nucleophiles; BQ is believed to be the al. (1991); proximate toxicant responsible for benzene-/ Schlosser hydroquinone-induced myelotoxicity et al. (1990) GSH conjugate (in vivo) male Sprague-Dawley rats were administered nephrotoxicity occurred with tris-(GSyl)-HQ > Lau et al. various (GSyl)-HQ conjugates i.v.; di-(GSyl)-HQ conjugate; mono and tetra (1988) nephro- and hepatotoxicity were measured conjugates were not toxic; toxicity of the tris as increased plasma blood urea nitrogen conjugate was depressed by AT-125, a gamma (BUN) and serum glutamate pyruvate glutamyl trans-peptidase (gamma-GT) inhibitor; transaminase (SGPT), respectively it is suggested that gamma-GT is probably required for the transport of the latent quinone into proximal tubular cells as the corresponding cysteine conjugate; alkylation and/or oxidation are suggested mechanisms of molecular toxicity Table 9. (contd). Index studied Method Result Reference GSH conjugate (in vitro) O2 consumption measurement of in situ autooxidation was stimulated by SOD Brunmark & formed glutathionyl-p-benzoquinone HQ Cadenas (1988); cf. conjugate in potassium phosphate buffer Eckert et al. at pH 7.65 in the absence and presence of (1990) superoxide dismutase (SOD) a NADPH = reduced nicotinamide adenine dinucleotide cAMP = cyclic adenosine monophosphate cGMP = cyclic guanosine monophosphate 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO SYSTEMS 7.1 Single exposure The acute toxicity of hydroquinone has been studied in several animal species (Tables 10 and 11). Oral LD50 values for different strains of rats range from 720 to 1300 mg/kg body weight (Carlson & Brewer, 1953; Mozhaev et al., 1966). Fasting the animals for 18 h previous to the administration of hydroquinone produced a two- to three-fold increase in the observed toxicity (Carlson & Brewer, 1953). The LD50 of unfed rats was 310 mg/kg, in contrast to an average of 1064 mg/kg observed in non-fasted animals. Oral LD50 values range from 340 mg/kg to 400 mg/kg body weights for mice, and are 550 mg/kg body weight for guinea-pigs, 540 mg/kg body weight for rabbits and 299 mg/kg body weight for dogs. Cats have a greater sensitivity (LD50 values of 42-86 mg/kg body weight). Signs of hydroquinone intoxication were developed 30-90 min after single oral doses and consisted of hyperexcitability, tremors, convulsions, dyspnoea and cyanosis. They also included salivation in dogs and cats, emesis in dogs and pigeons, swelling of the tissues around the eyes, and incoordination of the hind limbs of dogs (Woodard 1951; Christian et al., 1976; Deichmann & Keplinger, 1981). These signs were followed by complete exhaustion, hypothermia, paralysis, loss of reflexes, coma, respiratory failure and death. When the dose was sublethal, recovery was complete within three days (Christian et al., 1976). Single-dose acute dermal toxicity studies have not been reported. However, the acute dermal LD50 can be estimated to be > 3840 mg/kg for mice and 74 800 mg/kg for rats based on effects observed in two-week dermal studies (NTP, 1989). Hydroquinone as a 2% solution in dimethyl phthalate caused no adverse local or systemic effects in rabbits (Draize et al., 1944). In the rat, the LD50 values for intraperitoneal administration varied between 160 and 194 mg/kg body weight and the LD50 for intravenous administration was 115 mg/kg body weight (Woodard, 1951). In rabbits, intravenous injections of hydroquinone caused death at doses of 100-150 mg/kg body weight (Delcambre et al., 1962). Table 10. Acute oral toxicity of hydroquinone in experimental animals Species Material tested LD50 (mg/kg Comments Reference body weight) Rat 2% aqueous solution 320 rapid onset of symptoms: twitchings, Woodard (1951) Osborne-Mendel tremors, convulsions, respiratory failure and death within a few hours Rat Priestly glycerine 1005-1295 unfasted rats; the mean LD50 value Carlson & Brewer (1953) Sprague-Dawley propylene-glycol 1090 was 1050 mg/kg Sprague-Dawley distilled water 1182 Sprague-Dawley glycerine 1081 Wistar propylene-glycol 731 Sprague-Dawley propylene-glycol 323 fasted rats; the mean LD50 value Carlson & Brewer (1953) Wistar 298 was 310 mg/kg Rat water 743 (m) when the dose was sufficiently large, Christian et al. (1976) 627 (f) death occurred during a severe tonic spasm within 2 h; when the dose was sublethal, recovery was complete within 3 days Mouse 2% aqueous solution 400 the symptoms are similar to those Woodard (1951) Swiss in rats Guinea-pig 2% aqueous solution 550 the symptoms are similar to those Woodard (1951) in rats Cat 2% aqueous solution 70 the symptoms are similar to those in Woodard (1951) rats except for salivation and swelling of the area around the eye noted in cats Cat sugar-coated tablets 42-86 Carlson & Brewer (1953) Table 10. (contd). Species Material tested LD50 (mg/kg Comments Reference body weight) Dog sugar-coated tablets 299 similar symptoms to those of cats Carlson & Brewer (1953) Dog 2% aqueous solution 200 hyperexcitability, tremors, convulsions, Woodard (1951) salivation, emesis, incoordination of the hind legs, respiratory failure, and death; 100 mg/kg caused mild to severe swelling of the area around the eye, of the nictating membrane and of the upper lip Rabbit 2% aqueous solution 540 the symptoms are similar to those in rats Woodard (1951) Pigeon 2% aqueous solution 300 the symptoms are similar to those in Woodard (1951) rats, except for emesis noted in pigeons Table 11. Acute parenteral toxicity of hydroquinone in experimental animals Species Material Administration LD50 (mg/kg body Comments Reference tested route weight) (except where otherwise stated) Rat (Osborne-Mendel) 2 or 3% aqueous intraperitoneal 160 Woodard (1951) solution Rat (Wistar) intraperitoneal 194 Delcambre et al. (1962) Rat (Osborne-Mendel) 2 or 3% aqueous intravenous 115 Woodard (1951) solution Rabbit intravenous 150a death Delcambre et al. (1962) 100 tremor, hypotonia, death 10-20 hypertension, hyperkalaemia Mouse subcutaneous 160-170 (LLD)b Busatto (1940) Mouse 1% solution subcutaneous 182 Marquardt et al. (1947) Mouse subcutaneous 190 most animals died within 24 h Nomiyama et al. (1967) a Single dose b LLD = lowest lethal dose Single subcutaneous injections of up to 500 mg of hydroquinone/kg body weight in white mice caused symptoms in the central nervous system: markedly increased motor activity, hyperactive reflexes, sensitivity to light and sound, laboured breathing and cyanosis, followed by clonic convulsions, complete motor exhaustion, paralysis, a nearly complete loss of sensitivity and reflexes, semicoma and death (Busatto, 1940). The lowest lethal dose was 160-170 mg/kg. The subcutaneous LD50 for hydroquinone has been found to be 182-190 mg/kg in mice (Marquardt et al., 1947; Nomiyama et al., 1967). No experimental data on inhalation exposure have been located. 7.2 Skin and eye irritation; sensitization 7.2.1 Skin irritation Hydroquinone was applied to both epilated and unepilated skin of eight black guinea-pigs at concentrations of 1, 3, 5, 7 and 10% in three vanishing creams (Bleehen et al., 1968). The number of animals per dose group was not reported. Six animals served as controls. The material was applied once daily, five times a week, for one month. Hydroquinone was irritating only at concentrations of 5% or more. Weak to moderate depigmentation occurred in all areas of skin to which creams containing 1-10% hydroquinone were applied. Hydroquinone (0.001, 0.01 and 0.1% in 0.1 ml water) was not found to be a primary irritant when administered intracutaneously in 18 female guinea-pigs during a period of 10 days (Rajka & Blohm, 1970). Jimbow et al. (1974) reported depigmentation in the epilated skin of 24 black guinea-pigs (both males and females) after topical applications of hydroquinone. Creams containing 2 or 5% hydroquinone in an oil-water emulsion were applied daily, 6 days a week, for 3 weeks. The depigmentation was first seen within 8-10 days and was greatest between 14 and 20 days. It was more marked at the higher concentration. Inflammatory changes and thickening of the epidermis were also reported. When hydroquinone was applied topically for three weeks, biopsy specimens showed that it had caused a marked reduction both in the number of melanized melanosomes in the cells and the number of actively functioning melanocytes. In a preliminary screening study with eight guinea-pigs, an aqueous solution of hydroquinone was slightly irritating at 10% but not at 0.5, 1.0 or 5.0% (Springborn Institute for Bioresearch, 1984). The potential of hydroquinone to produce skin depigmentation and irritation has also recently been studied in male and female black guinea-pigs (Maibach & Patrick, 1989). The study indicated that females may be more sensitive than males. Groups of five male and five female animals were administered hydroquinone (0.1 ml in a hydrophilic ointment) at concentrations of 0.1, 1.0, and 5.0% on an epilated area of the back five days a week for 13 weeks. The lowest concentration caused marginal irritation without depigmentation, while the medium concentration resulted in a slight to marginal irritation in 30% of the animals (mainly females) and to weak depigmentation in females. Moderate to severe irritation and severe ulcerated inflammatory responses occurred with the highest concentration. Moderate depigmen-tation was observed in approximately 40% of the animals dosed (only females). Hyperpigmentation was noticed in 80-100% of the animals in all dose groups, but this was not considered to be attributable to the treatment. 7.2.2 Eye irritation Powdered hydroquinone (2-5 mg) instilled twice daily (5 days per week for 9 weeks) into the eyes of dogs caused immediate but transient irritation and lacrimation (Dreyer, 1940). Opacity of the cornea, lacrimation and redness of the conjunctiva were produced within 4 days, but no ulcers were seen. The eye returned to normal within two days after cessation of treatment. In guinea-pigs powdered hydroquinone (1-3 mg, twice daily for 9 weeks) also caused immediate but transient irritation. During the second day of application a slight corneal opacity was observed in some animals and on the third day opacity of varying degrees occurred in most of the animals. Ulcers appeared in two animals. The eyes had fully recovered 3 days after cessation of treatment. Following an injection of 0.1 ml of a solution (vehicle not specified) of hydroquinone (0.012-0.05 mol/litre) into the cornea of rabbits, the resultant reaction was graded 5 out of the possible maximum of 100 (Hughes, 1948). Finely powdered hydroquinone (amounts not specified) was applied daily, from 2-4 months, to the eyes of rabbits in 6 groups, which were, respectively, kept in the dark, in sunlight, in normal light, irradiated with UV light, or pre-sensitized with haematoporphyrin and then kept under either reduced light or sunlight. Most rabbits developed pigmentation, first in the conjunctiva and then in the cornea. Degenerative alterations of the corneal parenchyma were also observed. Pigment formation appeared earlier in animals exposed to light. Older animals seemed more prone to develop pigment than younger ones. Pigment was deposited in albino rabbit eyes as well as in those of rabbits with normal pigmentation (Ferraris de Gaspare, 1949). Hydroquinone in aqueous solution, e.g., in tears, is oxidized by air, forming a brown colour partly due to conversion to 1,4-benzoquinone (Grant, 1986). No disturbance of the inner parts of the eye is known to have been produced by exposure to hydroquinone (Grant, 1986). 7.2.3 Sensitization Several sensitization studies have been carried out with hydroquinone; methods and results are summarized in Table 12. The skin sensitizing potential of hydroquinone for female guinea-pigs was investigated by Rajka & Blohm (1970). Hydroquinone elicited "weak" sensitivity after sensitization with a 0.001% solution injected intracutaneously and challenge with an equipotent solution of hydroquinone. The ability of guinea-pigs to detect known human contact sensitizers was explored by Goodwin et al. (1981). Sensitization induced by hydroquinone was "strong" when assayed by the Magnusson & Kligman maximization test, "moderate" by the single injection adjuvant test and "weak" by the modified Draize procedure. Hydroquinone was found to be a "moderate" sensitizer in female guinea-pigs in both the guinea-pig maximization test and Freund's complete adjuvant test as performed by Van der Walle et al. (1982a,b). Hydroquinone produced identical sensitization potentials in the Freund's complete adjuvant test using induction concentrations of 0.5 mol/litre and 0.45 µmol/litre. The study also showed almost 100% cross reactivity of hydroquinone and p-methoxyphenol. Both substances are used as inhibitors in acrylic monomers to prevent unwanted polymerization. More recently, Basketter & Goodwin (1988) used three sensitization test methods representing both topical and intradermal routes of application. Groups of 10 guinea-pigs were sensitized by using the guinea-pig maximization test, a modified single injection adjuvant test, and a cumulative contact enhancement test. The sensitization potential of hydroquinone was assessed as "strong", "weak", and "moderate", respectively, in these three tests. Subsequent cross-challenges with p-phenylenediamine, sulfanilic acid, and p-benzoquinone gave only "restricted evidence" of cross-reactions. Table 12. Contact allergy predictive tests with hydroquinone in guinea-pigs Test Induction dose Challenge dose Sensitization Reference (number positive/number tested or percentage) Intracutaneous sensitization 0.001% (0.1 ml, injection) 0.001% (injection) 4/18 Rajka & Blohm (1970) Guinea-pig 2.0% (0.1 ml, injection) 5.0% (patch) 70%, "strong" sensitizer Goodwin et al. (1981) maximization test 10.0% (patch) 5.0% (patch) Guinea-pig 0.5 mol/litre (day 0) (patch) 0.125 mol/litre 5/10 (day 21); Van der Walle maximization test 1 mol/litre (day 7) (patch) 0.250 mol/litre 5/10 (day 35) et al. (1982a,b) Guinea-pig 2.0% (0.1 ml, injection) 0.5% (patch) "strong" sensitizer Basketter & maximization test 1.0% (patch) 0.5% (patch) Goodwin (1988) Single injection 2.0% (injection) 5.0% (patch) 40%, "moderate" sensitizer Goodwin et al. (1981) adjuvant test Modified single injection 2.0% (0.1 ml, injection) 10% (patch) "weak" sensitizer Basketter & adjuvant test Goodwin (1988) Modified Draize test 2.5% (injection) 1.0% (injection); 0%a, 30%b, "weak" Goodwin et al. (1981) 20% (application) sensitizer Freund's complete 5 x 0.45 µmol/litre 0.115 µmol/litre 3/8 (day 21) Van der Walle adjuvant test (0.1 ml, injection) (patch) 4/8 (day 35); et al. (1982a,b) 5 x 0.5 mol/litre 0.125 mol/litre 4/8 (day 21) (0.1 ml, injection) (patch) 4/8 (day 35) Cumulative contact 1.0% (patch) 20% (patch) "moderate" sensitizer Basketter & enhancement test Goodwin (1988) a the proportion of guinea-pigs sensitized after one induction treatment b the proportion of guinea-pigs sensitized after two induction treatments 7.3 Short-term exposure The short-term toxicity of hydroquinone has been studied in rats and mice. The effects are summarized in Table 13. Two groups of rats (14 animals/group, sex and strain not reported) were fed a diet containing 0 or 5% HQ for nine weeks (Carlson & Brewer, 1953). Findings at the end of the study consisted of atrophy of the liver cord cells, adipose tissue, striated muscle and lymphoid tissue of the spleen, as well as an average decrease of 66% in cellularity of the bone marrow (considered as aplastic anaemia by the authors). No information was reported on mortality but the fact that animal lost 46% of their body weight during the course of the study makes the findings difficult to interpret. Aqueous solutions of hydroquinone (0, 7.5 or 15 mg/kg, 6 days per week) administered by oral gavage to male Wistar rats for 40 days resulted in haematological changes, including anisocytosis, polychromatophilia and acidophilic erythroblasts, at the highest dose level (Delcambre et al., 1962). Administration of 0, 5 or 10 mg/kg for four months to groups of 15 male Wistar rats resulted in mortality in the highest dose group; 5 mg/kg was well tolerated. "Mild hyperplastic and hyperkeratotic areas near the oesophageal entry" occurred in groups of male and female Wistar rats fed powdered diets containing 2% hydroquinone. However, there were some deficiencies in the reporting of experimental design (Altmann et al., 1985). No other treatment-related changes were found, nor were there any sex-related changes concerning forestomach lesions. A two-week oral study on Fischer-344 rats and B6C3F1 mice was carried out by the National Toxicology Program (NTP, 1989; Kari et al., 1992). The animals were given hydroquinone in corn oil by gavage 5 days per week (12 doses over 14 days). Five rats per sex and dose group were administered 0, 63, 125, 250, 500 or 1000 mg/kg body weight and five mice per sex and dose group 0, 31, 63, 125, 250 or 500 mg/kg. All rats given 1000 mg/kg died during the dosing period; deaths were also reported at the 500 mg/kg dose level. Clinical signs of treatment-related toxicity included tremors and convulsions at the 500 and 1000 mg/kg dose levels. Tremors, convulsions and deaths also occurred in mice during the study period at the 250 and 500 mg/kg dose levels. Table 13. Effects of short-term exposure to hydroquinone via the oral route Species Concentration Means of Duration Observation Reference administration Rat 5% diet 9 weeks reduced weight; aplastic anaemia; decreased Carlson & Brewer bone marrow cellularity; atrophy of liver,(1953) spleen, adipose tissue and striated muscle; ulceration and haemorrhage of the stomach mucosa Rat 500, 750, 1000, stomach tube 12 days increased mortality Carlson & Brewer 1250, 1500, (1953) 1750 mg/kg Rat 7.5, 15 mg/kg intubation 40 days 15 mg/kg: anisocytosis, polychromatophilia, Delcambre et al. acidophilic erythroblasts (1962) Rat 5, 10 mg/kg intubation 4 months 10 mg/kg: deaths (7/15) Delcambre et al. (1962) Rat 2% diet 4 weeks; mild hyperplasia and hyperkeratosis Altmann et al. 8 weeks of forestomach (1985) Rat 20, 64, 200 mg/kg gavage 90 days 64 mg/kg: tremors, reduced activity; Eastman Kodak 200 mg/kg: tremors, reduced activity, reduced Company (1988) body weight and feed consumption (males) Rat 63, 125, 250, gavage 14 days 1000 mg/kg: tremors, convulsions andNTP (1989); Kari 500, 1000 mg/kg death (10/10); et al. (1992) 500 mg/kg: tremors, convulsions and death (1/5 male and 4/5 female) Table 13. (contd). Species Concentration Means of Duration Observation Reference administration Rat 25, 50, 100, 200, gavage 13 weeks 25 mg/kg: decreased relative liver weight NTP (1989) 400 mg/kg (males); 50 and 100 mg/kg: decreased (males) and increased (females) relative liver weight; 200 mg/kg: lethargy, decreased body weight gain and increased relative liver weight; tremors, convulsions and deaths (females); nephropathy; inflammation and/or epithelial hyperplasia of the forestomach; 400 mg/kg: tremors, convulsions and deaths Rat 2.5, 25, 50 mg/kg gavage 1,3,6 weeks 50 mg/kg: increased urinary enzyme excretion; English et al. (1992) renal tubular degeneration/regeneration; and increased renal tubular cell proliferation in male F-344 rats; female F-344 rats and male SD rats: no effects Mouse 25, 50, 100, 200, gavage 13 weeks 25 and 50 mg/kg: lethargy (males); increased NTP (1989) 400 mg/kg relative liver weight (males); 100 mg/kg: lethargy; increased relative liver weight (males); 200 mg/kg: lethargy; increased relative liver weight (males); tremors (males); lesions in the forestomach (one female); deaths (males); 400 mg/kg: lethargy, tremors, convulsions, lesions in the forestomach, deaths Mouse 31, 63, 125, 250, gavage 14 days 500 mg/kg: tremors, convulsions and death NTP (1989) 500 mg/kg (4/5 male and 5/5 female); 250 mg/kg: tremors, convulsions and death (3/5 male) Hydroquinone in 95% ethanol was dermally applied (12 doses over 14 days) to Fischer-344 rats and B6C3F1 mice (NTP, 1989). Rats (five per sex and dose group) received 0, 240, 480, 1920 or 3840 mg/kg and mice (five per sex and dose group) 0, 300, 600, 1200, 2400 or 4800 mg/kg. The survival rate was not affected. The only findings were a 6% lower body weight in male rats administered 3840 mg/kg and crystals on the skin and fur of animals at 3840 mg/kg. No histopathological examination of the tissues was carried out. In a 13-week oral toxicity study, 10 male and 10 female CD(SD)BR rats (initially, approximately 7 weeks old) in each of four groups were administered hydroquinone (0, 20, 64 or 200 mg/kg per day) by gavage on 5 days/week (Eastman Kodak Company, 1988). Brown urine was seen in rats of both sexes from all dose groups. Males also showed lower feed consumption; however, this was only significantly (P < 0.05) reduced during the first week of the study. Female body weight gain and food consumption were not significantly altered in any dose group during the study. Signs of behavioural effects were observed at both the 64 and 200 mg/kg dose levels (see section 7.8.3). The animals were sacrificed after the exposure period, and six males and six females from each group were perfused for neuropathological examination (see section 7.8.3). No treatment-related changes were observed at gross necropsy. Administration of 200 mg hydroquinone/kg reduced body weight gain in male rats so that the final body weight of the treated rats was 7% less than the mean weight for the controls. Thirteen-week studies in rodents have also been presented by the NTP (1989). Groups of 10 males and 10 females of each species (F-344/N rats, initially 4 to 5 weeks old, and B6C3F1 mice, initially 5 to 6 weeks old) were administered hydroquinone (0, 25, 50, 100, 200, or 400 mg/kg) in corn oil by gavage, five days per week. A dose level of 400 mg/kg was lethal to all rats. Tremors and convulsions were observed in most rats at this dose level and in several female rats receiving 200 mg/kg. Doses of 100 mg/kg or less did not cause signs of central nervous system (CNS) stimulation. Rats receiving 200 mg/kg also showed reduced body weight gain, nephropathy, and inflammation and/or epithelial hyperplasia of the forestomach. The kidney lesions in male rats were judged to be more severe than in females. In mice, a dose level of 400 mg/kg caused mortality in both males (8/l0) and females (8/10). In the 200 mg/kg male group two animals died (one due to gavage error). Lethargy was seen in all dosed males and in females in the three highest-dose groups of each sex. Tremors, often followed by convulsions, were noted in the highest-dose group. Liver-to-body weight ratios for all dosed males were significantly (P < 0.01) greater than for vehicle controls. Ulceration, inflammation or epithelial hyperplasia of the forestomach occurred in mice receiving 400 mg/kg (3/10 males and 2/10 females) and in one female mouse receiving 200 mg/kg. Male and female F-344 rats were given hydroquinone (0, 2.5, 25 or 50 mg/kg) 5 days/week in water by oral gavage for 1, 3 or 6 weeks (English et al., 1992). At each time point, 5 rats per sex and dose group were examined for urinalysis changes, renal tubular cell proliferation and histopathology. Body and kidney weights were not altered by hydroquinone exposure. Increased excretion of urinary enzymes was observed in male F-344 rats given 50 mg/kg as early as one week into the exposure. The incidence of tubular degeneration and regeneration was mild in the 50 mg/kg male group, and cell proliferation was increased by 87%, 50% and 34% in the P1, P2 and P3 tubular segments, respectively. Female rats and males in the lower dose level groups were not affected by hydroquinone exposure. Male Sprague-Dawley rats given 50 mg/kg for 6 weeks were also not affected by hydroquinone exposure. 7.4 Long-term exposure The effects of long-term exposure to hydroquinone in experimental animals are shown in Table 14. In addition to the acute (see section 7.1) and subacute (see section 7.3) toxicity tests, Carlson & Brewer (1953) carried out a "cumulative" toxicity study and a series of long-term experiments with rats and dogs. In the cumulative toxicity study a group of 16 rats (strain and sex not reported) received 500 mg hydroquinone/kg by stomach tube 101 times in 151 days. More than 50% of the rats died during the study period. However, the survivors grew at the same rate as the controls and were autopsied at the end of the experiment. However, no information on autopsy findings was reported. In the first long-term study, four groups of male and four groups of female Sprague-Dawley rats (23 to 24 days old, 10 rats in each group) were given a basic diet containing 0, 0.1, 0.5 or 1.0% hydroquinone (Carlson & Brewer, 1953). In the second experiment the hydroquinone was heated together with the lard in the feed. Eight groups of rats (16 to 23 in each group) were fed the basic diet containing 0, 0.1, 0.25 or 0.5% hydroquinone. In the third experiment eight groups of rats (20 rats in each group) were given a basic diet containing 0, 0.1, 0.5 or 1.0% hydroquinone with 0.1% citric acid added. The experiments lasted for 103 weeks. During the first month of the study the animals given 0.5 or 1.0% hydroquinone in their diets gained weight at a slower rate than did control animals. A similar reduction was not found in the rats given hydroquinone previously heated with lard before feeding. However, the final body weights of the dose groups were not significantly different from those of the controls. Haematological analysis (red blood cell count, % haemoglobin and differential white blood cell count) showed no statistically significant deviations from the control values. The microscopic examinations (liver, omentum, kidney, spleen, heart, lung, bone marrow, stomach wall, pancreas, adrenal, subperitoneal and intramuscular abdominal fat) also failed to reveal compound-related changes. Some of the males and females (number and dose groups not specified) in each experiment were mated after six months of dosing to produce two successive litters. The average numbers of offspring for the two successive litters showed no difference between experimental and control groups. The offspring given diets containing 0.1, 0.25 or 0.5% hydroquinone previously heated with lard grew at the same rate as the controls. Carlson & Brewer (1953) also studied the long-term effects of hydroquinone in male and female dogs (four months of age at the beginning of the study). One dog was maintained on a diet containing 16 mg of hydroquinone/kg per day for 80 weeks, while two dogs received 1.6 mg of hydroquinone/kg per day for 31 weeks and 40 mg/kg per day for the succeeding 49 weeks. The compound was administered in sugar-coated tablets mixed with the food. Two dogs served as controls. The sex distribution in the different groups was not reported. In addition, five adult male dogs were fed 100 mg of hydroquinone/kg per day for 26 weeks. Routine blood and urine analyses (not specified) were made periodically. After the experiment, the dogs were killed and autopsied. The dogs given hydroquinone in their diet from four months of age grew at the same rate as controls. The adults maintained their body weights. Haematological analyses and urinalyses showed no differences between exposed rats and controls. No effects on gross pathology or histopathology were observed. Fifteen-month oral toxicity studies on rats and mice were also included in two-year studies presented by the National Toxicology Program (NTP, 1989) (see also section 7.). Groups of F-44/N rats and B6C3F1 mice (64 or 65 males and 65 females of each species) were administered 0, 25 or 50 mg hydroquinone/kg and 0, 50 or 100 mg/kg, respectively, in deionized water by gavage 5 days per week. No compound-related clinical signs were observed during the study period. At 15 months, ten animals from each group were selected for haematological and clinical chemical analyses, killed and necropsied. In male rats significantly (P < 0.01) higher mean relative kidney and liver weights were observed in the high-dose group and there was also a compound-related increase in the severity of nephropathy. For high-dose female rats, the haematocrit value, haemoglobin concentration and erythrocyte counts were decreased. In mice the relative liver weights were significantly (P < 0.01) higher for high-dose males and females than for vehicle controls. A significantly (P < 0.05) higher relative brain weight was noted for high-dose female mice and kidney weights were significantly (P < 0.01) increased for dosed females. In dosed males, but not in females, compound-related lesions in the liver were seen, including centrilobular fatty changes, cytomegaly and syncytial cells. Table 14. Effects of long-term oral administration of hydroquinone in experimental animals Species Concentration Duration Observation Reference Rat 500 mg/kg body weight 151 days > 50% died Carlson & (by stomach tube) (101 dosings) Brewer (1953) Rat 0, 0.1, 0.5, 1.0% 103 weeks decreased weight gain for the first months at 0.5 Carlson & (in the diet) and 1.0%; final body weights did not differ Brewer (1953) Rat 0, 0.1, 0.25, 0.5% 103 weeks no adverse effects Carlson & (heated together with Brewer (1953) lard in the diet) Rat 0, 0.1, 0.5, 1.0% + 103 weeks no adverse effects Carlson & 0.1% citric acid (in the diet) Brewer (1953) Rat 0, 25 or 50 mg/kg in 15 months significantly higher relative kidney weight in high-dose NTP (1989) deionized water (by gavage) males and increased severity of nephropathy in dosed males; decreased haematocrit value, haemoglobin concentration and erythrocyte count in females Mouse 0, 50 or 100 mg/kg in 15 months significantly higher relative liver weights for high- NTP (1989) deionized water (by gavage) dose males and females; liver lesions in males Dog 16 mg/kg per day 80 weeks no adverse effects Carlson & (in the diet) Brewer (1953) Dog 1.6 mg/kg per day; 31 weeks no adverse effects during the total experimental Carlson & 40 mg/kg per day 49 succeeding period Brewer (1953) (sugar coated tablets) weeks 7.5 Reproduction, embryotoxicity and teratogenicity 7.5.1 Effects on male reproduction In a study by Skalka (1964), hydroquinone was injected subcutaneously into 16 male rats (100 mg/kg body weight per day for 51 days), while 17 male rats served as controls. The average weights of the testes, epididymides, seminal vesicles and adrenals were decreased after the treatment period. The fertility was reduced by 33% in the males and the number of pregnancies in mated females was reduced by 19% compared with the corresponding results for the control animals. Histological examinations indicated a disruption in sperm production. Diminished content of DNA in sperm heads was also noted. In 13-week and two-year oral studies in rats and mice, no effects on testicular or epididymal weights or on the histopathology of these organs were observed (NTP, 1989). Hydroquinone in corn oil was given by gavage to groups of males F-344/N rats and to male B6C3F1 mice for 13 weeks. The dose ranged from 0 to 400 mg/kg body weight for both animal species. In the two-year studies hydroquinone in water was given in doses of 0-50 mg/kg body weight to rats and in doses of 0-100 mg/kg to mice. In a dominant lethal assay in male rats (CRL:COBS CD (SD) BR) (see also section 7.6), the males (25 per dose group) were given doses of 0, 30, 100 or 300 mg hydroquinone/kg by gavage 5 days per week for ten weeks (Krasavage, 1984b). The controls consisted of two groups, one positive and one negative. During the 2 weeks immediately following the final treatment, the males were mated (1:1) with untreated females. All females were killed on day 14 of gestation. In the high-dose group (300 mg/kg) the mean body weight and the feed intake of the males were significantly reduced compared to the negative controls (P < 0.05). The high-dose males also exhibited clinical signs of toxicity such as brown urine, sialorrhoea, swollen eyelids, tremors, convulsions and death. No compound-related effects were seen on male fertility and no dominant lethality was observed. There were no compound-related effects on the reproductive parameters studied in the mated females (insemination rate, pregnancy rate, mean number of corpora lutea, implantation sites, viable implants, early and late deaths, and pre- and postimplantation losses). In a two-generation study oral administration of hydroquinone did not appear to affect the reproduction of Fo and F1 parental rats at dose levels up to 150 mg/kg per day (see also section 7.5.3) (Bio/dynamics Inc., 1989b). Male fertility indices, mating indices and pregnancy rates did not differ significantly between the control and the hydroquinone-treated groups. 7.5.2 Effects on female reproduction Hydroquinone was shown to affect the rat estrus cycle when given parenterally (Rosen & Millman, 1955). Three rats were given 10 mg hydroquinone/day subcutaneously for 11 days, and vaginal smears were used to indicate estrus or diestrus. Following an induction period of about three days, the estrus cycle was interrupted for 5 days, after which normal cycling was observed. Similar results were obtained in a study performed by Racz et al. (1958). One group of rats was given 200 mg hydroquinone/kg body weight per day by gavage for 14 days and one group was given 100 mg hydroquinone plus 100 mg phloridzin/kg per day for 14 days. Ten animals were used per group. Some of the rats treated with hydroquinone remained in diestrus. In the group treated with both hydroquinone and phloridzin no effects on the estrus cycle were shown. As the compound caused effects on the central nervous system and mortalities occurred after 4-5 days in the 200 mg/kg dose group, the dose of hydroquinone was reduced to 50 and 100 mg/kg per day. At autopsy no mature Graafian follicles were seen, but some were growing. Hydroquinone in stock diets (0.003 and 0.3%) fed to female rats (10/group) for 10 days prior to insemination had no effect on gestation length, maternal mortality or on other reproductive parameters studied (fertility index, litter efficiency, mean litter size, fetal viability or lactation index (Ames et al., 1956). However, it is not clearly stated if the dosing also included the gestation period. Hydroquinone did not produce adverse affects on female reproduction in a two-generation study in rats (see section 7.5.3) after daily oral administration of doses up to 150 mg/kg per day (Bio/dynamics Inc., 1989b). 7.5.3 Embryotoxicity and teratogenicity The earliest experimental study of the developmental toxicity of hydroquinone and other antioxidants was performed by Telford et al. (1962). They reported that hydroquinone added to the diet caused increased resorption rates. Ten Walter Reed-Carworth Farm strain rats (first gestation animals), weighing about 200 g at the time of breeding, were given 0.5 g hydroquinone in their diet (about 100 mg/kg body weight per day); however, the treatment period was not documented. Based on the number of implantation sites, the resorption rate was 26.8% compared to 10.6% for controls (126 untreated, pregnant rats). The report made no mention of the numbers of corpora lutea. Hydroquinone caused no maternal toxicity. Burnett et al. (1976) reported the findings of a teratology study on twelve hair dye composite formulations. The study groups were tested along with one positive and three negative control groups. Mated Charles River CD female rats (20 per group) were treated by topical application with 2 ml/kg (0.2% hydroquinone) on pregnancy days 1, 4, 7, 10, 13, 16, and 19. No signs of toxicity were seen throughout the study. There were no differences between untreated controls and the hydroquinone-treated group in any reported parameter (maternal toxicity, body weight and food consumption, mean number of corpora lutea, implantation sites, resorption sites, mean resorptions per pregnancy, live fetuses and sex ratio) and no significant soft tissue or skeletal changes. Hydroquinone has been found to induce micronuclei transplacentally in fetal liver cells (Ciranni et al., 1988a) (see also section 7.6). The compound was given by gastric intubation at a dose level of 80 mg/kg to four pregnant Swiss CD-1 mice on the 13th day of gestation. Micronuclei were detected from 9 h after the administration. In a pilot study on developmental toxicity in rats, hydroquinone (5% in distilled water) was administered daily by gavage to groups of ten pregnant COBSCD(SD)BR rats on gestation days 6 to 15 at dose levels of 0, 50, 100 or 200 mg/kg (Krasavage, 1984a). The animals were sacrificed and autopsied on gestation day 16. No significant maternal toxicity or embryo-toxicity was produced in any dose group during the treatment period with the exception of slightly reduced weight gain and feed consumption in the highest-dose. A dose-dependent brownish discolouration of the urine was observed in all treated groups. No other treatment-related changes were found. In the subsequent developmental toxicity study, hydroquinone (5% in distilled water) was administered daily by gavage to groups of 30 plug-positive female (COBSCD(SD)BR rats (initially 6 weeks old) on gestation days 6 to 15 (0, 30, 100 or 300 mg/kg) (Krasavage et al., 1985; Krasavage et al., 1992). Maternal toxicity, manifested as a slight but statistically significant (P < 0.05) reduction in body weight gain and feed intake, was observed in those dams given 300 mg/kg. A reduction in the fetal body weight was seen at 300 mg/kg (statistically significant (P < 0.05) only for the females). No compound-related teratogenic effects were recorded. Dilated renal pelvis, hydronephrosis and hydroureter were seen more frequently in the treated groups than in the controls; however, the changes did not appear to be dose-related and were not significantly different from the controls. Skeletal variations were seen in fetuses from all dose groups and from the control group. The total number of fetuses with a vertebral variation was statistically increased in the highest dose group compared with the controls, but analyses of individual skeletal variations and statistical analyses of total number of fetuses with a skeletal variation indicated no significant effects. The authors concluded that the no-observed-effect level (NOEL) for maternal and developmental toxicity in rats was 100 mg/kg body weight, and the no-observed-adverse-effect level (NOAEL) for fetal development was set at 300 mg/kg (Krasavage et al., 1992). In a pilot study for developmental toxicity in rabbits, hydroquinone (0, 50, 100, 200, 300, 400 or 500 mg/kg per day) was administered by gavage to mated New Zealand White rabbits (five rabbits/group) from days 6 to 18 of gestation (Bio/dynamics Inc., 1988). Dose levels of 300, 400 and 500 mg/kg per day caused maternal death. At dose levels of 50, 100 and 200 mg/kg per day, dose-related reductions in body weight and food consumption were recorded. In the 200 mg/kg per day dose group, an increased number of resorptions were observed, suggesting an embryotoxic effect. Mean fetal weights were unaffected at 50 and 100 mg/kg per day, but at 200 mg/kg per day they were lower than those of the controls (by 20.7%). No fetuses were recovered at the higher dose levels as no females survived throughout the study. The fetal external examinations showed no treatment-related adverse effects. In the subsequent developmental toxicity study, New Zealand White rabbits (18 mated females/group) were given hydroquinone by gavage at dose levels of 0, 25, 75 or 150 mg/kg per day on gestation days 6 to 18 (Bio/dynamics Inc., 1989a; Murphy et al., 1992). The exposure of rabbits to 25 or 75 mg hydroquinone/kg per day had no effect on any maternal or fetal parameter. However, there was a reduction in mean food consumption during the 11-14 day gestational interval at 75 mg/kg per day, but only on days 11 and 12 were these differences statistically (P < 0.05) different from control data. In the 150 mg/kg per day dose group there were statistically significant (P < 0.01) reductions in the body weight and food consumption during the dosing period, suggesting maternal toxicity. No other parameter (clinical observations, uterine implantations, liver or kidney weights or gross pathology) showed any treatment-related adverse effect on the females. No embryotoxicity was found from uterine implantation data. An increased incidence (not statistically significant) of external, visceral and skeletal malformations was noticed in the fetuses from the 150 mg/kg per day group. The NOEL for maternal toxicity was 25 mg/kg per day and the NOEL for developmental toxicity was 75 mg/kg per day. The results of a two-generation reproduction study in rats revealed no hydroquinone-related reproductive toxicity (Bio/dynamics Inc., 1989b). Charles River CD rats (180 Fo and 180 F1) were exposed via gavage to hydroquinone at concentrations of 15, 50 or 150 mg/kg per day (30 rats per sex and group; 120 rats served as controls). The two lower dose levels did not affect mortality rates, body weight, or feed consumption either in the Fo or the F1 parental animals. One Fo male given 50 mg/kg per day had tremors during the post-dosing period. There were no adverse effects on mean litter size, body weight, sex distribution or survival, or in postmortem evaluations of pups from females given 15 or 50 mg/kg per day. At the highest-dose level, tremors were observed in Fo and F1 parental animals of both sexes following dosing. Reproductive indices and pregnancy rates for the Fo and F1 parents were not considered to have been adversely affected by hydroquinone treatment, nor were there any adverse effects of treatment for pups from Fo and F1 parental animals. The NOAEL for parental toxicity was estimated to be 15 mg/kg per day and for reproductive effects through two generations to be 150 mg/kg per day. A quantitative approach to relate the physico-chemical properties of a series of substituted phenols (including hydroquinone) to maternal and developmental toxicity in rats was conducted by Kavlock (1990) and by Kavlock et al. (1991). However, due to theoretical and statistical mistakes, no formal conclusions can be made on the possibility of modelling these particular end-points quantitatively. A review of the different reproduction studies is given in Table 15. 7.6 Mutagenicity and related end-points The results of genotoxicity studies, with or without S9 metabolic activation, are indicated in Table 16. Hydroquinone has been found to be non-mutagenic in Salmonella typhimurium tester strains TA97, TA98, TA100, TA1535, TA1537 and TA1538, with and without metabolic activation, at doses up to 1000 µg/plate (Bulman & Wampler, 1979; Bulman & Van der Sluis, 1980; Florin et al., 1980; Gocke et al., 1981; Serva & Bulman, 1981; Bulman & Serva, 1982; Haworth et al., 1983; Sakai et al., 1985). However, in one study hydroquinone was reported to be clearly mutagenic without metabolic activation in strain 1535A (a strain that the authors suggested might harbour an undefined genetic alteration from TA1535 because of differences in length of storage) with ZLM minimal medium (a modified minimal medium for Escherichia coli) but not with the standard VB (Vogel-Bonner) minimal medium (Gocke et al., 1981). The concentration of citrate was 3.5 times higher in VB medium than in ZLM medium. In a fluctuation test using the Salmonella typhimurium tester strain TA100, hydroquinone was shown to be mutagenic at the concentrations 100 and 200 ng/well with metabolic activation (Koike et al., 1988). In Escherichia coli hydroquinone causes differential killing of DNA-repair-deficient (Pol A-) and -proficient (Pol A+) strains without a supplementary metabolic activation system, indicating induction of repairable DNA damage (Bilimoria, 1975; Van der Sluis, 1980; Wampler, 1980). In Salmonella typhimurium no SOS-inducing activity of hydroquinone (3300 mg/litre liquid medium) could be demonstrated (Nakamura et al., 1987). Table 15. Studies on reproductive effects in laboratory animals Species Route of Number of Dosage Time of Result Reference exposure animals treatment Rat subcutaneous 16 males 100 mg/kg body 51 days 33% reduced fertility, diminished Skalka (1964) weight content of DNA in sperm heads; 19% reduced number of pregnancies in mated females Rat subcutaneous 3 10 mg/day 11 days interrupted estrus cycle Rosen & Millman (1955) Rat oral (gavage) 10/group 200 mg/kg body 14 days some rats remained in diestrus, Racz et al. weight per daya CNS effects and death within 5 days (1958) Rat oral (diet) 10 females/ 0.003% 10 days prior to no effects on gestation and Ames et al. group 0.3% insemination maternal mortality (1956) Rat oral (diet) 10 0.5 g not clearly stated increased resorption rate Telford et al. (1962) Rat oral (gavage) 10 females/ 0, 50, 100 or 200 gestation days reduced feed consumption and body Krasavage (1984a) group mg/kg body weight 6-15 weight gain at 200 mg/kg Rat oral (gavage) 25/group 0, 30, 100 or 300 5 days per week no treatment-related effects on Krasavage (1984b) (male) mg/kg body weight for 10 weeks reproductive parameters studied Rat oral (gavage) 30/group 0, 30, 100 or 300 gestation days reduced fetal body weight at Krasavage et al. mg/kg 6-15 300 mg/kg (1985) Rat oral (gastric 30/group 0, 15, 50 or 150 two-generation no effect on reproduction at Bio/dynamics intubation) mg/kg body weight study 150 mg/kg Inc. (1989b) Table 15. (contd). Species Route of Number of Dosage Time of Result Reference exposure animals treatment Rat oral 16/group 0, 100, 333, 667 gestation day 11 reduced weight gain at 667 and 1000 Kavlock (1990) (intubation) or 1000 mg/kg mg/kg; increased mortality at 1000 mg/kg; malformations of the limbs, tail and urogenital system Rat embryo < 0.5 mmol/litre from day 10 growth retardation, structural Kavlock et al. culture (approx. 12 somite) abnormalities of hind limb and tail (1991) system Mouse oral (gastric 4 80 mg/kg body gestation day 13 micronuclei in fetal liver cells Ciranni et al. intubation) weight (1988a) Rabbit oral (gastric 5/group 0, 50, 100, 200, gestation days increased number of resorptions Bio/dynamics intubation) 300, 400 or 500 6-18 and lower fetal weights at 200 mg/kg; Inc. (1988) mg/kg body weight no females survived 300-500 mg/kg Rabbit oral (gastric 18/group 0, 25, 75 or 150 gestation days an increased incidence (not Bio/dynamics Inc. intubation) mg/kg body weight 6-18 significant) of malformations at (1989a); Murphy 150 mg/kg et al. (1992) a Due to high mortality at 200 mg/kg, the dose was reduced for the rest of the 14-day period. In Aspergillus nidulans hydroquinone was found to induce mitotic segregation at 110-330 mg/litre (Crebelli et al., 1987) and 264-396 mg/litre (Crebelli et al., 1991) and mitotic crossing-over at 250-750 mg/litre (Kappas, 1989) and 264-396 mg/litre (Crebelli et al., 1991). The studies were performed without a supplementary metabolic activation system. Hydroquinone has been shown to induce chromosome aberrations or karyotypic alterations in several plant species, e.g., Allium cepa (Levan & Tjio, 1948a,b), Allium sativum (Sharma & Chatterjee, 1964; Valadaud & Izard, 1971), Callisia fragrans (Roy, 1973), Chara zeylanica (Chatterjee & Sharma, 1972), Nigella sativa (Sharma & Chatterjee, 1964), Trigonella foenum-graecum (Sharma & Chatterjee, 1964), and Vicia faba (Sharma & Chatterjee, 1964; Valadaud & Izard, 1971; Valadaud-Barrieu & Izard, 1973). Hydroquinone produced an equivocal increase in recessive lethal mutations in the X-chromosome of Drosophila melanogaster after feeding adult males with a sucrose solution containing 26.4 or 30.0 mg hydroquinone/litre (NTP, 1989). No increase in the frequency of recessive lethal mutations could be established in similar feeding studies with hydroquinone at concentrations of 5500 to 11 000 mg/litre (Gocke et al., 1981) and 1000 mg/litre (Serva & Murphy, 1981), or in a study where males were injected with a dose of 1.5 mg/litre (NTP, 1989) (see also section 7.5.1). At concentrations of 1.25 mg/litre (without a supplementary metabolic activation system) and 2.5 mg/litre (with a supplementary metabolic activation system), hydroquinone induced forward mutations in vitro in the thymidine kinase locus of the mouse lymphoma cell line L5178Y (McGregor et al., 1988a,b). Gene mutations to 6-thioguanine resistance were induced in vitro in V79 Chinese hamster cells exposed to hydroquinone at 350 µg/litre (Glatt et al., 1989). No supplementary metabolic activation system was used. Structural chromosome aberrations were induced in vitro in Chinese hamster ovary cells treated with hydroquinone, but only in the presence of a supplementary metabolic activation system (Galloway et al., 1987). Table 16. Studies on genotoxicity Genetic end-point Resulta References +S9 -S9 Gene mutations Salmonella typhimurium - Bulman & Wampler (1979) Salmonella typhimurium - - Bulman & Van der Sluis (1980) Salmonella typhimurium - - Florin et al. (1980) Salmonella typhimurium - - Gocke et al. (1981) Salmonella typhimurium - - Serva & Bulman (1981) Salmonella typhimurium - - Bulman & Serva (1982) Salmonella typhimurium - - Haworth et al. (1983) Salmonella typhimurium - - Sakai et al. (1985) Salmonella typhimurium - + Gocke et al. (1981) TA1535A, ZLM medium Salmonella typhimurium + - Koike et al. (1988) TA100, fluctuation test Mouse lymphoma cells L5178Y + + McGregor et al. (1988a,b) Chinese hamster cells V79 nd + Glatt et al. (1989) Drosophila melanogaster, (+) NTP (1989) feeding Drosophila melanogaster, - Gocke et al. (1981) feeding Drosophila melanogaster, - Serva & Murphy (1981) feeding Drosophila melanogaster, - NTP (1989) injection Mouse, spot test - Gocke et al. (1983) Rat, dominant lethals - Krasavage (1984b) Chromosomal aberrations Aspergillus nidulans nd + Crebelli et al. (1987, 1991) Chinese hamster ovary cells + - Galloway et al. (1987) Allium cepa + Levan & Tjio (1948a,b) Allium sativum + Sharma & Chatterjee (1964) Allium sativum + Valadaud & Izard (1971) Callisia fragrans + Roy (1973) Chara zeylanica + Chatterjee & Sharma (1972) Nigella sativa + Sharma & Chatterjee (1964) Trigonella foenum-graecum + Sharma & Chatterjee (1964) Vicia faba + Sharma & Chatterjee (1964) Vicia faba + Valadaud & Izard (1971) Vicia faba + Valadaud-Barrieu & Izard (1973) Mouse, bone marrow + Xu & Adler (1990) Table 16. (contd). Genetic end-point Resulta References +S9 -S9 Mouse, bone marrow + Pacchierotti et al. (1991) Mouse, spermatocytes and + Ciranni & Adler (1991) differentiating spermatogonia Rat, dominant lethals - Krasavage (1984b) Micronuclei Chinese hamster cells V79nd + Glatt et al. (1989, 1990) Rat intestinal cells nd + Glatt et al. (1990) Embryonal human liver cells nd + Glatt et al. (1990) Human lymphocytes nd + Yager et al. (1990) Human lymphocytes nd + Robertson et al. (1991) Chinese hamster embryonicnd + Antoccia et al. (1991) lung cells Mouse, bone marrow + Gocke et al. (1981) Mouse, bone marrow + Gad-El-Karim et al. (1985) Mouse, bone marrow + Ciranni et al. (1988a,b) Mouse, bone marrow + Adler & Kliesch (1990) Mouse, bone marrow + Barale et al. (1990) Mouse, bone marrow + Adler et al. (1991) Mouse, bone marrow + Pachierotti et al. (1991) Mouse, fetal liver + Ciranni et al. (1988a) Chinese hamster cells V79 nd + Glatt et al. (1989) Chinese hamster ovary cells + + Galloway et al. (1987) Human lymphocytes nd + Erexson et al. (1985) Human lymphocytes nd + Morimoto & Wolff (1980) Human lymphocytes nd ± Knadle (1985) Human lymphocytes + nd Morimoto et al. (1983) Mouse, bone marrow - Pacchierotti et al. (1991) Mitotic crossing-over Aspergillus nidulans nd + Kappas (1989) Aspergillus nidulans nd + Crebelli et al. (1991) C-mitotic effects Mouse, small intestine + Parmentier & Dustin (1948) Mouse, bone marrow + Miller & Adler (1989) DNA damage Mouse lymphoma cells L5178YS, nd - Pellack-Walker & Blumer breaks (1986) Phi X-174 DNA, breaks + Lewis et al. (1988a) Table 16. (contd). Genetic end-point Resulta References +S9 -S9 Rat, liver (hepatectomy), + Stenius et al. (1989) single-strand breaks DNA repair Escherichia coli nd + Bilimoria (1975) Escherichia coli nd + Van der Sluis (1980) Escherichia coli nd + Wampler (1980) Salmonella typhimurium nd + Nakamura et al. (1987) HeLa cells + + Painter & Howard (1982) Mouse lymphoma cells L5178YS nd + Pellack-Walker et al. (1985) DNA adducts In vitro + Rushmore et al. (1984) In vitro + Jowa et al. (1986) In vitro + Reddy et al. (1989) In vitro + Jowa et al. (1990) In vitro + Leanderson & Tagesson (1990) In vitro + Reddy et al. (1990) In vitro + Levay et al. (1991) a + = positive; - = negative; (+) = equivocal; ± = induction in some cases but not in others; nd = not determined The induction of sister-chromatid exchanges in vitro after exposure to hydroquinone without the use of a supplementary metabolic activation system has been demonstrated in V79 Chinese hamster cells at 2.2 mg/litre (Glatt et al., 1989) and human lymphocytes at 0.55-11 mg/litre (Erexson et al., 1985), 4.4-22 mg/litre (Morimoto & Wolff, 1980), and 4.4 mg/litre in cells from some individuals but not from others (Knadle, 1985), with a supplementary metabolic activation system in human lymphocytes at 110 mg/litre (Morimoto et al., 1983), and both with and without a supplementary metabolic activation system in Chinese hamster ovary cells (Galloway et al., 1987). Hydroquinone has been found to induce micronuclei in vitro in V79 Chinese hamster cells (Glatt et al., 1989, 1990), rat intestinal cells IEC-17 and IEC-18, and embryonal human liver cells HuFoe-15 (Glatt et al., 1990). In cultured human lymphocytes an 11-fold and 3-fold increase in the frequency of micronuclei, was reported after exposure to hydroquinone at 13.75 mg/litre (Yager et al., 1990) and 8.25 mg/litre (Robertson et al., 1991), respectively. Hydroquinone induced micronuclei in vitro in Cl-1 Chinese hamster embryonic lung cells at concentrations of 1, 3, and 4.5 mg/litre (Antoccia et al., 1991). The use of an antikinetochore antibody in the three latter studies indicated the occurrence of numerical as well as structural chromosome aberrations. All studies were performed without a supplementary metabolic activation system. With the in vitro porcine brain tubulin assembly assay, hydroquinone had no effect with respect to lag-phase, polymerization velocity or end absorption at doses up to 2750 mg/litre. The disassembly process was stimulated at doses higher than 1100 mg/litre (Brunner et al., 1991). In another study, hydroquinone exhibited a weak inhibition of microtubule assembly and resulted in abnormal microtubule formation at a concentration of 110 mg/litre (Wallin & Hartley-Asp, 1993). Although hydroquinone itself appears to be a poor inhibitor of microtubule assembly, its oxidation products have been shown to bind to both alpha- and beta-tubulin and to inhibit microtubule assembly at low concentrations (5 µmol/litre or less) (Epe et al., 1990). In another study it was demonstrated that polymerization of tubulin was inhibited by hydroquinone in a concentration-dependent manner (Irons et al., 1981). An effect on DNA synthesis, indicating DNA damage, following in vitro exposure to hydroquinone has been demonstrated both with and without a supplementary metabolic activation system in HeLa cells (Painter & Howard, 1982) and without the use of a metabolic activation system in the mouse lymphoma cell line L5178YS (Pellack-Walker et al., 1985). Female mice (C57BL/6JHan), mated with T-stock males, were injected intraperitoneally with hydroquinone (110 mg/kg) on the 10th day of pregnancy and subsequently analysed in accordance with the mammalian spot test, which detects somatic gene mutations in mouse embryos. No substance-related effect on the mutation frequency was detected (Gocke et al., 1983). In a study by Xu & Adler (1990), mice [(101/E1 x C3H/E1)F1], 10-14 weeks old and weighing 25-28 g, were injected intraperito-neally with hydroquinone (40, 75 and 100 mg/kg). Bone marrow cells, sampled 6 and 24 h later in the case of the two lower doses and 6, 12, 18, 24 and 36 h later in the case of the highest dose, were analysed for the presence of structural chromosome aber-rations. At the highest dose level hydroquinone significantly increased the frequency of aberrations 6-24 h after the treatment, while at 75 mg/kg such an effect was detectable only 24 h after treatment. Male mice [(101/E1 x C3H/E1)F1], 10-14 weeks old and weighing 25-28 g, were injected intraperitoneally with hydroquinone (80, 100 and 150 mg/kg), and bone marrow cells, sampled 2 h later, were analysed for the induction of c-mitotic effects. At dose levels of 100 and 150 mg/kg hydroquinone significantly increased the frequency of metaphases with spread chromosomes (Miller & Adler, 1989). Colchicine-like accumulation of metaphases and unusual "three-group metaphases" in the small intestine were described in mice injected with hydroquinone (125 mg/kg) (Parmentier & Dustin, 1948, 1951). Similar results were observed in the intestine and bone marrow cells of rats and hamsters following hydroquinone administration (Parmentier, 1952, 1953). In a study by Pacchierotti et al. (1991), male mice [(C57Bl/Cne x C3H/Cne)F1], 12 weeks old, were injected with hydroquinone (40, 80 and 120 mg/kg). Bone marrow cells, sampled 18 and 24 h later were analysed for the presence of numerical chromosome changes, micronuclei and sister-chromatid exchanges. At a dose level of 80 mg/kg, hydroquinone significantly increased the frequency of hyperploid cells with 41-42 chromosomes after 18 h. At all concentrations tested the frequency of micronuclei was significantly increased after 24 h, while after 18 h such an effect was only detectable at the concentration of 80 mg/kg. An increase in sister-chromatid exchange frequencies over control values was not detected in any treated animal. Mice [(101/E1 x C3H/E1)F1], 10-14 weeks old and weighing 25-28 g, were injected intraperitoneally with hydroquinone (30, 50, 75 and 100 mg/kg), and bone marrow cells, sampled 18, 24 and 30 h later (75 mg/kg), 6 and 24 h later (50 mg/kg) and 24 h later (30 and 100 mg/kg), were analysed for the presence of micronuclei. At a dose level of 75 mg/kg, hydroquinone significantly increased the frequency of micronuclei in cells at all sampling intervals. Treatment with hydroquinone at 50 and 100 mg/kg significantly increased the frequency of micronuclei after 24 h (Adler & Kliesch, 1990). The authors also reported results from daily treatments (up to 3 days) of male mice of the same strain with hydroquinone (15 or 75 mg/kg) by the intraperitoneal route of administration. Bone marrow cells were sampled 24 h after the first, second and third injections, respectively. The frequency of micronuclei increased in the case of the lower dose and decreased in the case of the higher dose with increasing number of treatments. The induction of micronuclei in mouse bone marrow cells after intraperitoneal injection of hydroquinone has also been demonstrated by Gocke et al. (1981), Ciranni et al. (1988a,b), Barale et al. (1990) and Adler et al. (1991). Oral administration of hydroquinone (200 mg/kg) induced an increase in the frequency of micronuclei in mice (Gad-El-Karim et al., 1985). Ciranni et al. (1988b) demonstrated that oral administration of hydroquinone (80 mg/kg) produced a weak increase in the frequency of micronuclei compared to the effect found following intraperitoneal administration. In male mice (outbred NMRI) daily subcutaneous injections of hydroquinone on six consecutive days induced micronuclei in the bone marrow at dose levels of 25 to 100 mg/kg (Tunek et al., 1982). When pregnant mice (Swiss CD-1, three months old) were given hydroquinone (80 mg/kg) by gastric intubation on the 13th day of gestation, micronuclei were induced in fetal liver cells (Ciranni et al., 1988a). In male rats (Sprague-Dawley), weighing 200 g and subjected to 70% partial hepatectomy, single-strand breaks were induced in hepatic DNA after the rats had received daily hydroquinone doses of 200 mg/kg, given in a liquid casein-based diet, for 7 weeks (Stenius et al., 1989). Male mice [(102/ElxC3H/El)F1], 12-14 weeks old and weighing 25-28 g, were injected intraperitoneally with hydroquinone (40, 80 and 120 mg/kg). In spermatocytes sampled 12 days after treatment (representing cells treated at preleptotene), the frequency of chromosomal aberrations excluding gaps was significantly increased at 40 and 80 mg/kg but not at 120 mg/kg. In differentiating spermatogonia sampled 24 h after treatment, the frequency of chromosomal aberrations excluding gaps was significantly increased at all dose levels (Ciranni & Adler, 1991). No increased frequency of dominant lethal mutations was detected in male rats [CRL:COBS"CD"(SD)BR] given repeated doses of hydroquinone (30, 100 or 300 mg/kg, 5 days/week for 10 weeks) by gavage (Krasavage, 1984b). The induction of sperm-head abnormalities could not be demonstrated in male mice after intraperitoneal injection of hydroquinone (55-220 mg/kg) (Wild et al., 1981). The ability of hydroquinone to produce adducts with DNA or its nucleotides in vitro has been shown by Rushmore et al. (1984), Jowa et al. (1986), Reddy et al. (1989), Jowa et al. (1990), Leanderson & Tagesson (1990), Reddy et al. (1990) and Levay et al. (1991). There is less evidence for the hydroquinone-induced formation of DNA adducts in vivo. Using the 32P postlabelling assay, no treatment-related adducts were detected in the kidneys of either male or female F-344 rats following the oral administration of hydroquinone at dose levels of up to 50 mg/kg for 6 weeks (English et al., 1992). In additional studies by Reddy et al. (1990), also using the postlabelling assay, no treatment-related DNA adducts were detected in the bone marrow, zymbal gland or liver of female F-344 rats following the oral co-administration of phenol and hydroquinone at either 75 or 150 mg/kg for 4 days (Reddy et al., 1990). Hydroquinone has been shown to be capable of producing breaks in phi X-174 DNA (Lewis et al., 1988a). No increase in the frequency of breaks was detected in DNA from hydroquinone-treated mouse lymphoma cells (L5178YS) at concentrations of up to 11 mg/litre (Pellack-Walker & Blumer, 1986). 7.7 Carcinogenicity The available studies on the carcinogenicity of hydroquinone are summarized in Table 17. 7.7.1 Long-term bioassays In an NTP study (NTP, 1989; Kari et al., 1992), groups of 65 F-344/N rats of each sex were given hydroquinone (0, 25 or 50 mg/kg body weight) in deionized water by gavage 5 days/week for up to 103 weeks, and groups of 65 B6C3F1 mice of each sex were administered 0, 50 or 100 mg/kg body weight according to the same schedule. A 15-month interim kill of ten animals from each group showed that the kidney of male rats was a target organ forthe toxicity (see also section 7.4), since there was a compound-related increased severity of nephropathy. The lesions were less severe in female rats, in which a mild regenerative anaemia was also found (slightly decreased haematocrit, haemoglobin and erythrocyte count). After termination of the experiment, a dose-related increase in the incidence of renal tubular cell adenomas was observed in male rats (controls 0/55, low dose 4/55, high dose 8/55; P = 0.003). The incidence of adenomas was closely associated with the severity of chronic nephropathy. No renal adenomas were observed in animals examined at 15 months, when the severity of nephropathy was less severe, or in female rats, which developed nephropathy to a lesser degree. In the male rats, 9/12 adenomas were seen in kidneys with marked nephropathy, two were seen in animals with moderate nephropathy, and only one was seen in an animal with mild nephropathy. In the high-dose group single tubules exhibited tubular cell hyperplasia. No renal tumours were seen in females. A dose-related increase in the incidence of mononuclear cell leukaemia was found in female rats (controls 9/55, low dose 15/55, high dose 22/55) (P < 0.01 in the high-dose group versus controls). However, this was not observed in the animals killed at 15 months. The incidence in controls was lower than the historical control mean incidence but was within the historical control group range. Table 17. Carcinogenicity studies in animals Species Route of Number of Dosage Time of Result Remarks Reference exposure animals treatment Long-term bioassays Mouse oral 64 or 65 of 50 or 100 103 weeks liver lesions (males), some evidence of NTP (1989); each sex mg/kg hepatocellular adenomas carcinogenic activity Kari et al. per group 5 days/week (females) for female mice (1992) Mouse oral 30 m, 30 f 0.8% in 96 weeks squamous cell hyperplasia of potential of Shibata et al. the diet the forestomach epithelium; hepatocarcinogenicity (1991) renal tubular hyperplasia and in male mice adenomas (males); increased incidence of liver foci and hepatocellular adenomas (males) Rat oral 65 of each 25 or 50 103 weeks nephropathy (more severe in some evidence of NTP (1989); sex per mg/kg males), renal tubular cell carcinogenic activity Kari et al. group 5 days/week hyperplasia and adenomas for male and female (1992) (males), leukaemia (females) rats Rat oral 30 m, 30 f 0.8% in 104 weeks renal tubular hyperplasia, potential of renal Shibata et al. the diet adenomas and epithelial carcinogencity in (1991) hyperplasia of the renal male rats papilla (males); decreased incidence of liver foci Table 17. (contd). Species Route of Number of Dosage Time of Result Remarks Reference exposure animals treatment Carcinogenicity-related studies Mouse skin 24 m 0.3 ml of 6.7% one skin papilloma (1/24) no initiating Roe & Salaman application solution; application; activity (1955) 0.3 ml of then three 0.5% croton weeks later, oil 18 weekly applications Mouse skin 50 f 5 mg three 368 days papilloma (7/50), squamous no co-carcinogenic van Duuren & application times carcinoma (3/50) or tumour-promoting Goldschmidt weeklya activity; partial (1976) inhibition of BP carcinogenicity Mouse implantation not stated 2 mg 25 weeks carcinomas (6/19) Boyland et al. in urinary (1964) bladder Rat oral 20 f 0.8% in 32 weeks no preneoplastic lesions Kurata et al. basal dietb or papillomas of the (1990) urinary bladder Rat oral 15-16 m 0.8% in 51 weeks no increase in forestomach or Hirose et al. dietc glandular stomach neoplasms (1989) Rat oral 5 m 8 weeks no proliferative changes Shibata et al. in forestomach or glandular (1990) stomach Table 17. (contd). Species Route of Number of Dosage Time of Result Remarks Reference exposure animals treatment Rat oral 7-10 m per 100 mg/kg 7 weeks increased number of liver foci relatively weak Stenius et al. group diet per dayd decreased number of liver foci inducer of enzyme- (1989) 200 mg/kg compared to the 100 mg/kg altered liver foci diet per dayd dose Hamster oral 15 m 0.5% in basal 20 weeks no proliferative changes in Hirose et al. diet forestomach (1986) a after initiating dose of benzo[ a] pyrene (BP) b after initiating with N-butyl-2 N-(4-hydroxybutyl) nitrosamine for four weeks c one week after 150 mg/kg body weight d after partial hepatectomy In male mice centrilobular fatty changes and cytomegaly were found in the animals killed at 15 months, but these findings were not seen in mice killed at 2 years. The authors reported that hydroquinone dosing stopped two weeks before necropsy and that the microscopic lesions were likely to be reversible after cessation of treatment. There was a significantly (P=0.0005) increased incidence of hepatocellular adenomas in female mice given hydroquinone for 2 years (controls 2/55, low dose 15/55, high dose 12/55) and the incidences of hepatocellular carcinomas were 1/55, 2/55 and 2/55, respectively. In males the incidence of adenomas was increased in treated mice but the incidence of hepatocellular carcinomas was decreased. Preneoplastic changes (anisokaryosis, multinucleated hepatocytes, and basophilic foci) were increased in high-dose male mice. Treatment-related, but not statistically significant, follicular cell hyperplasia of the thyroid gland was observed in both male and female mice (NTP, 1989; Kari et al., 1992). The NTP concluded that there was "some evidence of carcinogenic activity" of hydroquinone for male F-344/N rats (tubular cell adenomas of the kidney) and also for female F-344/N rats (mononuclear cell leukaemia). There was "no evidence of carcinogenic activity" for male B6C3F1 mice and "some evidence of carcinogenic activity" for female B6C3F1 mice (hepatocellular adenomas and carcinomas combined). Shibata et al. (1991) administered hydroquinone at dietary levels of 0.% or 8 g/kg to groups of 30 Fischer-344 rats and B6C3F1 mice of each sex. The rats were dosed for 104 weeks and the mice for 96 weeks. Average daily intakes were reported to be 351 and 368 mg/kg body weight per day in male and female rats, respectively, and 1046 and 1486 mg/kg per day in male and female mice, respectively. No treatment-related clinical signs and no significant differences in mortality were found between treated and control animals of either species. The final body weight was significantly (P < 0.05) lower in treated female rats than in corresponding controls. In male rats the absolute and relative liver and kidney weights were significantly (P < 0.01) increased, but in females this applied only to the relative kidney weights (P < 0.05). Histologically, chronic nephropathy was seen in both control and treated groups of male rats. However, treated males were more severely affected than the controls, while treated females showed only slight nephropathy. The incidence of epithelial hyperplasia of the renal papilla was significantly (P < 0.05) increased in treated male rats as was the incidence of renal tubular hyperplasia (30/30) and renal tubular adenomas (14/30). The authors found that renal cell tumour development in male rats under the long-term influence of hydroquinone was not associated with alpha2u-globulin nephropathy. The incidence of liver foci showed a tendency to decrease in treated males. A quantitative analysis showed a statistically significant (P < 0.05 in males, P< 0.01 in females) reduction in both sexes given hydroquinone. The authors did not find an increased incidence of mononuclear cell leukaemia in female rats (personal communication). In mice, the final body weight was significantly (P < 0.05) lower in females given hydroquinone; the relative liver and kidney weights were significantly (P < 0.05) increased. Histologically, the incidence of squamous cell hyperplasia of the forestomach epithelium was significantly (P < 0.01) increased in both sexes. A significant increase in the incidence of renal tubular hyperplasia (P < 0.01) and three renal cell adenomas were seen in 30 males given hydroquinone. In treated males the incidence of liver foci and hepatocellular adenomas (14/30) was also significantly (P < 0.05) increased. 7.7.2 Carcinogenicity-related studies 188.8.131.52 Skin In a study by Roe & Salaman (1955), stock albino mice (24 males, "S" strain) were given a single skin application of 0.3 ml of a 6.7% solution of hydroquinone in acetone (total dose 20.0 mg). Three weeks later the mice received 18 weekly applications of 0.3 ml of 0.5% croton oil in acetone as a promoter on the same area of the skin. Of the 24 treated animals, two died during the experiment and one mouse developed a skin papilloma. In a two-stage carcinogenesis test on mouse skin using benzo[ a]pyrene (BP) as the initiating agent, no tumour-promoting activity was shown (Van Duuren & Goldschmidt, 1976). Hydroquinone (5 mg) was applied to mouse skin (50 female ICR/Ha Swiss mice/group; both positive and negative controls) three times weekly for 368 days, together with 5 µg BP. Hydroquinone showed no potential as a co-carcinogen when applied simultaneously with BP; in fact, it partially inhibited BP carcinogenicity. 184.108.40.206 Bladder Implantation of cholesterol pellets containing hydroquinone into the urinary bladder of mice (strain and sex unspecified) has been studied by Boyland et al. (1964). The amount of hydroquinone was 20% in 10 mg cholesterol pellets (2 mg hydroquinone per mouse). Bladder carcinomas were produced in 6 out of 19 mice (32%) surviving 25 weeks. The incidence of urinary bladder carcinomas in survivors of the dosed group was significantly (P=0.03) higher than in controls (11.7%) given cholesterol pellets only. However, the number of animals surviving the study was low, and the original number of animals and their sex distribution were not specified. In a study by Kurata et al. (1990), groups of 20 male Fischer-344 rats received 0.05% N-butyl- N-(4-hydroxybutyl) nitrosamine in the drinking-water for four weeks (as initiation) followed by 8 g hydroquinone/kg in the basal diet for 32 weeks. No increase in the incidence of preneoplastic lesions or papillomas/carcinomas of the urinary bladder was observed when compared to the incidences in rats given nitrosamine alone. 220.127.116.11 Stomach Hirose et al. (1989) examined the promotion activity and the carcinogenic potential of some dihydroxybenzenes, such as hydroquinone, in the glandular stomach and forestomach of F-344 rats. Groups of 15-16 male rats were given a single intragastric dose of 150 mg/kg body weight N-methyl- N'-nitro- N- nitrosoguanidine (MNNG), followed one week later by powdered diet containing hydroquinone (8g/kg) or basal diet alone for 51 weeks. Further groups of 10 and 15 animals, respectively, were administered the basal diet alone or a diet containing hydroquinone (8 g/kg) for 51 weeks without pretreatment with MNNG. Hydroquinone did not cause an increased incidence of forestomach or glandular stomach lesions, either with or without pretreatment with MNNG, in comparison with the control groups. In studies performed by Hirose et al. (1986), hydroquinone did not produce proliferative lesions in the stomach of hamsters. Male Syrian golden hamsters (15/group, seven weeks old at the beginning of the study) were given basal diet with hydroquinone (5 g/kg) added or basal diet alone for 20 weeks. The dose was chosen as approximately a quarter of the LD50. Tissues from forestomach and glandular stomach showed mild to moderate hyperplasia in the group given hydroquinone, but at the same incidence as in the controls. Similar results were obtained by Shibata et al. (1990) in an 8-week oral study using five male F-344 rats. Hydroquinone did not induce any proliferative changes in the forestomach or the glandular stomach epithelium. 18.104.22.168 Liver Hydroquinone has been shown to be a relatively weak inducer of enzyme-altered foci in rat liver when tested for tumour-promoting activity in a liver focus test (Stenius et al., 1989). Male Sprague-Dawley rats (7-10/group) given diethylnitrosamine (30 mg/kg intraperitoneally) after partial hepatectomy were treated with hydroquinone (0, 100 and 200 mg/kg per day) in their diet for 7 weeks. At 100 mg/kg there was a significantly (P < 0.01) increased number of liver foci and an increased focus volume. The 200-mg dose caused less foci (0.34 ± 0.16 per cm2) than the 100-mg dose (0.65 ± 0.25 per cm2), but the incidence was higher than in the control group (0.08 ± 0.08 per cm2). A study by Kurata et al. (1990) yielded similar results concerning the tumour-promoting potential of hydroquinone in rats. Dietary administration of hydroquinone (8g/kg in basal diet) for 32 weeks, after initiation for four weeks with N-butyl- N- (4-hydroxybutyl) nitrosamine, caused no preneoplastic lesions or papillomas of the urinary bladder. 7.8 Special studies 7.8.1 Effects on spleen and bone marrow cells; immunotoxicity The bone marrow is the target in benzene toxicity; among the many metabolites of benzene, hydroquinone has received increased scrutiny as one of the possible contributing factors. Intravenous or intraperitoneal administration of hydroquinone (100 mg/kg) for three consecutive days to male C57BL/6 CRIBR mice significantly (P < 0.05) reduced the spleen and bone marrow cellularity, with bone marrow demonstrating the greatest sensitivity (Wierda & Irons, 1982). Laskin et al. (1989) found that after injection in Balb/c mice hydroquinone (50 mg/kg) caused a 30-40% decrease in bone marrow cellularity. In vitro studies have demonstrated direct myelotoxic effects of hydroquinone toward mouse bone marrow stromal cells (Gaido & Wierda, 1984; Gaido & Wierda 1987). Hydroquinone inhibited stromal cell colony growth along with the ability of these cells to support granulocyte/monocyte colony formation in co-culture. The bone marrow stroma predominantly consists of macrophages and fibroblastoid stromal cells which interact to regulate myelopoiesis. Treatment with hydroquinone thus results in reduced capacity of the stroma to support myelopoiesis. In addition to this cytotoxic effect, Wierda & Irons (1982) found in in vivo studies that hydroquinone also affected the immune function by reducing the number of progenitor B-lymphocytes in the spleen and bone marrow in mice, thus demonstrating an immunosuppressive potential. The rapid generation and maturation of progenitor B cells renders them highly susceptible to toxic agents that affect dividing cells. Evidence has accumulated concerning the effect of hydroquinone on the cellular activity of the immune system in vitro. Exposure of lymphocytes in vitro to hydroquinone has been shown to result in a dose-dependent inhibition of RNA synthesis in the lymphocytes (Post et al., 1985). A hydroquinone concentration of 1-2 x 10-5 mol/litre inhibited the RNA synthesis by 50%. In vitro exposure (one hour) of mouse bone marrow cells to hydroquinone (10-7-10-5 mol/litre) inhibited the maturation of B-lymphocytes from pre B-cells after 24 and 48 h in culture (King et al., 1987). More recent data have demonstrated that hydroquinone-induced inhibition of pre-B cell maturation results from toxicity to adherent stromal cells, and that bone marrow macrophages may be the primary target for hydroquinone myelotoxicity, rather than fibroblastic stromal cells or pre-B cells (King et al., 1989; Thomas et al., 1989a). Results also indicate a dose-related reduction of macrophage interleukin-1 (IL-1) secretion in cultures of bone marrow macrophages exposed to hydroquinone (King et al., 1989; Thomas et al., 1989b). IL-1 is necessary for the induction of interleukin-4 (IL-4), which is produced by fibroblastic stromal cells and is required for maturation of pre-B cells to B cells (King et al., 1989). Fan et al. (1989) demonstrated that hydroquinone can inhibit the natural killer activity of mouse spleen cells in vitro at low concentrations. Concentrations of 1 x 10-5 mol/litre and 1 x 10-6 mol/litre inhibited 29 and 22% of the activity, respectively. Lewis et al. (1988b) found that hydroquinone had a selective effect on macrophage functions important in host defense. At concentrations of 3-100 µmol/litre, hydroquinone significantly (P < 0.05) inhibited the release of hydrogen peroxide and at 100 µmol/litre it significantly (P < 0.05) inhibited priming by interferon for tumour cell cytolysis. Cheung et al. (1989) have shown a concentration-dependent inhibition of interferon-alpha/ß production following exposure to hydroquinone in murine L-929 cell cultures. 7.8.2 Effects on tumour cells The cytotoxic activity of hydroquinone has been tested on different tumour cells. Chavin et al. (1980) studied the effect on melanoma transplants in female BALB/c mice. The incidence of melanoma transplants was reduced and the survival significantly (P < 0.0005) increased in mice that received hydroquinone treatment (80 mg/kg). Vladescu & Apetroae (1983) studied the molecular mechanisms of antitumour action and the possibilities of using hydroquinone as a toxic agent against cancer cells. In H 18R tumour-bearing male Wistar rats treated with hydroquinone (5 mg/kg per day) for seven days, the catalase activity was markedly depressed in liver, spleen, blood and H 18R tumour. In vitro studies on tumour and liver homogenates from normal and tumour-bearing rats showed a marked inhibition of catalase activity in the tumour, which was less evident in the liver. The activity was less reduced in normal liver homogenates. It was suggested that the mechanism of action of hydroquinone as an antitumour agent is achieved mainly via peroxide production. When tested on cultured rat hepatoma cells hydroquinone showed a dose-dependent cytotoxic activity (Assaf et al. 1987). A dose of 33 mg/litre (300 µmol/litre) caused cellular mortality of 40% after 24 h of incubation and 66 mg/litre (600 µmol/litre) resulted in 100% cellular mortality. 7.8.3 Neurotoxicity Hydroquinone, given as single oral or subcutaneous lethal doses, causes nonspecific effects on the nervous system such as hyperexcitability, tremor and convulsions in several experimental animal species (see section 7.1). Animals given sublethal oral doses recover within a few days. These central nervous system stimulation effects were confirmed in a 90-day oral study on rats (Eastman Kodak Company, 1988) (see also section 7.3). Male and female weanling rats (CD(SD)BR), initially seven weeks old, were treated with hydroquinone (20, 64 or 200 mg/kg per day) dissolved in water at a concentration of 5%. Doses were given by gavage 5 days per week. Functional-observational battery examinations were performed throughout the study. The battery included observations of body position, activity level, coordination of movement and gait, behaviour, presence of convulsions, tremors, lacrimation, salivation, piloerection, pupillary dilatation or constriction, respiration, diarrhoea, urination, vocalization, forelimb/hindlimb grip strength and sensory function. Tremors and depression of general activity were observed in both sexes shortly after dosing with 64 or 200 mg hydroquinone/kg. Functional-observational battery examinations did not result in any evidence of neurotoxicity as assessed by quantitative grip strength measurement, brain weight or neuropathological examination. The NOEL was considered to be 20 mg hydroquinone/kg body weight. Otsuka & Nonomura (1963) reported that hydroquinone reversed curare blockage at neuromuscular junctions in frog sciatic nerve - sartorius muscle preparations. The authors suggested that this effect was due to an increased release of transmitter at the neuromuscular junction induced by hydroquinone. 7.8.4 Nephrotoxicity Until recently, exposure to hydroquinone has not been associated with nephrotoxicity. Nephrotoxicity has not been reported following either occupational exposure to hydroquinone or acute exposures in humans. Carlson & Brewer (1953) gave human volunteers daily hydroquinone doses of 300 or 500 mg/day for periods of up to 20 weeks without effects on urinalysis parameters. Exposure of five male mixed-breed dogs to 100 mg hydroquinone/kg per day for 26 weeks had no effect on urinalysis parameters or renal histopathology (Carlson & Brewer, 1953). Christian et al. (1976) reported that exposure of Carworth rats to hydroquinone in the drinking-water at concentrations of up to 10 g/litre (6 rats of each sex per group for 8 weeks) or up to 4 g/litre (20 rats of each sex per group for 15 weeks) resulted in slight changes in kidney weight but no histopathological changes. Carlson & Brewer (1953) also reported no evidence of renal histopathological changes in Sprague-Dawley rats fed diets containing 10g hydroquinone/kg for 104 weeks. NTP (1989) reported that oral gavage of hydroquinone (0, 25, 50, 100, 200 or 400 mg/kg) in corn oil for 13 weeks resulted in toxic nephropathy in F-344 rats at the two highest dose levels (200 mg/kg: 7/10 males, 6/10 females; 100 mg/kg: 1/10 females). Oral gavage of 0, 25 or 50 mg/kg in water for 15 months resulted in an increased incidence of chronic nephropathy in male F-344 rats (25 mg/kg: 5/5 males; 50 mg/kg: 6/10 males). When male F-344 rats were dosed at 0, 25 or 50 mg/kg for two years, there was an increased severity of chronic progressive nephropathy in 20/55 animals given 50 mg/kg. At a dosage level of 50 mg/kg for either 15 months or 2 years, male rats had heavier relative kidney weights. Shibata et al. (1991) also reported that F-344 rats developed chronic nephropathy when fed 8g hydroquinone/kg diet for 2 years. Male rats showed increased relative and absolute kidney weight, as well as an increased severity of chronic nephropathy (14/30 animals). Female rats showed an increased relative kidney weight, but only a minimal increase in severity of chronic nephropathy in 7/30 animals. Boatman et al. (1992) reported on the urinalysis changes observed in male and female F-344 rats and Sprague-Dawley rats given single doses of 0, 200 or 400 mg hydroquinone/kg in water by oral gavage. B6C3F1 mice were examined after receiving doses of 0 or 350 mg/kg in a similar fashion. The placement of venous catheters in F-344 rats increased their response to hydroquinone. At 400 mg/kg, male and female F-344 rats, but not Sprague-Dawley rats, displayed pronounced enzymuria and glucosuria, which resolved in 72-96 h. At 200 mg/kg, enzymuria and glucosuria were present in female F-344 rats but not males. Epithelial cell counts in the urine were statistically significantly increased (P < 0.05) at 400 mg/kg (male and female F-344 rats only) and 200 mg/kg (female F-344 rats only). Statistically significant (P < 0.05) decreases in osmolality were reported at 400 mg/kg for F-344 (both sexes) and female Sprague-Dawley rats. Diuresis (ml urine/h) was statistically significant (P < 0.005) only for female F-344 rats at 200 mg/kg and 400 mg/kg. Although differences were observed in some of the urinary parameters measured, mice were generally not responsive to hydroquinone. To characterize the early development of renal toxicity in rats, cell proliferation was quantified within the proximal (P1, P2 and P3) and distal tubular segments of the kidney in rats given 0, 2.5, 25 or 50 mg hydroquinone/kg by oral gavage. Male and female F-344 rats were treated for 1, 3 or 6 weeks, and male Sprague-Dawley rats were treated for 6 weeks. At 6 weeks, an 87% increase in cell proliferation was measured in the P1 segment, a 50% increase in the P2 segment, and a 34% increase in the P3 segment from kidneys of male F-344 rats dosed with 50 mg/kg. Urinalysis indicated increased enzymuria in this same dose group, and mild histological changes were present in the kidneys. Animals examined at other time points or from other dose groups were not affected by hydroquinone. The increased incidence of renal adenomas only in male F-344 rats (NTP, 1989) has led to speculation that the tumours observed may be related to alpha2u-globulin-induced nephropathy. This mechanism of action for induction of kidney tumours does not appear to be relevant for hydroquinone as none of the studies cited above has reported finding evidence of hyalin droplet nephropathy following subacute, subchronic or chronic hydroquinone exposure. Glutathione metabolites, which are at least partially formed in the liver and transported to the kidney, are reported to be involved in the nephrotoxicity observed. Some of the potential glutathione conjugates of hydroquinone have been shown to be more nephrotoxic when injected parenterally than the parent chemical (Boatman et al., 1992; Hill et al., 1992a,b; Kleiner et al., 1992). Administration of hydroquinone by parenteral injection, a route which is likely to increase hydroquinone glutathione conjugates, induces nephrotoxicity in otherwise non-responsive male Sprague-Dawley rats (Hill et al., 1992a,b; Kleiner et al., 1992). The formation of glutathione metabolites and an increased susceptibility of the male F-344 rat to the conjugates appear to be mechanistically linked to the nephrotoxicity observed in these rats. 7.8.5 Interaction with phenols Recently there have been a number of studies reporting interactive effects between hydroquinone and other phenolic compounds. Initially, Eastmond et al. (1987) showed that the co-administration of hydroquinone and phenol (75 mg/kg), when given by intraperitoneal injection twice per day, produced a synergistic decrease in bone marrow cellularity in B6C3F1 mice that was similar to that induced by benzene. This combined treatment was significantly more myelotoxic than that observed when either hydroquinone or phenol was administered separately. Associated in vitro studies suggested that this interactive effect was due to a phenol-induced stimulation of the myeloperoxidase-mediated conversion of hydroquinone to 1,4-benzoquinone in the bone marrow (Eastmond et al., 1987; Smith et al., 1989; Subrahmanyam et al., 1991). Subsequent studies have indicated that interactions between hydroquinone and other phenolic compounds can result in a variety of cytotoxic, immunotoxic and genotoxic effects. Some of the adverse interactive effects that have been reported are outlined below: a) Decreased uptake of 59Fe, an indicator of toxicity to the bone marrow, has been reported with the combined administration of hydroquinone and various phenolic metabolites (Guy et al., 1990, 1991). b) The combined administration of phenol and radiolabelled hydroquinone results in increased binding of hydroquinone equivalents in the bone marrow (Subrahmanyam et al., 1990). c) Decreased bone marrow cellularity and increased production of reactive oxygen species in phagocytes when stimulated with a phorbol ester tumour promoter have been observed following hydroquinone and phenol co-administration (Laskin et al., 1989). d) Synergistic increases in the formation of micronuclei have been observed in mice and human lymphocytes obtained from one individual following exposure to hydroquinone and other phenolic metabolites (Barale et al., 1990; Robertson et al., 1991). e) Three- to six-fold increases in DNA adduct formation (over that observed using the sum of the individual metabolites) were observed in HL-60 cells treated with the combination of hydroquinone and either catechol or 1,2,4-trihydroxybenzene. In addition, the combined treatment of hydroquinone and 1,2,4-trihydroxybenzene produced DNA adducts not detected after treatment with either metabolite alone (Levay & Bodell, 1992). f) Co-treatment of phenol and hydroquinone was reported to shift the optimal concentration of hydroquinone inducing the maximal recombinant granulocyte/macrophage colony-stimulating factor response from 1 µmol/litre to 100 pmol/litre (Irons et al., 1992). g) The co-administration of hydroquinone with either phenol or catechol in vivo to B6C3F1 mice increased the formation of oxidative DNA damage as measured by the formation of 8-hydroxy-2'-deoxyguanosine which occurred in the bone marrow of B6C3F1 mice (Kolachana et al., in press). 8. EFFECTS ON HUMANS 8.1 General population exposure 8.1.1 Acute toxicity - poisoning incidents There have been reports of poisoning due to accidental or suicidal ingestion of hydroquinone alone (Mitchell & Webster, 1919; Rémond & Colombies, 1927) or of photographic developers containing hydroquinone (Busatto, 1939; Zeidman & Deutl, 1945; Grudzinski, 1969; Larcan et al., 1974). Deaths have been reported after ingestion of photographic developers containing hydroquinone in amounts of 3-12 g (80-200 mg/kg body weight). The main symptoms of intoxication by hydroquinone include tremors, vomiting, abdominal pain, headache, tachycardia, convulsions, loss of reflexes, dark urine, dyspnoea, cyanosis and coma. No adverse systemic effects have been reported after acute inhalation of hydroquinone dust (Anderson, 1947; Oglesby et al., 1947; Sterner et al., 1947). 8.1.2 Short-term controlled human studies In a controlled oral study, ingestion of 500 mg hydroquinone daily for 5 months (two males) or 300 mg/day for 3-5 months (17 volunteers, both males and females) produced no observable pathological changes in blood and urine (Carlson & Brewer, 1953). No further data were given. 8.1.3 Dermal effects; sensitization Skin lighteners often contain hydroquinone (1.5 to 2%) as the bleaching agent, which inhibits the production of melanin. Prolonged use (about three years) of strong (>5%) hydroquinone bleaching creams has been reported to cause ochronosis and pigmented colloid milium in South African black women (Findlay et al., 1975; Findlay & de Beer, 1980). Sporadic skin reactions have also occurred among amateurs who develop their own films manually (Fisher, 1986). In a study to assess the safety of hydroquinone in cosmetic skin-lightening products, 840 male volunteers from different human races such as Blacks (Zulu), Asians (Indians), and Coloureds (mixed ethnic origins) were treated with various concentrations of hydroquinone in different bases (Bentley-Philips & Bayles, 1975). They were subjected to open-patch tests, normal usage tests, and standard 48 h closed-patch tests. The results of the study showed that concentrations of hydroquinone below 3% produced negligible adverse effects, irrespective of the base used or the colour of the user's skin. In earlier, less extensive studies, Fitzpatrick et al. (1966) found that a 5% cream of hydroquinone caused a high incidence of primary irritant reactions such as erythema and tingling at the site of application. Fisher (1982) reported four cases of leucoderma following the use of bleaching creams containing 2% hydroquinone. The leucoderma was not preceded by inflammation and the patients had no positive patch-test reaction to 1% hydroquinone in petrolatum after 72 h. Hypopigmentation produced by 1% hydroquinone occurred in one patient (Fisher, 1986). Spencer (1965) found that higher concentrations (5%) of hydroquinone might cause sensitization. Van Ketel (1984) reported a case of probable sensitization to hydroquinone with cross-sensitization to hydroquinone monobenzyl ether. Two days after the first application of a cream containing 5% hydroquinone monobenzyl ether (after a treatment period of three months with 2% hydroquinone in a cream base), an acute dermatitis developed. Patch testing was positive for the two substances. Sensitization to hydroquinone monobenzyl ether occurs fairly frequently (Fisher, 1986), while hydroquinone is regarded as a weak sensitizer. There have been several cases in Europe and the USA of ochronosis in people who have used creams containing 2% hydroquinone or less (Connor & Braunstein, 1987; Lawrence et al., 1988). Hardwick et al. (1989) established a causal link between hydroquinone and exogenous ochronosis. Hydroquinone, like its monobenzyl ether or monoethyl ether, has been reported to cause severe patchy depigmentation disorders in a confetti-like pattern in a single black man (Markey et al., 1989). These authors also noted that four other cases had been reported. Several cases of brown discoloration of the finger-nails due to hydroquinone-containing skin-lightening creams have been reviewed by Mann & Harman (1983). The colour change is considered to be due to hydroquinone oxidation products resulting from exposure to sunlight. 8.2 Occupational exposure 8.2.1 Dermal effects There have been case reports of occupational depigmentation of the skin where a causal relationship between photographic developers containing hydroquinone and depigmentation (leucoderma or vitiligo) has been suggested (Frenk & Loi-Zedda, 1980; Kersey & Stevenson, 1981). In one case a vitiligo-like depigmentation was induced in a black man by contact with a dilute hydroquinone solution (0.06%) after 8-9 months (Frenk & Loi-Zedda, 1980). The depigmentation occurred without any inflammatory skin changes and without itching. Biopsy revealed lack of epidermal melanin pigment and melanocytes. However, in the upper dermis there were numerous melanin-laden macrophages. Developer containing 7% hydroquinone has also caused vitiligo in a man wearing protective gloves (Kersey & Stevenson, 1981). The gloves were considered to have functioned as an occlusive bandage. Lidén (1989) carried out dermatological examination and patch testing on 78 employees exposed to film chemicals at a film laboratory. Hydroquinone (1% aqueous solution) was found to cause irritation (erythema or staining) in previously non-exposed, healthy volunteers. Contact allergy to hydroquinone (1% in water and petrolatum) was diagnosed in four out of seven tested employees. 8.2.2 Ocular effects Ocular lesions of various degrees have been observed in workers exposed to quinone vapour and hydroquinone dust in the manufacture of hydroquinone, and the clinical characteristics are well described (Sterner et al., 1947; Oglesby et al., 1947; Anderson & Oglesby, 1958). Airborne concentrations of hydroquinone dust were occasionally 20-35 mg/m3 (Oglesby et al., 1947). In the presence of air and moisture hydroquinone is rapidly converted into the more volatile quinone. Acute exposure to high (not specified) vapour concentrations resulted in irritation, sensitivity to light, lacrimation, injury of the corneal epithelium, and corneal ulceration. Acute exposure to hydroquinone dust caused eye irritation. Chronic exposure to hydroquinone dust led to corneal staining (greenish-brown), corneal opacity, and conjunctival staining (brownish to brownish-black), with a distribution corresponding to the palpebral fissure (Anderson, 1947; Sterner et al., 1947). In some cases, an appreciable loss of vision due to permanent fine opacities, astigmatism and irregularity occurred in the cornea (Anderson & Oglesby, 1958). Eye irritation occurred following exposure to 2.25 mg/m3 (0.5 ppm) and became marked at 13.5 mg/m3 (3 ppm). Slowly developing inflammation and discoloration of the cornea and conjunctiva followed daily exposures of 0.05 to 14.4 mg hydroquinone/m3 (0.01 to 3.2 ppm) for two or more years (Oglesby et al. 1947). The degree of eye injury showed a positive correlation only with length of employment (Anderson, 1947; Sterner et al., 1947; Anderson & Oglesby, 1958). The ocular lesions developed gradually over a period of several years of exposure; no serious cases were seen until after five or more years of exposure. Removal from exposure resulted in considerable improvement of the staining, but improvement of the corneal opacities was questionable. The relative contribution of quinone vapour and hydroquinone dust to eye injury was not assessed. Three cases of corneal pigmentation were described among men working in a hydroquinone factory (Naumann, 1966). At a later date, corneal damage became apparent even though exposure had been discontinued, and progressive deterioration of vision was reported. Histopathological examinations revealed pigmentary and degenerative changes. Two distinct forms of pigment were distinguished. One was intraepithelial and contained iron, the other was confined to a band-like zone between normal and altered stroma and was considered to be quinone. An operation nurse repeatedly developed a corneal ulcer while mixing bone cement containing methyl methacrylate and hydroquinone (Norrelykke Nissen & Corydon, 1985). It was suggested that the eye symptoms developed because of a composite effect of methyl methacrylate and hydroquinone vapours. However, the presence of hydroquinone or quinone vapour in the air was not determined. 8.2.3 Systemic effects Clinical and laboratory evaluation of 88 workers in 1943 and 101 workers in 1945, who had been exposed to high airborne concentrations of both quinone and hydroquinone for periods up to 15 years, showed no evidence of any systemic toxicity (Sterner et al., 1947). 8.2.4 Epidemiological studies 22.214.171.124 Respiratory effects In a study on airway responses to hydroquinone (Choudat et al., 1988), 33 workers exposed to hydroquinone, trimethyl- hydroquinone, and retinene-hydroquinone were compared to a reference group of 55 matched, non-exposed workers regarding the potential allergic effect of the exposure. No further information on exposure conditions (nature, extent and duration) and possible exposure to other chemicals was reported. The prevalence of respiratory symptoms was increased in workers exposed to hydroquinone and its derivatives. The exposed workers had a significantly (P < 0.01) higher prevalence of cough induced by a smoky atmosphere or by cold air. The prevalences of eczema and coughing at work were also higher. Pulmonary function values were significantly (P < 0.01) lower in the exposed than in the non-exposed group. According to the results from a bronchodilator test, hydroquinone or its derivatives also seemed to induce intermittent dyspnoea and reversible obstruction. The exposed workers had higher levels of IgG (P < 0.002) and IgE (not significant) than the non-exposed workers. However, because of the higher chemical reactivity of trimethyl- hydroquinone compared with hydroquinone, it is difficult to determine the effects of hydroquinone alone (O'Brien, 1991). 126.96.36.199 Carcinogenicity studies Greenwald et al. (1981) performed a nested case-referent study of brain cancer in a group of employees from the Eastman Kodak Company. This study was prompted by preliminary data suggesting a possible increase of brain cancer incidence in New York state, USA, and included a case group of 56 employees who had died with primary brain tumours during the period 1956-1975. An elevated relative risk was found among photographic processing workers exposed to colour developers, but the results were not statistically significant. The authors stated that the excess of brain neoplasms noted may have resulted from a diagnostic sensitivity bias arising from more complete medical evaluation of Kodak employees than of other plant employees. A cohort study of 478 photographic processors in nine Eastman Kodak Color Print and Processing laboratories was undertaken as a follow-up study. The primary objectives of the study were to evaluate mortality, cancer incidence, and absence due to sickness of employees exposed to photographic chemicals. Both industrial and general population references were used. There was no significant increase in the incidence of the parameters investigated. Two cases classified as malignant neoplasms on the brain and CNS were observed versus 0.4 expected (Friedlander et al., 1982), but this difference was not statistically significant. Individual exposures were not examined, but hydroquinone was identified among the many possible exposures. One air sample analysed for hydroquinone contained < 0.01 mg/m3. Work-related mortality among male employees at Tennessee Eastman Company was studied by Pifer et al. (1986). The exposure included hydroquinone among other chemical agents. Cancer mortality was lower than that of the general population; the standard mortality ratio was 56. No information on levels of hydroquinone exposure or total number of workers exposed to hydroquinone was reported. However, work is underway to explore the feasibility of conducting a mortality and, possibly, a morbidity study of hydroquinone workers at this facility (personal communication to the IPCS, 1992). 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD Hydroquinone has been shown to be an outlier in numerous QSAR (Quantitative Structure-Activity Relationship) studies (e.g., Devillers et al., 1986, 1987; Hodson et al., 1988; Nendza & Seydel, 1988a,b,c). Due to this fact Devillers et al. (1990) reviewed the environmental and health risks of hydroquinone. Ecotoxicity data for hydroquinone are listed in Table 18. From these data, it appears that hydroquinone is highly toxic for most of the organisms studied. However, a difference in sensitivity exists among the taxons. It should be noted that the results need to be analysed in relation to the experimental conditions under which they were obtained (e.g., pH, light). Thus, numerous studies have been performed under static condition. In only a few experiments has the concentration of hydroquinone been monitored. Table 18. Ecotoxicity data for hydroquinone Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Bacteria Beneckea harveyi 50% inhibition of luminescence 10 sec 82.6 Stom et al. 100% inhibition of luminescence 10 sec 550.6 (1986) 50% inhibition of dehydrogenase 1 h 110 activity Escherichia coli toxicity threshold concentration 6 h 50 Bringmann & for inhibition of acid production Kühn (1959a) from glucose 50% inhibition of cell 6-8 h 34.0 Devillers et multiplication al. (1990) Photobacterium EC10, luminescence 30 min 0.022 Devillers et phosphoreum EC50 0.072 al. (1990) EC90 0.210 EC50, luminescence 5 min 0.042 Ribo & Kaiser 10 min 0.038 (1983) 30 min 0.038 Pseudomonas zone of growth inhibition on 24 h 200 the zone of inhibition Trevors & fluorescens agar was 14 mm; after exposure Basaraba the % survival of resting (1980) cells was 0.11 Pseudomonas putida toxicity threshold concentration 16 h 58 Bringmann & for inhibition of cell Kühn (1977a) multiplication Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Dinoflagellates Crypthecodinium 50% mortality 40 h 50.0 Devillers et cohnii al. (1990) Prorocentrum micans 50% immobilization 1 h 0.750 Devillers et 50% immobilization 2 h 0.300 al. (1990) 100% immobilization 18 h 5.00 Cyanobacteria (Blue-green algae) Anabaena flos-aque lowest concentration for no 14 days 39.8 irradiance of 2 W/m2 Wängberg & detectable growth Blanck (1988) Anabaena sp. lowest concentration for no 14 days 10.0 irradiance of 2 W/m2 Wängberg & detectable growth Blanck (1988) "LPP sp" 1 PCC6402 lowest concentration for no 14 days 5.01 irradiance of 10 W/m2 Wängberg & detectable growth 20.0 irradiance of 2 W/m2 Blanck (1988) "LPP sp" 2 PCC73110 lowest concentration for no 14 days 5.01 irradiance of 2 W/m2 Wängberg & detectable growth Blanck (1988) Microcystis toxicity threshold concentration 8 days 1.1 Bringmann & aeruginosa for inhibition of cell Kühn (1978) multiplication EC100 24 h 0.5 Fitzgerald et al. (1952) Synechococcus lowest concentration for no 14 days 10.0 irradiance of 10 W/m2 Wängberg & leopoliensis detectable growth 10.0 irradiance of 2 W/m2 Blanck (1988) Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Algae Bumilleriopsis lowest concentration for no 14 days 10.0 irradiance of 10 W/m2 Wängberg & filiformis detectable growth Blanck (1988) Chlamydomonas lowest concentration for no 14 days 79.4 irradiance of 10 W/m2 Wängberg & dysosmos detectable growth Blanck (1988) Chlamydomonas 100% inhibition of motility 15 min 55.1 Stom & Roth reinhardii (1981) Chlorella emersonii lowest concentration for no 14 days 79.4 irradiance of 10 W/m2 Wängberg & detectable growth Blanck (1988) slight activation of growth 24 h 10.0 Devillers et al. (1990) Dunaliella marina activation of growth 24 h 10.0 Devillers et al. (1990) Dunaliella salina 100% inhibition of motility 15 min 330.3 Stom & Roth (1981) Euglena gracilis 100% inhibition of motility 15 min 7708 Stom & Roth (1981) Kirchneriella contorta lowest concentration for no 14 days 79.4 irradiance of 10 W/m2 Wängberg & detectable growth Blanck (1988) Klebsormidium lowest concentration for no 14 days 20.0 irradiance of 10 W/m2 Wängberg & marinum detectable growth Blanck (1988) Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Monodus lowest concentration for no 14 days 20.0 irradiance of 10 W/m2 Wängberg & subterraneus detectable growth Blanck (1988) Monoraphidium lowest concentration for no 14 days 20.0 irradiance of 10 W/m2 Wängberg & pusillum detectable growth 79.4 irradiance of 2 W/m2 Blanck (1988) Nitella sp. 100% inhibition of cytoplasmic 15 min 2753 Stom & Roth streaming (1981) Raphidonema lowest concentration for no 14 days 0.316 irradiance of 10 W/m2 Wängberg & longiseta detectable growth Blanck (1988) Scenedesmus lowest concentration for no 14 days 39.8 irradiance of 10 W/m2 Wängberg & obtusiusculus detectable growth Blanck (1988) Scenedesmus toxicity threshold concentration for 96 h 4 Bringmann & quadricauda inhibition of cell multiplication Kühn (1959a) toxicity threshold concentration for 7 days 0.93 Bringmann & inhibition of cell multiplication Kühn (1978) Selenastrum EC50, growth 3 days 0.335 irradiance of 17 W/m2 Devillers et capricornutum al. (1990) lowest concentration for no 14 days 79.4 irradiance of 10 W/m2 Wängberg & detectable growth Blanck (1988) Tribonema aequale lowest concentration for no 14 days 2.51 irradiance of 10 W/m2 Wängberg & detectable growth Blanck (1988) Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Yeasts Candida albicans activation of growth 24 h 500 Devillers et 50% inhibition of growth 24 h 3750 al. (1990) Candida tropicalis R2 100% inhibition of growth 24 h 1000 Devillers et al. (1990) Saccharomyces 50% inhibition of growth 24 h 2750 Devillers et cerevisiae al. (1990) Torulopsis glabrata 50% inhibition of growth 24 h 1000 Devillers et 100% inhibition of growth 3000 al. (1990) Fungi Fusarium oxysporum no significant inhibition of spore 1000 Ismail et al. f sp lycopersici germination (1987) no significant inhibition of length 1000 of germ tube Plants Elodea canadensis 50% inhibition of growth 9 days 42.9 Stom & Roth (1981) Lemna minor 50% inhibition of plant 12 days 7.71 Stom & Roth multiplication (1981) Vallisneria spiralis 100% inhibition of cytoplasmic 15 min 2753 in leaves Stom & Roth streaming 275.3 in roots (1981) Protozoa Chilomonas toxicity threshold concentration for 48 h 22 Bringmann & paramaecium inhibition of cell multiplication Kühn (1981) Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Colpidium campylum EC50, growth 24 h 73.3 Devillers et al. (1990) Entosiphon sulcatum toxicity threshold concentration for 72 h 11 Bringmann inhibition of cell multiplication (1978) Microregma toxicity threshold concentration for 28 h 2 Bringmann & nutrient uptake Kühn (1959b) Tetrahymena EC50, growth 60 h 95.0 Schultz et al. pyriformis (1987) Uronema parduczi toxicity threshold concentration for 20 h 21 Bringmann & inhibition of cell multiplication Kühn (1980) Mollusc Deroceras reticulatum 0% mortality 4 days 0.020a Briggs & 20% mortality 4 days 0.200a Henderson (1987) Crustacea Artemia salina LC50 2 h 321 Devillers et 4 h 67.5 al. (1990) 6 h 57.5 24 h 20.7 Crangon LT50 84 h 0.83 time to 50% mortality McLeese et al. septemspinosa (1979) Daphnia magna toxicity threshold concentration for 48 h 0.60 Bringmann & inhibition of mobility Kühn (1959a) Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Daphnia magna LC0 24 h 0.04 Bringmann & LC50 0.09 Kühn (1977b) LC100 0.31 EC0, inhibition of mobility 24 h 0.05 Bringmann & EC50 0.12 Kühn (1982) EC100 0.19 EC50, inhibition of mobility 24 h 0.137 Devillers et al. (1987) EC0, inhibition of mobility 24 h 0.13 Kühn et al. EC50 0.32 (1989) EC100 0.71 EC0 48 h 0.13 Kühn et al. EC50 0.29 (1989) EC100 0.71 EC50, inhibition of mobility 24 h 0.15 Tissot et al. (1985) Daphnia pulicaria LC50 48 h 0.162 DeGraeve et al. (1980) Daphnia pulex LC100 6 min 8809 Stom et al. (1986) Gammarus toxicity threshold concentration 1.5 Bandt (1955) Insect Apis mellifera LD50 24 h 0.200 concentration in mg per bee Devillers et al. (1990) Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Fish Brachydanio rerio LC50 24 h 0.265 Devillers et al. (1988) LC0 96 h 0.12 Wellens (1982) LC50 0.17 LC100 0.25 Carassius auratus 100% mortality 22 h 5 US EPA (1987) LC100 48 h 0.287 Sollmann (1949) Lepomis macrochirus 100% mortality 22 h 5 US EPA (1987) Leuciscus idus LC0 48 h 0.1 Juhnke & melanotus LC50 0.15 Lüdemann LC100 0.2 (1978) LC0 0.1 LC50 0.16 LC100 0.25 Oncorhynchus mykiss 100% mortality 2 h 7.69 Devillers et 4 h 4.50 al. (1990) LC50 96 h 0.097 DeGraeve et al. (1980) LC50 96 h 0.639 Hodson et al. LD50, intraperitoneal injection 24.2 concentration in mg per kg (1984) Table 18. (contd). Species Effect Test Hydroquinone Comments Reference duration concentration (mg/litre) Pimephales promelas LC50 96 h 0.044 DeGraeve et al. (1980) LT50 11 h 0.2 time to 50% mortality Terhaar et al. (1972) Salmo trutta 100% mortality 22 h 5 US EPA (1987) a Slugs injected with 2 µl of glycerol formal containing 20 or 200 µg of hydroquinone. 10. EVALUATION OF HUMAN HEALTH RISKS AND ON THE ENVIRONMENT 10.1 Toxicokinetics The property of hydroquinone most pertinent to its toxicity is its ability to undergo reversible redox reactions. Autooxidation of hydroquinone leads to p-benzoquinone and/or p-benzosemiquinone. These two strong electrophiles do not redox-cycle under physiological conditions to form active oxygen species, but readily arylate nucleophiles. They are probably the two major toxic metabolites of hydroquinone. Toxicokinetic studies with hydroquinone show that although it is readily absorbed from the gut of animals it has a low potential for bioaccumulation < 2% distributed out of total administered dose). Extensive conjugation and rapid excretion, primarily via the urine, suggests that hydroquinone is effectively detoxified. However, because hydroquinone is oxidized to p-benzosemiquinone and/or p-benzoquinone, which are able to readily react with nucleophilic body components, it represents a potentially harmful toxicant. Indeed, hydroquinone and/or its metabolites covalently bind to cellular components in vitro. It is, therefore, possible that although the bioaccumulation potential of hydroquinone is low critical body components may still be adversely affected. 10.2 Animal and in vitro studies Hydroquinone exhibits moderately high acute oral toxicity for animals, with LD50 values generally being in the range of 300 to 1300 mg/kg. However, cats are more sensitive, LD50 values of 40-85 mg/kg having been reported. The principle toxic effects of a single oral lethal dose of hydroquinone are increased motor activity, dyspnoea and cyanosis followed by convulsions, paralysis, coma and death. Repeated oral dosing has caused tremors and reduced activity (> 64 mg/kg), reduced body weight gain (> 200 mg/kg), convulsions (> 400 mg/kg) and adverse effects on the liver and kidneys (> 200 mg/kg). Dermal exposure of black guinea-pigs to hydroquinone (2 or 5%) has been found to cause depigmentation, inflammatory changes and thickening of the epidermis. Slight skin irritation has been recorded following topical application of hydrophillic ointment containing 1% hydroquinone. The results also showed that female guinea-pigs were more sensitive than males. Limited data suggest that powdered hydroquinone causes transient eye irritation and corneal opacity in dogs and guinea-pigs; in rabbits powdered hydroquinone induced brownish pigmentation of conjunctiva and cornea, but only after a period of at least 2-4 months. Hydroquinone is a skin sensitizer in rabbits. The ability to induce sensitization has been found to vary from "weak" to "strong" depending on the test procedure and vehicle used. The cross- reactivity of hydroquinone and p-methoxyphenol has been reported to be almost 100%. Early reproductive studies indicated reduced fertility in male rats and a disturbed sexual cycle in female rats when hydro-quinone was administered parentally. However, this was not confirmed in more recent studies in rats, i.e. a dominant lethality study and a two-generation study with oral doses of hydroquinone up to 300 mg/kg per day and 150 mg/kg per day, respectively. Oral dosing of 100 or 300 mg hydroquinone/kg to pregnant rats on days 6-15 of gestation caused maternal toxicity at the higher dose level (a statistically significant reduction in body weight gain and feed consumption). A reduction in mean fetal body weight was correlated with the reduced maternal body weight. No compound-related teratogenic effects were produced at this dose level; thus, 100 mg/kg was considered the NOEL for maternal and developmental toxicity in rats. Findings of increased resorption rates in rats given hydroquinone orally at about 100 mg/kg per day were not confirmed in this study, and, consequently, the NOAEL for maternal reproductive effects and teratogenicity was 300 mg/kg. In rabbits, 150 mg/kg caused reductions in body weight and feed intake and an increased (but not statistically significant) increase of malformations in the fetuses. The malformations may have been associated with maternal toxicity. The dose level of 200 mg/kg produced an increased number of resorptions, indicating embryotoxicity. The NOEL for developmental toxicity in rabbits was 25 mg/kg per day. In a two-generation reproduction study in rats the NOAEL for reproductive effects through two generations was 150 mg/kg per day (the highest tested dose). In mice, 80 mg hydroquinone/kg given orally on the 13th day of gestation transplacentally induced micronuclei in fetal liver cells. A single oral dose of 1000 mg/kg on gestation day 11 caused maternal toxicity (decreased weight gain) and an increased incidence of mortality. Reduced litter size and perinatal loss occurred in the treated groups (333, 667 and 1000 mg/kg), together with dose-related malformations of limbs, tail and urogenital system. In a rat embryo culture system, hydroquinone (effective concentration < 55 mg/litre, < 0.5 mmol/litre) caused growth retardation and an increased incidence of structural abnormalities involving tail and hindlimb buds. Genotoxicity data indicates that hydroquinone induces micronuclei, structural chromosome aberrations, and c-mitotic effects in vivo in mouse bone-marrow cells. in vitro studies with various cell lines showed that hydroquinone was capable of inducing gene mutations, structural chromosome aberrations, sister-chromatid exchange and DNA damage. Hydroquinone induces chromosome aberrations or karyotypic alterations in plant species and mitotic crossing-over in fungi. Hydroquinone is not mutagenic in the Salmonella/microsome test, but induces repairable DNA damage in Escherichia coli. It produces adducts with DNA. Hydroquinone induces chromosome aberrations in germ cells of male mice. Cholesterol pellets containing 20% hydroquinone implanted into the bladders of mice produced bladder tumours in 6 out of 19 mice surviving 25 weeks. However, the study was incompletely reported and the method is not generally recognized as a valid measure of carcinogenic potential as small pellets of cholesterol are known to induce transitional carcinoma of the bladder in both rats and mice. There is no support for hydroquinone being a stomach carcinogen in experimental animals after oral dosing. Hydroquinone produced an increase in the number of liver foci in rats at a dose level of 100 mg/kg per day for 7 weeks. However, increased dose levels (200 mg/kg per day for 7 weeks or 8 g/kg diet for about two years) caused a reduction in the number of foci of cellular alteration of the liver. In mice, the incidence of liver foci was increased when hydroquinone was added to the diet at 8 g/kg. Hydroquinone did not show any potential as an initiator or a co-carcinogen when dermally applied to mice before application of a tumour promoter (croton oil or BP) or following co-exposure with BP. Orally administered hydroquinone showed no promotion activity after MNNG initiation or carcinogenic potential in the forestomach and glandular stomach of rats. Most of the earlier carcinogenicity studies on hydroquinone lasted for less than one year, which might be considered too short for the assessment of carcinogenicity. Two-year studies performed recently give support for hydroquinone being a carcinogen in F-344 rats and B6C3F1 mice. In an NTP study, renal tubular cell adenomas occurred in male rats and leukaemia in females, and hepatocellular neoplasms, mainly adenomas, in female mice. The NTP concluded that these data indicated "some evidence of carcinogenic activity" in male and female rats and in female mice. In another study, renal tubular cell adenomas were again noted in male rats; hepatocellular adenomas occurred in male mice along with a biologically significant increase in the incidence of renal tubular cell adenomas. Both in vivo and in vitro studies have shown that hydroquinone causes direct myelotoxic effects in mouse bone marrow stromal cells by reducing bone marrow cellularity. In reducing the number of progenitor B-lymphocytes in mouse spleen and bone marrow, hydroquinone also demonstrates an immunosuppressive potential. Moreover, hydroquinone may inhibit the natural killer activity of mouse spleen cells in vitro, and have a selective effect on macrophage functions important in host defence. Hydroquinone has also demonstrated cytotoxic activity on various tumour cells such as cells from melanoma transplants and rat hepatoma cells. Central nervous system stimulatory effects have been produced in animal studies. However, functional-observational battery and neuropathological examinations failed to give any evidence of neurotoxicity after repeated dosing for 90 days. The NOEL was 20 mg hydroquinone/kg per day. 10.3 Evaluation of human health risks 10.3.1 Exposure Potential exposure of the general population to hydroquinone may occur through the consumption of foods that contain hydroquinone as a natural component, through smoking or exposure to cigarette smoke, or from the use of cosmetics and skin-lightening creams. People who use skin lighteners with concentrations of hydroquinone exceeding 2%, apply creams over large areas of the body or use creams for long time periods represent one group with significant and sometimes excessive exposure. Heavy cigarette smokers and those living and working in environments contaminated by cigarette smoke represent another group that experiences significant exposure to hydroquinone. Photohobbyists who develop their film manually may also be exposed to hydroquinone solutions through skin contact and inhalation. Exposure to hydroquinone may occur in a variety of occupations, particularly among those involved in its manufacture. 10.3.2 Human health effects Ingestion of large quantities of hydroquinone may produce tremors, vomiting, convulsions, dyspnoea, cyanosis and coma. Deaths have been reported to occur after ingestion estimated at 3-12 g of hydroquinone in developing agents. In studies with human volunteers, ingestion of up to 500 mg hydroquinone per day over a 20-week period resulted in no observable pathological changes in the blood and urine. Dermal exposure to hydroquinone causes skin depigmentation; cases of ochronosis, patchy depigmentation and brown staining of the nails after repeated usage of skin-lightening products have been reported. Hydroquinone has shown a sensitizing potential in both animals and humans. Eye irritation, sensitivity to light, staining of the cornea and conjunctiva, corneal opacities and visual disturbances are associated with long-term occupational exposure to airborne hydroquinone. Isolated cases of corneal ulceration have also been described. Effects on the central nervous system have been seen in cases of acute human poisoning. Similar symptoms have been observed in animal studies; these effects were reversible when exposure was discontinued. Nephrotoxicity has been seen in F-344 rats dosed repeatedly with hydroquinone. Male rats are more susceptible to these effects than females. Nephrotoxic effects due to hydroquinone have not been observed in humans. Myelotoxic and immunotoxic effects have been observed in animals exposed to hydroquinone. However, the routes of exposure differed from those via which humans are normally exposed. Studies conducted by routes of exposure similar to those by which humans are exposed have not revealed specific reproductive and developmental effects. Numerous studies using cell culture systems and in vivo rodent experiments have shown that co-exposure to hydroquinone and various phenolic compounds can result in toxic effects that are substantially greater than the sum of the effects of the individual compounds. The relevance of these interactive effects in understanding and predicting the toxic effects of human exposure to hydroquinone is uncertain. However, since many of the human exposures to hydroquinone occur under conditions in which other phenolic compounds are present, the possibility that significant interactive effects may occur should be considered. Several experiments with hydroquinone in vivo and in vitro have shown mutagenic effects; the relevance of these results to human risk is uncertain. The evidence for carcinogenicity in animals is limited. Adequate epidemiological studies are lacking, and, at present, the experimental data are insufficient to allow a thorough assessment of the carcinogenic potential for humans. 10.4 Evaluation of effects on the environment When hydroquinone is released into the environment it is distributed mainly to the water compartment due to its physicochemical properties. However, hydroquinone can be degraded both photochemically and biologically and will therefore not persist in the environment. The ecotoxicity of hydroquinone, which can be also related to its physicochemical properties, is generally high but varies from species to species. Therefore, a battery of tests using organisms occupying different trophic levels in the ecosystems is required to assess thoroughly the adverse effects of hydroquinone in the environment. 11. RECOMMENDATIONS a) In view of the widespread inappropriate use of skin-lightening creams, it is recommended that over-the-counter sales of creams containing hydroquinone be restricted. Health Education Programmes should be developed to discourage the use of hydroquinone-containing creams for whole body skin lightening. b) Further investigations into the safety of long-term use of creams containing 1-2% hydroquinone is needed. c) Hydroquinone in waste water effluent should be allowed sufficient time for degradation before reaching recipient water. d) Multispecies toxicity testing performed under controlled experimental conditions is required to make a thorough assessment of the environmental effects of hydroquinone and its derivatives. e) Epidemiological studies, including precise exposure data, would assist in an assessment of the occupational hazards from hydroquinone. 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J Water Pollut Control Fed, 40: 354-370. Zeidman I & Deutl R (1945) Poisoning by hydroquinone and mono-methyl-paraaminophenol sulfate. Am J Med Sci, 210: 328-333. APPENDIX 1. Bibliographic data bases consulted Agricola Aqualine Aquatic Science and Fisheries Abstracts BIBLINE BIBRA Biosis Previews CA Search CAB Abstracts Cancerlit CHEMLIST CHRIS ECDIN Enviroline EXICHEM FEDREG Food Science and Technical Abstracts HSDB Medline Oceanic Abstracts OHMTADS RISKLINE RTECS ROADMAPS SERIX Toxline TOXLIT TSCATS RESUME 1. Identité, propriétés physiques et chimiques et méthodes d'analyse L'hydroquinone (1,4-benzènediol; C6H4(OH)2) se présente à l'état pur sous la forme d'un solide cristallin blanc dont le point de fusion est égal à 173-174°C. Sa densité est de 1,332 à 15°C et sa tension de vapeur de 2,4 x 10-3 Pa (1,8 x 10-5 mmHg) à 25°C. Elle est extrêmement soluble dans l'eau (70g/litre à 25°C) et le logarithme de son coefficient de partage n-octanol/eau est de 0,59. Sa solubilité dans les solvants organiques varie de 57% dans l'éthanol à moins de 0,1% dans le benzène. L'hydroquinone est combustible à condition de subir un chauffage préalable. C'est un réducteur qui est oxydé réversiblement en semiquinone correspondante et en quinone. L'échantillonnage de l'hydroquinone dans l'air s'effectue soit par piégeage dans un solvant soit par filtration sur membrane d'ester cellulosique mixte. Le dosage de l'hydroquinone s'effectue soit par titrimétrie soit par spectrophotométrie ou plus couramment par chromatographie. 2. Sources d'exposition humaine et environnementale L'hydroquinone existe sous forme libre ou conjuguée dans les bactéries, les plantes et certains animaux. Plusieurs pays la produisent en quantités industrielles. En 1979, la capacité mondiale totale de production dépassait 40 000 tonnes et était retombée à environ 35 000 tonnes en 1992. L'hydroquinone est très largement utilisée comme réducteur, comme développateur en photographie ainsi que antioxydant ou comme stabilisant pour certaines substances qui se polymérisent en présence de radicaux libres; elle sert encore d'intermédiaire dans la production des antioxydants, antiozonants, produits agrochimiques et polymères. L'hydroquinone est également utilisée pour la fabrication de cosmétiques et de préparations médicinales. 3. Transport, distribution et transformation dans l'environnement L'hydroquinone qui est présente dans l'environnement est le produit de l'activité humaine mais elle se trouve également dans des substances naturelles d'origine végétale ou animale. En raison de ses propriétés physico-chimiques, l'hydroquinone se répartit essentiellement dans le compartiment aquatique lorsqu'elle est libérée dans l'environnement. Sa décomposition résulte principalement de processus photochimiques et biologiques; elle n'est donc pas persistante et ne manifeste aucune tendance à la bioaccumulation. 4. Concentrations dans l'environnement et exposition humaine On ne dispose d'aucune données sur la concentration de l'hydroquinone dans l'air, le sol ou l'eau. Toutefois le dosage de l'hydroquinone dans la fumée de cigarettes (courant principal) sans bout-filtre a donné des résultats allant de 110 à 300 ug/cigarette, résultats qui valent également pour la fumée du courant latéral. On trouve également de l'hydroquinone dans des denrées alimentaires d'origine végétale (par exemple le germe de blé), dans le café infusé, dans les thés préparés à partir des feuilles de certaines baies, avec des concentrations pouvant dépasser quelquefois 1%. Les photographes amateurs peuvent être exposés à l'hydroquinone par voie percutanée ou respiratoire. Toutefois on ne dispose d'aucune donnée sur l'importance de cette exposition. L'exposition percutanée peut également résulter de l'utilisation de produits cosmétiques ou médicinaux contenant de l'hydroquinone, tels que les éclaircissants. Dans les pays de la Communauté européenne, la teneur des cosmétiques en hydroquinone est limitée à 2% au maximum. Aux Etats-Unis d'Amérique, la Food and Drug Administration a proposé une concentration comprise entre 1,5 et 2% pour les éclaircissants cutanés. La concentration peut atteindre 4% dans certains médicaments délivrés sur ordonnance. Dans certains pays, les éclaircissants cutanés peuvent en contenir des quantités encore plus importantes. Du point de vue hygiène et sécurité, on ne dispose que de peu de données concernant la surveillance de l'hydroquinone. On indique des concentrations moyennes dans l'air au cours de la fabrication de l'hydroquinone et des divers traitements subis par cette substance, qui se situeraient dans la gamme de 0,13 à 0,79 mg/m3. Les limites d'exposition professionnelles dans l'air (moyenne pondérée par rapport au temps) dans les différents pays vont de 0,5 à 2 mg/m3. 5. Cinétique et métabolisme L'hydroquinone est rapidement et largement résorbée chez l'animal au niveau de l'intestin et de la trachée. L'absorption percutanée est plus lente mais elle peut s'accélérer en présence de véhicules tels que les alcools. L'hydroquinone se répartit rapidement et largement dans les différents tissus. Elle est métabolisée en p-benzoquinone et autres produits d'oxydation et sa détoxification s'effectue par conjugaison sous forme de monoglucuronide, monosulfate et mercapturates. L'excrétion de l'hydroquinone et de ses métabolites est rapide et s'effectue principalement par la voie urinaire. L'hydroquinone et/ou ses dérivés réagissent avec différents constituants biologiques tels que les macromolécules et les molécules de faible masse moléculaire et ils exercent des effets sur la métabolisme cellulaire. 6. Effets sur les mammifères de laboratoire et les systèmes d'épreuve in vitro Les valeurs de la DL50 par voie orale varient de 300 à 1300 mg/kg de poids corporel pour un certain nombre d'espèces animales. Toutefois pour le chat, elle se situe entre 42 et 86 mg/kg de poids corporel. Une intoxication aiguë par de fortes concentrations d'hydroquinone entraîne de graves effets sur le système nerveux central et notamment une hyperexcitabilité, des tremblements, des convulsions, le coma et la mort. A des doses sublétales, ces effets sont réversibles. Pour les rongeurs, on estime la DL50 par voie percutanée à > 3800 mg/kg. On ne dispose d'aucun renseignement sur les valeurs de la CL50. L'épidermo-réaction effectuée en une seule fois au moyen d'une préparation à 2% d'hydroquinone a provoqué une irritation chez le lapin notée à 1,22 sur une échelle allant de 0 à 4. Des applications topiques quotidiennes effectuées pendant trois semaines avec de l'hydroquinone à 2 ou 5% dans une émulsion huile/eau, sur la peau rasée de cobayes noirs, a entraîné une dépigmentation, des altérations inflammatoires et un épaississement de l'épiderme. La dépigmentation était plus marquée à fortes concentrations et les femelles se sont révélées plus sensibles que les mâles. Les tests de sensibilisation effectués sur des cobayes suscitent des réactions faibles à fortes selon la méthode ou le véhicule utilisé. Les réactions les plus fortes ont été obtenues avec le test de sensibilisation maximale sur le cobaye. On a également observé une sensibilisation croisée de presque 100 pour cent entre l'hydroquinone et le p-méthoxyphénol chez le cobaye mais les éléments qui pourraient militer en faveur de l'existence de réactions analogues avec la p-phénylénediamine, l'acide sulfanilique et la p-benzoquinone restent limités. Une étude de toxicité par voie orale de six semaines chez des rats mâles F-344 a permis de mettre en évidence des néphropathies et une prolifération des cellules rénales. Après 13 semaines de gavage, on a mis en observation des rats F-344 et des souris B6C3F1. Des signes de néphrotoxicité se sont manifestés chez les rats aux doses respectives de 100 et 200 mg/kg, avec des tremblements et des convulsions à cette dernière dose; chez les deux espèces, on a observé une diminution du gain de poids. L'administration d'une dose de 400 mg/kg a entraîné la mort des rats. Chez les souris qui avaient reçu cette même dose pendant 13 semaines, on a relevé des tremblements, des convulsions et des lésions affectant l'épithélium gastrique. Des rats Sprague Dawley exposés pendant 13 semaines à de l'hydroquinone ont présenté une réduction du gain de poids et des signes neurologiques témoignant d'une atteinte centrale à 200 mg/kg. Ces signes ont également été observés à la dose de 64 mg/kg de poids corporel mais ils étaient absents à 20 mg/kg. Après injection sous-cutanée d'hydroquinone à des rats, on a observé une diminution de la fécondité chez les mâles et un allongement du cycle oestral chez les femelles. Toutefois ces effets n'ont pas été observés dans les études portant sur une administration par voie orale (étude de létalité dominante et étude sur deux générations). Une étude portant sur le développement de rats ayant reçu par voie orale des doses de 300 mg/kg de poids corporel a révélé une légère toxicité pour les femelles gravides et une réduction du poids du foetus. Chez le lapin, la dose sans effets toxiques observables sur la mère était de 25 mg/kg/jour; la dose sans effets toxiques sur le développement était de 75 mg/kg/jour. Lors d'une étude de deux générations portant sur la reproduction de rats, l'administration d'hydroquinone n'a entraîné aucun effet nocif sur la reproduction à des doses orales quotidiennes allant jusqu'à 150 mg/kg de poids corporel. La dose sans effets toxiques observables pour les géniteurs a été fixée par 15 mg/kg/jour; en ce qui concerne les effets sur la reproduction observés en l'espace de deux générations, on a obtenu une valeur de 150 mg/kg/jour. L'hydroquinone provoque la formation de micronoyaux in vivo et in vitro. Des aberrations portant sur la structure et le nombre des chromosomes ont été observées in vitro ainsi qu'après administration intrapéritonéale in vivo. En outre, on a pu mettre en évidence in vitro des mutations géniques, des échanges entre chromatides soeurs et des lésions de l'ADN. Après injection intrapéritonéale d'hydroquinone à des souris, on a observé, dans les cellules germinales des mâles, de aberrations chromosomiques d'une ampleur comparable à celles que l'on observait dans les cellules de la moelle osseuse. Une épreuve de létalité dominante effectuée sur des rats mâles recevant de l'hydroquinone par voie orale n'a pas permis d'établir l'existence de mutations au niveau des cellules germinales. Lors d'une étude de deux ans, au cours de laquelle on a administré à des rats F-344/N de l'hydroquinone par voie orale, on a observé, chez les mâles, des adénomes affectant les tubules rénaux dont la fréquence était liée à la dose. L'incidence de ces adénomes était statistiquement significative dans le groupe qui recevait une forte dose. Dans ce même groupe, on a également observé une hyperplasie des cellules tubulaires rénales. Chez les femelles, on a observé un accroissement de l'incidence, lié à la dose, des leucémies à monocytes. Chez des souris B6C3F1, on a observé une incidence sensiblement accrue des adénomes hépatocellulaires. Dans une autre étude, l'hydroquinone administrée dans la proportion de 0,8% de la nourriture, a entraîné une augmentation significative de l'incidence de l'hyperplasie épithéliale des papilles rénales et une augmentation également significative des hyperplasies et des adénomes au niveau des tubules rénaux chez les rats mâles. En revanche, on n'a pas observé d'augmentation dans l'incidence des leucémies à monocytes chez les femelles. Chez les souris, l'incidence de l'hyperplasie spinocellulaire affectant l'épithélium de la portion cardiaque de l'estomac présentait une augmentation significative dans les deux sexes. Chez les mâles, on notait également une incidence sensiblement accrue des adénomes hépatocellulaires et des hyperplasies tubulaires rénales. Quelques adénomes rénaux ont été observés. Des études sur des souris, effectuées in vivo (injection intrapéritonéale) et in vitro montrent que l'hydroquinone a un effet cytotoxique, à savoir qu'elle diminue la cellularité médullaire et splénique et qu'elle possède également un pouvoir immunodépresseur puisqu'elle inhibe la maturation des lymphocytes-B et bloque l'activité des cellules tueuses naturelles. Les résultats obtenus indiquent également que les macrophages de la moelle osseuse pourraient être les principales cibles des effets myélotoxiques exercés par l'hydroquinone. Cependant, une étude biologique au long cours sur des rongeurs n'a pas révélé l'existence d'effets myélotoxiques. Une étude de 90 jours sur des rats au cours de laquelle on a utilisé une batterie de tests fonctionnels et observationnels, a montré qu'à des doses respectives de 64 et 200 mg d'hydroquinone/kg, les animaux étaient pris de tremblements et qu'à la dose de 200 mg/kg, il y avait réduction de l'activité générale. L'examen anatomopathologique du système nerveux n'a rien donné. 7. Effets sur l'homme On a fait état de cas d'intoxication consécutifs à l'ingestion d'hydroquinone seule ou de développateurs photographiques contenant de l'hydroquinone. Les principaux signes de ces intoxications étaient les suivants: urines foncées, vomissements, douleurs abdominales, tachycardie, tremblements, convulsions et coma. On a également signalé des décès après l'ingestion de développateurs photographiques à base d'hydroquinone. Lors d'une étude contrôlée au cours de laquelle des volontaires humains ont ingéré quotidiennement pendant 3 à 5 mois 300 à 500 mg d'hydroquinone, on n'a pas relevé le moindre signe pathologique dans le sang et les urines. L'application cutanée d'hydroquinone dans divers excipients à des concentrations inférieures à 3% n'a causé que des effets négligeables sur des volontaires humains appartenant à diverses races. Cependant, on dispose de rapports selon lesquels des crèmes destinées à éclaircir la peau et contenant 2% d'hydroquinone ont produit une leucodermie ainsi qu'une ochronose. L'hydroquinone (solution aqueuse à 1% ou crème à 5%) peut provoquer des irritations (érythème ou taches épidermiques). On a également observé des dermatites de contact d'origine allergique dues à l'hydroquinone. Une double exposition à de l'air chargé d'hydroquinone et de quinone entraîne une irritation oculaire, une photophobie, des lésions de l'épithélium cornéen, voire des ulcères cornéens et des troubles visuels. On connaît des cas où l'acuité visuelle a sensiblement baissé. Une irritation peut se manifester à partir de 2,25 mg/m3. Une exposition de longue durée peut faire apparaître des taches sur la conjonctive et la cornée et provoquer l'opacification de cette dernière. Après exposition quotidienne pendant au moins deux ans à 0,05-14,4 mg d'hydroquinone/m3, on a observé une inflammation et une dyschromie de la cornée et de la conjonctive; on n'a pas observé de cas graves avant cinq ans au moins d'exposition. On dispose d'un rapport qui décrit des cas de lésions cornéennes apparues plusieurs années après cessation de l'exposition à l'hydroquinone. On ne dispose pas de données épidémiologiques suffisantes pour évaluer la cancérogénicité de l'hydroquinone chez l'homme. 8. Effets sur les autres êtres vivants au laboratoire et dans leur milieu naturel Pour expliquer le comportement écotoxicologique de l'hydroquinone il faut se rapporter à ses propriétés physico-chimiques, et notamment à sa sensibilité à la lumière, au pH et à l'oxygène dissous. L'écotoxicité de l'hydroquinone, qui est généralement élevée (par exemple < 1 mg/litre pour les organismes aquatiques), varie d'une espèce à l'autre. Les algues, les levures, les champignons et les plantes en général sont moins sensibles à l'hydroquinone que les autres organismes habituellement utilisés pour les épreuves toxicologiques. Toutefois,au sein d'un même groupe toxonomique, la sensibilité des différentes espèces à l'hydroquinone peut varier d'un facteur 1000. RESUMEN 1. Identidad, propiedades físicas y químicas y métodos analíticos La hidroquinona (1,4-bencenodiol; C6H4(OH)2) es una sustancia cristalina blanca en estado puro, cuyo punto de fusión es de 173-174 °C. Su peso específico es de 1,332 a 15 °C, y su presión de vapor, de 2,4 x 10-3Pa (1,8 x 10-5 mmHg) a 25 °C. Es muy hidrosoluble (70 g/litro a 25 °C), y el logaritmo de su coeficiente de reparto n-octanol/agua es de 0,59. Por lo que se refiere a los disolventes orgánicos, su solubilidad varía entre el 57% para el etanol, y menos del 0,1% para el benceno. La hidroquinona es combustible si se calienta previamente. Es un agente reductor, que se oxida reversiblemente transformándose en semiquinona y quinona. Se pueden obtener muestras de la hidroquinona presente en el aire capturándola, bien sea en un disolvente o sobre una membrana- filtro de éster de celulosa mixto. Para analizar los niveles de hidroquinona se utilizan técnicas de valorimetría, espectrofotometría o, muy a menudo, cromatografía. 2. Fuentes de exposición humana y ambiental La hidroquinona está presente en forma tanto libre como conjugada en bacterias, plantas y algunos animales. Varios países la producen industrialmente. En 1979, la capacidad mundial total de producción de esta sustancia superaba las 40 000 toneladas, mientras que en 1992 fue de aproximadamente 35 000 toneladas. Es ampliamente utilizada como agente reductor, como medio de revelado fotográfico, como antioxidante o estabilizador de ciertos materiales que se polimerizan en presencia de radicales libres, y como producto químico intermedio en la producción de antioxidantes, agentes antiozono, productos agroquímicos y polímeros. La hidroquinona se emplea también en la elaboración de productos cosméticos y preparados médicos. 3. Transporte, distribución y transformación en el medio ambiente La hidroquinona presente en el medio ambiente procede de la actividad del hombre o forma parte de productos naturales de las plantas y animales. Debido a sus propiedades fisicoquímicas, la hidroquinona liberada en el medio penetra sobre todo en los compartimientos de agua. Se degrada como resultado de procesos tanto fotoquímicos como biológicos, por lo que no persiste en el medio. No se produce bioacumulación. 4. Niveles ambientales y exposición humana No se han hallado datos sobre las concentraciones de hidroquinona en la atmósfera, el suelo o el agua. Sin embargo, se ha analizado la hidroquinona presente en la corriente principal de humo emitida por cigarrillos sin filtro, en la que se han hallado entre 110 y 300 µg por cigarrillo, así como en el humo lateral. Se ha hallado hidroquinona en alimentos derivados de las plantas (por ejemplo, el germen de trigo), en el café listo para beber y en los tes preparados a partir de las hojas de algunas bayas en que la concentración supera a veces el 1%. Los aficionados a la fotografía pueden verse expuestos a la hidroquinona por vía cutánea o por inhalación; sin embargo, no se dispone de datos sobre niveles de exposición. Otra causa de exposición cutánea es el uso de productos cosméticos o médicos con hidroquinona, como los utilizados para aclarar el color de la piel. Los países de la Comunidad Europea (CE) han restringido su empleo en los productos cosméticos a un máximo del 2%. En los Estados Unidos, la Administración de Alimentos y Medicamentos ha propuesto concentraciones de entre 1,5% y 2% para los productos de maquillaje decolorante. Algunos medicamentos de venta con receta contienen concentraciones de hasta un 4%, y en ciertos países se venden cosméticos decolorantes que contienen concentraciones incluso mayores. Son pocos los datos disponibles sobre la hidroquinona por lo que se refiere a la vigilancia de la higiene industrial. Según se ha señalado, el valor promedio de las concentraciones que este producto alcanza en el aire durante su fabricación y transformación oscila entre 0,13 y 0,79 mg/m3. El límite de la exposición atmosférica ocupacional (promedio ponderado por el tiempo) oscila entre 0,5 y 2 mg/m3, según los países. 5. Cinética y metabolismo La hidroquinona es rápida y ampliamente absorbida en el tubo digestivo y la tráquea de los animales. La absorción cutánea es más lenta, pero se ve acelerada por vehículos como los alcoholes. La hidroquinona se distribuye de forma rápida y generalizada por los tejidos. Es metabolizada en p-benzoquinona y otros productos de oxidación, y la detoxificación se produce por conjugación, formándose los derivados monoglucurónido, monosulfato y mercaptúrico. La excreción de la hidroquinona y de sus metabolitos es rápida y se produce principalmente a través de la orina. La hidroquinona y/o sus derivados reaccionan con distintos componentes biológicos, como macromoléculas o moléculas de bajo peso molecular, y tienen efectos sobre el metabolismo celular. 6. Efectos en mamíferos de laboratorio y en los sistemas in vitro Los valores de la DL50 por vía oral en varias especies animales oscilan entre 300 y 1300 mg/kg de peso corporal. En el gato, sin embargo, la DL50 varía entre 42 y 86 mg/kg de peso corporal. La exposición aguda a altos niveles de hidroquinona tiene efectos graves sobre el sistema nervioso central (SNC), que abarcan desde la hiperexcitabilidad, pasando por temblores, convulsiones y coma, hasta la muerte. A dosis subletales esos efectos son reversibles. Se ha calculado que la DL50 por vía cutánea es de > 3800 mg/kg en roedores. No se dispone de los valores de la CL50 por inhalación. Un preparado de hidroquinona al 2% aplicado a conejos mediante una única prueba del parche tuvo unos efectos irritantes de 1,22 (en una escala de 0 a 4). La aplicación tópica diaria durante tres semanas de hidroquinona al 2% o 5% en una emulsión de aceite/agua sobre la piel depilada de cobayos negros provocó despigmentación, cambios inflamatorios y espesamiento de la epidermis. La despigmentación fue más marcada con las concentraciones mayores, y los cobayos hembra se revelaron más sensibles que los machos. Las pruebas de sensibilización realizadas en cobayos han provocado respuestas de carácter entre débil y fuerte, según los métodos o vehículos empleados. Las reacciones más intensas fueron las observadas en la prueba de maximización aplicada a cobayos. Se observó también en estos animales una sensibilización cruzada de casi el 100% entre la hidroquinona y el p-metoxifenol, pero los indicios de reacción cruzada con la p-fenilendiamina, el ácido sulfanílico y la p-benzoquinona fueron sólo limitados. Un estudio de toxicidad oral realizado durante seis semanas con ratas macho F-344 reveló la aparición de nefropatía y proliferación de las células renales. En estudios de sobrealimentación forzada mediante sonda esofágica llevados a cabo durante 13 semanas con ratas F-344 y ratones B6C3F1, se observaron en las ratas signos de nefrotoxicidad a dosis de 100 y 200 mg/kg, temblores y convulsiones a dosis de 200 mg/kg, así como una disminución del aumento del peso corporal tanto en las ratas como en los ratones. Las dosis de 400 mg/kg resultaron letales para las ratas. En los ratones sometidos durante 13 semanas a dosis de 400 mg/kg se observaron temblores, convulsiones y lesiones del epitelio gástrico. La exposición de ratas Sprague Dawley a hidroquinona durante 13 semanas dio lugar a una atenuación del aumento del peso corporal y la aparición de signos de afección del SNC a la dosis de 200 mg/kg. Estos signos neurológicos se observaron también a la dosis de 64 mg/kg de peso corporal, pero no así a la de 20 mg/kg. La hidroquinona inyectada por vía subcutánea redujo la fecundidad de las ratas macho y prolongó el ciclo menstrual de las ratas hembra. Esa observación no se reprodujo en cambio en los estudios de administración oral (un estudio de dominancia letal y otro efectuado con dos generaciones). En un estudio sobre el desarrollo de la rata, dosis orales de 300 mg/kg de peso corporal tuvieron un ligero efecto tóxico en las madres y provocaron una disminución del peso corporal del feto. En el conejo, el nivel sin efectos observados (NOEL) por lo que se refiere a la toxicidad materna fue de 25 mg/kg al día, y de 75 mg/kg al día en lo que respecta a la toxicidad ontogénica. En un estudio de los efectos sobre la reproducción realizado con dos generaciones de ratas, la hidroquinona no tuvo efecto alguno a dosis orales de hasta 150 mg/kg de peso corporal al día. El nivel sin efectos adversos observados (NOAEL) fue de 15 mg/kg al día por lo que hace a la toxicidad materna, y de 150 mg/kg al día en lo referente a los efectos sobre la reproducción observados a lo largo de dos generaciones. La hidroquinona tiene un efecto inductor sobre los micronúcleos in vivo e in vitro. Se han observado aberraciones cromosómicas estructurales y numéricas in vitro y tras la administración intraperitoneal in vivo. Se ha demostrado además la inducción de mutaciones genéticas, intercambio de cromátides hermanas y lesiones del ADN in vitro. La hidroquinona causó aberraciones cromosómicas en las células germinales de ratones macho, del mismo orden de magnitud que en las células de médula ósea de esa misma especie tras inyección intraperitoneal. En una prueba de dominancia letal llevada a cabo con ratas macho tratadas por vía oral no se observó ningún efecto de inducción de mutaciones de las células germinales. En un estudio de dos años de duración, la administración oral de hidroquinona provocó una incidencia dosisdependiente de adenomas de las células tubulares renales en ratas macho F-344/N. La incidencia fue estadísticamente significativa en el grupo tratado con la dosis más alta; en los machos sometidos a esa dosis se halló también hiperplasia de las células tubulares renales. En las ratas hembra se observó un aumento dosisdependiente de la incidencia de leucemia de células mononucleares. En los ratones hembra B6C3F1 se observó una incidencia significativamente mayor de adenomas hepatocelulares. En otro estudio, la hidroquinona (presente a un nivel del 0,8% en la ingesta alimentaria) provocó un aumento significativo de la incidencia de hiperplasia epitelial de la papila renal y un aumento significativo de la incidencia de adenomas e hiperplasia de los túbulos renales en las ratas macho. En las ratas hembra no se observó ningún incremento de la incidencia de leucemia de células mononucleares. En los ratones, la incidencia de hiperplasia de las células escamosas del epitelio del antro cardiaco aumentó significativamente en los dos sexos. En los ratones macho se observó un aumento significativo de la incidencia de adenomas hepatocelulares, así como de hiperplasia tubular renal. Se observó también un reducido número de adenomas de células renales. Los estudios realizados in vivo (inyección intraperitoneal) e in vitro con ratones han demostrado que el efecto citotóxico de la hidroquinona se debe a que reduce la celularidad de la médula ósea y del bazo, y a su posible efecto inmunosupresor, mediado por la inhibición de la maduración de los linfocitos B y de la actividad natural de las células asesinas. Los resultados indican además que las células más afectadas por la mielotoxicidad de la hidroquinona son quizá los macrófagos de la médula ósea. En una biovaloración prolongada realizada con roedores no se observaron esos efectos mielotóxicos. En un estudio realizado durante 90 días con ratas mediante una batería de pruebas funcionales y de observación, se advirtieron temblores a dosis de 64 y 200 mg de hidroquinona/kg, dosis esta última que además provocó una disminución de la actividad general. El resultado de los análisis neuropatológicos fue negativo. 7. Efectos en el hombre Se han notificado casos de intoxicación tras la ingestión oral de hidroquinona sola o de productos de revelado fotográfico que contenían dicho producto. Los principales signos de intoxicación fueron el oscurecimiento de la orina, vómitos, dolor abdominal, taquicardia, temblores, convulsiones y coma. Se han notificado muertes provocadas por la ingestión de productos de revelado fotográfico que contenían hidroquinona. En un estudio controlado realizado con voluntarios, la ingestión de 300-500 mg de hidroquinona diarios durante 3-5 meses no produjo cambios patológicos observables en la sangre y la orina. La aplicación cutánea de distintas bases que contenían concentraciones de hidroquinona inferiores al 3% tuvo efectos insignificantes en hombres voluntarios de distintas razas. Sin embargo, se han notificado algunos casos que llevan a pensar que hay cremas cutáneas decolorantes con hidroquinona al 2% que han provocado la aparición de leucoderma, así como de ocronosis. Se han producido casos de irritación (eritema o coloración) por hidroquinona (en solución acuosa al 1% o en forma de crema al 5%). También se han diagnosticado casos de dermatitis alérgica de contacto por hidroquinona. La exposición simultánea a hidroquinona y quinona presentes en el aire causa irritación ocular, sensibilidad a la luz, lesiones del epitelio corneal, úlceras corneales y trastornos visuales. En algunos casos se han producido pérdidas considerables de visión. Se han observado efectos irritantes a niveles de exposición de 2,25 mg/m3 o más. La exposición prolongada provoca coloración de la conjuntiva y la córnea, así como opacidad. La exposición diaria durante al menos dos años a concentraciones de hidroquinona de 0,05- 14,4 mg/m3 ha dado lugar al lento desarrollo de inflamación y decoloración de la córnea y la conjuntiva; pero no se han observado casos graves hasta transcurridos al menos 5 años. En un estudio se describieron casos de aparición de lesiones de la córnea al cabo de varios años de interrumpida la exposición a la hidroquinona. No se dispone de datos epidemiológicos adecuados para evaluar la carcinogenicidad de la hidroquinona en el hombre. 8. Efectos en otros organismos en el laboratorio y sobre el terreno Los efectos ecotoxicológicos de la hidroquinona guardan relación con sus propiedades fisicoquímicas, entre ellas su sensibilidad a la luz, al pH y al oxígeno disuelto. Su ecotoxicidad, que por lo general es alta (por ejemplo, < 1 mg/litro para los organismos acuáticos), varía de una especie a otra. Las algas, levaduras, hongos y plantas son menos sensibles a la hidroquinona que los otros organismos empleados habitualmente para evaluar la toxicidad. No obstante, dentro de un mismo grupo taxonómico, la sensibilidad de las distintas especies a la hidroquinona puede variar de 1 a 1000.
See Also: Hydroquinone (CHEMINFO) Hydroquinone (ICSC)