INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 174 Isophorone 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 at the National Institute of Health Sciences, Tokyo, Japan, and the Institute of Terrestrial Ecology, Monk's Wood, United Kingdom Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Organization Geneva, 1995 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 Linear Alkylbenzene Sulfonates and Related Compounds. (Environmental health criteria ; 174) 1.Cyclohexanones 2.Environmental exposure 3.Occupational exposure 4.Solvents I.Series ISBN 92 4 157174 8 (NLM Classification: QV 633) 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 1995 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 expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any 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 ISOPHORONE Preamble 1. SUMMARY AND EVALUATION, CONCLUSIONS AND RECOMMENDATIONS 1.1. Summary and evaluation 1.1.1. Physical and chemical properties 1.1.2. Production and use 1.1.3. Environmental transport, distribution and transformation 1.1.4. Environmental levels and human exposure 1.1.5. Kinetics and metabolism in laboratory animals and humans 1.1.6. Effects on laboratory mammals and in vitro test systems 1.1.7. Effect on humans 1.1.8. Effects on other organisms in the laboratory and field 1.2. Conclusions 1.2.1. General population 1.2.2. Occupational exposure 1.2.3. The environment 1.3. Recommendations 1.3.1. Protection of human health and the environment 1.3.2. Further research 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.3. Conversion factors 2.4. Analytical methods 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Environmental distribution 4.2. Biotransformation and environmental fate 4.2.1. Atmospheric fate 4.2.2. Aquatic fate 4.2.3. Terrestrial fate 4.2.4. Biodegradation 4.2.5. Bioaccumulation 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air 5.1.2. Water 5.1.3. Soil and sediment 5.1.4. Terrestrial organisms 220.127.116.11 Plants 18.104.22.168 Animals 5.1.5. Aquatic organisms 5.2. General population exposure 5.3. Occupational exposure 6. KINETICS AND METABOLISM 6.1. Human 6.2. Laboratory mammals 7. EFFECTS ON LABORATORY MAMMALS, AND IN VITRO TEST SYSTEMS 7.1. Acute toxicity 7.1.1. Oral 22.214.171.124 ß-Isophorone 7.1.2. Dermal 7.1.3. Inhalation 7.2. Skin, eye and respiratory irritation, sensitization 7.2.1. Skin irritation 126.96.36.199 ß-Isophorone 7.2.2. Eye irritation 188.8.131.52 ß-Isophorone 7.2.3. Respiratory irritation 7.2.4. Sensitization 7.3. Subchronic toxicity 7.3.1. Inhalation 7.3.2. Oral 7.3.3. Dermal 7.4. Mutagenicity 7.4.1. Gene mutation in bacteria (Ames tests) 184.108.40.206 Dihydroisophorone 7.4.2. Gene mutation in mammalian cells 7.4.3. Chromosome aberrations and sister chromatid exchange 220.127.116.11 Chromosome aberrations 18.104.22.168 Sister chromatid exchange 7.4.4. Micronucleus test 7.4.5. Primary DNA damage 22.214.171.124 Bacterial tests 126.96.36.199 Unscheduled DNA synthesis 188.8.131.52 DNA binding 7.4.6. Morphological transformation 7.5. Chronic toxicity and carcinogenicity 7.6. Mechanisms of toxicity 7.7. Appraisal for mutagenicity/carcinogenicity 7.8. Reproduction, embryotoxicity, teratogenicity 7.9. Neurotoxicity 7.10. Other special studies 8. EFFECTS ON HUMANS 8.1. Acute 8.2. Sub-chronic 8.3. Irritation and sensitization 8.3.1. Eye and respiratory irritation 8.4. Chronic toxicity and carcinogenicity 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1. Microorganisms 9.2. Aquatic organisms 9.3. Terrestrial organisms REFERENCES RESUME ET EVALUATION, CONCLUSIONS ET RECOMMANDATIONS RESUMEN Y EVALUACION, CONCLUSIONES Y RECOMENDACIONES 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 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 ES02617-15 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA, and by financial support from the European Commission. Environmental Health Criteria PREAMBLE Objectives In 1973 the WHO Environmental Health Criteria Programme was initiated with the following objectives: (i) to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits; (ii) to identify new or potential pollutants; (iii) to identify gaps in knowledge concerning the health effects of pollutants; (iv) to promote the harmonization of toxicological and epidemio- logical methods in order to have internationally comparable results. The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976 and since that time an ever-increasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to evaluating toxicological methodology, e.g., for genetic, neurotoxic, teratogenic and nephrotoxic effects. 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It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation. All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed. WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR ISOPHORONE Members Dr L.A. Albert, Xalapa, Veracruz, Mexico (Vice-Chairman) Dr G.J. van Esch, Bilthoven, Netherlands Dr S.K. Kashyap, National Institute of Occupational Health Ahmedabad, India Mr H. Malcolm, Institute of Terrestrial Ecology, Monks Wood Experimental Station Huntingdon, United Kingdom (part-time) Dr K. Peltonen, Institute of Occupational Health, Helsinki, Finland Professor Wai-On Phoon, Worksafe Australia, and Department of Occupational Health, University of Sydney, Sydney, Australia (Chairman) Mr D.J. Reisman, US Environmental Protection Agency, Cincinnati, USA Dr E. Soderlund, National Institute of Public Health, Oslo, Norway (Rapporteur) Observer Dr H. Certa, Hüls AG, Marl, Germany Secretariat Dr K.W. Jager, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary) Mr J. Wilbourn, International Agency for Research on Cancer (IARC), Lyon, France ENVIRONMENTAL HEALTH CRITERIA FOR ISOPHORONE A WHO Task Group on Environmental Health Criteria for Isophorone met at the World Health Organization, Geneva, from 12 to 16 December 1994. Dr K.W. Jager of the IPCS, welcomed the participants on behalf of Dr M. Mercier, Director IPCS, and the three IPCS cooperating organizations (UNEP/ILO/WHO). The Task Group reviewed and revised the draft monograph and made an evaluation of the risks for human health and the environment from exposure to isophorone. The first draft of the monograph was prepared by Dr H.J. Wiegand (Hüls), Dr J.F. Regnier (Atochem) and Dr P.L. Mason (British Petroleum), and appeared as ECETOC-JACC Report No. 10. The second draft, incorporating comments received following circulation of the first draft to the IPCS contact points for Environmental Health Criteria, was prepared by the IPCS Secretariat. Dr K.W. Jager and Dr P.G. Jenkins, both of the IPCS Central Unit, were responsible for the scientific content of the monograph and the technical editing, respectively. The fact that industry made available to the IPCS and the Task Group their proprietary toxicological information on isophorone is gratefully acknowledged. This allowed the Task Group to make its evaluation on a more complete data base. The effort of all who helped in the preparation and the finalization of the document is gratefully acknowledged. 1. SUMMARY AND EVALUATION, CONCLUSIONS AND RECOMMENDATIONS 1.1 Summary and evaluatione 1.1.1 Physical and chemical properties Isophorone is a colourless liquid with a peppermint-like odour. It is soluble in water (12 g/litre) and miscible with most organic solvents. Its freezing point is -8.1°C and its boiling point 215°C. Its vapour pressure at 20°C is in the order of 40 Pa, and its vapour density (air = 1) is 4.7. It is a stable substance. Commercial samples of technical grade isophorone contain 1-3% of the isomer ß-isophorone (3,5,5-trimethyl-3-cyclohexene-1-one); the sum of alpha and isomers exceeds 99%. 1.1.2 Production and use Isophorone is widely used as a solvent for a number of synthetic resins and polymers, as well as in special application paints and printing inks. It is also a chemical intermediate and a solvent in certain pesticide formulations. Its worldwide production was estimated to be in the order of 92 000 tonnes per year in 1988. 1.1.3 Environmental transport, distribution and transformation Isophorone may enter the environment from numerous industries, waste and wastewater disposal and its use as a solvent and a pesticide carrier. Following release to water or soil, environmental concentrations will decrease as a result of volatilization and biodegradation. Isophorone in the atmosphere is removed by photochemical processes with an estimated half-life of about 30 min (based on a mathematical model). In a Die-away test, isophorone was biodegraded to the extent of approximately 70% within 14 days and 95% within 28 days. The results of biodegradation studies are variable and limited. Water solubility, soil adsorption coefficients and polarity indicate that significant adsorption by suspended solids and sediments is unlikely to occur. Although isophorone has been found in fish tissues, the data and the physical and chemical properties suggest that significant bioconcentration is unlikely. A half-life of one day has been measured in a single fish species. 1.1.4 Environmental levels and human exposure Isophorone has not been measured in ambient air. An isophorone concentration in coal fly ash of 490 µg/kg has been reported. Isophorone has been identified in surface waters (0.6 to 3 µg/litre), groundwater (10 µg/litre), urban run-off (10 µg/litre) and landfill leachate (29 µg/litre). Isophorone has been found in industrial wastewater at a concentration of 100 µg/litre. After classical secondary treatment, the concentration of isophorone in the effluent was 10 µg/litre. Isophorone has been identified in lake sediments (0.6 to 12 µg/kg dry weight) and in the tissues of several species of fish at concentrations up to 3.61 mg/kg wet weight. Isophorone was not detected in the edible parts of bean plants, rice or sugar beet following application as a pesticide carrier. 1.1.5 Kinetics and metabolism in laboratory animals and humans Distribution studies in rats using 14C-isophorone showed that 93% of orally administered radioactivity appeared mainly in urine and expired air within 24 h. The tissues retaining the highest concentration after this period were the liver, kidney and preputial glands. The metabolites from oral administration of isophorone identified in rabbits' urine resulted from oxidation of the 3-methyl group, reduction of the keto group and hydrogenation of the double bond of the cyclohexene ring, and were eliminated as such or as glucuronide derivatives in the case of the alcohols. Percutaneous LD50 values indicate that isophorone is rapidly absorbed through the skin. 1.1.6 Effects on laboratory mammals and in vitro test systems The acute toxicity of isophorone is low, with oral LD50 values being > 1500 mg/kg in the rat, > 2200 mg/kg in the mouse and > 2000 mg/kg in the rabbit. Dermal LD50 values were 1700 mg/kg in the rat and > 1200 mg/kg in the rabbit. Acute effects from dermal exposure in rats and rabbits ranged from mild erythema to scabs. Conjunctivitis and corneal damage have been reported following direct application to the eye or exposure to high concentrations of isophorone. No skin sensitization was reported in guinea-pigs using the Magnusson-Kligman test. In acute and short-term oral studies on rodents at high doses (> 1000 mg/kg), degenerative effects were seen in the liver as well as CNS depression and some deaths. In 90-day studies, a NOEL in rats and mice of 500 mg/kg body weight per day was determined. In a 90-day oral study in beagle dogs (with limited numbers), no effects were seen at doses of up to 150 mg/kg body weight per day. In the acute and short-term inhalation experiments which were reviewed, eye and respiratory irritation, haematological effects and decreased body weights were noted. Since the study designs were inadequate, no NOEL could be determined and no inference regarding human health can be made. Isophorone does not induce gene mutations in bacteria, chromosomal aberrations in vitro, DNA repair in primary rat hepatocytes, or bone-marrow micronuclei in mice. Positive effects were observed only in the absence of an exogenous metabolic system in L5178Y TK +/- mouse lymphoma mutagenesis assays as well as in a sister chromatid exchange assay. Isophorone induced morphological transformation in vitro in the absence of an exogenous metabolism system. It did not induce sex-linked recessive lethal mutations in Drosophila. The weight of evidence of all mutagenicity data supports the contention that isophorone is not a potent DNA-reactive compound. In an in vivo assay, no DNA binding was observed in the liver and kidneys (organs affected in the carcinogenicity bioassays). In long-term oral toxicity studies in mice and rats, male rats showed several lesions of the kidney, including nephropathy, tubular cell hyperplasia and low incidence of tubular cell adenomas and adenocarcinomas. The role of alpha2u-globulin accumulation in the etiology of these lesions has been recognized. Since significant amounts of alpha2u-globulin have not been detected in humans this mechanism of carcinogenesis appears not to be relevant to humans. Preputial gland carcinomas were observed in five high-dose male rats, and two clitoral gland adenomas were seen in low-dose female rats following exposure to isophorone. These tumours may also be related to alpha2u-globulin accumulation. Isophorone exposure was associated with some neoplastic lesions of the liver, and the integumentary and lymphoreticular systems of male mice, as well as non-neoplastic liver and adrenal cortex lesions, but this was not observed in dosed female mice. In the only available long-term inhalation study in rats and rabbits, irritation to eye and nasal mucosa, and lung and liver changes, were observed at approx. 1427 mg/m3 (approx. 250 ppm). However, it may have been due to limitations in the study. Very limited studies in rats and mice indicate that isophorone does not affect fertility nor does it cause developmental toxicity in experimental animals. The fact that central nervous system depression occurs in experimental animals could indicate a possible neurotoxic effect. Isophorone also elicited a positive effect in the behavioural despair swimming test. 1.1.7 Effect on humans The odour of isophorone can be detected at a concentration as low as 1.14 mg/m3 (0.2 ppm). Eye, nose and throat irritation has been reported at concentrations below 28.55 mg/m3 (5 ppm); above 1142 mg/m3 (200 ppm) nausea, headache, dizziness, faintness and inebriation have been reported. 1.1.8 Effects on other organisms in the laboratory and field No data on terrestrial animals were available. Acute LC50 values are available for several freshwater and marine species. The 96-h EC50 values (based on cell count and chlorophyll) range from 105 to 126 mg/litre. 48-h LC50 values for Daphnia magna range from 117 to 120 mg/litre, and 96-h LC50 values for freshwater fish range from 145 to 255 mg/litre. The 96-h LC50 values for marine invertebrates range from 12.9 to 430 mg/litre, while the 96-h LC50 for a single marine fish species was between 170 and 300 mg/litre. Data from studies with measured exposure concentrations did not differ from studies with nominal concentrations. NOEL values for Pimephales promelas tested in different laboratories ranged from 14 to 45.4 mg/litre. The available data suggest that isophorone has a low toxicity to aquatic organisms. 1.2 Conclusions 1.2.1 General population Isophorone is used as a solvent for resins, polymers and pesticides formulations. Dermal and inhalation exposure may occur, but will most likely be minimal. Data show that isophorone can occur in µg/litre (kg) concentrations in drinking-water and fish. In view of low toxicity in experimental studies and low levels of exposure from environmental sources, the risk to the general population appears to be minimal. 1.2.2 Occupational exposure In the absence of adequate engineering controls and industrial hygiene measures, occupational exposure to isophorone may exceed acceptable levels and cause eye, skin and respiratory irritation. At higher concentrations other health effects may occur. No studies on long-term health effects in workers were available for review by the Task Group. 1.2.3 The environment Isophorone may be released into the environment following its use as a pesticide carrier and its ubiquitous use as a solvent. Low concentrations have been identified in several environmental compartments, although it has a low environmental persistence due to biodegradation, volatilization and photochemical oxidation processes. The available data suggest that isophorone has low toxicity to aquatic organisms. 1.3 Recommendations 1.3.1 Protection of human health and the environment Care should be taken to prevent contamination of groundwater and air. Workers manufacturing or using isophorone should be protected from exposure by means of adequate engineering controls and appropriate industrial hygiene measures. Their occupational exposure should be kept within acceptable levels and monitored regularly. 1.3.2 Further research a) Health surveillance of exposed workers should be conducted. b) Actual levels of isophorone in the waters surrounding industrial areas should be determined. c) Adequate short-term/long-term inhalation studies in experimental animals should be conducted in order to determine safe levels of occupational exposure. d) Information on anaerobic biodegradation of isophorone is needed, especially as it has been identified in landfill leachate. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1 Identity Common name: Isophorone Synonyms: 2-cyclohexen-1-one, 3,5,5,-trimethyl; 3,5,5-trimethyl-2-cyclohexene-1-one; 1,1,3-trimethyl-3-cyclohexene-5-one; alpha-isophorone; isoacetophorone; isoforone; izoforon; 1,5,5-trimethyl-3-oxo-cyclohexene Empirical formula: C9H14O Chemical structure: Relative molecular mass: 138.2 CAS registry number: 78-59-1 RTECs registry number: GW7700000 EEC No: 606-012-00-8 EINECS No: 1011260 2.2 Physical and chemical properties Isophorone is a colourless liquid. Its odour has been described as being similar to peppermint and camphor. It is soluble in water and is miscible in all proportions with aliphatic and aromatic hydrocarbons, alcohols, ethers, esters, ketones and chlorinated hydrocarbons. Its physical and chemical data are summarized in Table 1. A typical commercial sample of isophorone may contain 1-3% of the isomer ß-isophorone (3,5,5-trimethyl-3-cyclohexene-1-one) with the sum of alpha- and ß-isomers exceeding 99% (Hüls, 1981; Atochem, 1986). Isophorone is stable and may be stored in steel or aluminium containers. Prolonged periods of storage may lead to slight yellowing. 2.3 Conversion factors The following conversion factors have been calculated for 22°C and 1013 hPa: 1 ppm = 5.71 mg/m3 1 mg/m3 = 0.175 ppm 2.4 Analytical methods The purity of technical isophorone may be determined by capillary gas chromatography (GC) with a flame ionization detector (FID). Recommended conditions are shown in Table 2. Earlier methods for determining isophorone in air were based on adsorption on charcoal (White et al., 1970; US NIOSH, 1977). However, it has been found that isophorone adsorbed on charcoal decomposes during storage. More recent methods involve adsorption on polymers such as XAD resins (Levin & Carleborg, 1987) or Tenax-GC (Brown & Purnell, 1979), followed by desorption and analysis by capillary GC with FID. The analysis of isophorone present in wastewater samples and fish tissues may be achieved by solvent extraction, clean up by gel permeation chromatography and analysis by GC/MS, in both the electron impact and chemical ionization modes (Jungclaus et al., 1976; US EPA, 1979; Sheldon & Hites, 1979). Table 1. Physical and chemical data of isophorone Value Reference Specific gravity (20°C/4°C) 0.922 Bartholomé et al. (1977) Boiling point at 1013 hPa 215°C Bartholomé et al. (1977) Freezing point -8.1°C Cheminfo (1988) Refractive index (n20D) 1.4775 Bartholomé et al. (1977) Viscosity at 20°C 2.6 mPa Hüls (1981) Coefficient of cubical expansion 0.00085°C-1 BP (1988b) at 20°C 0.00078°C-1 Atochem (1986) Surface tension at 20°C 30 mN/m BP (1988b) Vapour pressure 40 Pa (20°C) Bartholomé et al. (1977) 34.7 Pa (25°C) BIBRA (1991) Vapour density (air = 1) 4.7 Cheminfo (1988) Concentration in saturated air at 20°C and 1013 hPa 1941 mg/m3 Cheminfo (1988) Solubility at 20°C - Isophorone in water 12.0 g/litre Lyman et al. (1982) 17.5 g/litrea - Water in isophorone 53 g/litrea Log Kow (20°C) 1.67 (measured) Veith et al. (1978) 1.7 (estimated) Callahan et al. (1979) Table 1 (cont'd) Value Reference Solubility parameters (Hansen) delta 19.2 (J/cm3)1/2 Hüls (1981) deltaD 16.6 (J/cm3)1/2 Hüls (1981) deltaP 8.2 (J/cm3)1/2 Hüls (1981) deltaH 7.4 (J/cm3)1/2 Hüls (1981) Hydrogen bonding parameter, gamma 14.9 Hüls (1981) Flash point, closed cup 85°C Cheminfo (1988) Explosion limits in air 0.8-3.8 vol-% Bartholomé et al. (1977) Ignition temperature 470°C Bartholomé et al. (1977) 455°C BIBRA (1991) Heat of evaporation at 215°C 349.2 kJ/kg Bartholomé et al. (1977) Heat of combustion at 20°C 38 100 kJ/kg Bartholomé et al. (1977) Relative permittivity at 20°C 19.9 Hüls (1981) Specific resistivity 1 × 107 ohm × cm Atochem (1986) a Communication from Hüls AG, Marl, Germany, 1989. Table 2. Gas chromatographic conditions for the analysis of technical isophorone Column Fused silica capillarya Macroboreb Coating OV-1701 CP Wax 52CB Dimensions 60 m/0.25 mm 25 m/0.53 mm Injector temperature 240°C 250°C Temperature-programme 6 min 70°C to 10 min 105°C to 120°C 220°C at 4°/min at 6°/min 2 min 120°C to 150°C at 10 °/min From: a Hüls (1988c); b Atochem (1988) 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE Isophorone has tentatively been identified as a component of the essential oil of Thymus cariensis (Baser et al., 1992). Isophorone is produced commercially by catalytic condensation of acetone at elevated temperature and pressure and is purified by distillation. Worldwide annual production capacity was estimated to be 92 000 tonnes in 1988 (Personal communication from Hüls AG, Marl, Germany, 1989, to the IPCS). Isophorone is a solvent for a number of natural and synthetic resins and polymers such as polyvinyl chlorides and acetates, cellulose derivatives, epoxy and alkyd resins and polyacrylates. It is therefore used as a high boiling solvent in industrial air drying and stoving paints, nitro emulsion leather finishes and the manufacture of vinyl resin based printing inks for plastic surfaces. Isophorone is also used as a solvent for some pesticide formulations, especially for emulsifiable concentrates of anilides and carbamates. Isophorone is used as a chemical intermediate for the synthesis of a variety of organic chemicals (Hüls, 1981; Thier & Xu, 1990). Coal-burning power plants may be a source of atmospheric isophorone as it was detected in the fly ash from an electrostatic precipitator (Harrison et al., 1985). It has also been found in the breathing zones and area samples of work sites (US NIOSH, 1980, 1984) and in wastewater from industrial processes (Jungclaus et al., 1976). Thus, some manufacturing processes may be an environmental source of isophorone. 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1 Environmental distribution In view of its widespread use as a solvent for polymers, resins, waxes, oils and pesticides, there is a possibility of a wide distribution into the environment. Isophorone has been detected in river, surface, and groundwaters and in finished drinking-water (Thier & Xu, 1990) (see section 5.1.2.), in effluents from latex and chemical plants (Shackelford & Keith, 1976), in wastewater from a tyre manufacturing plant (Jungclaus et al., 1976), and in air during manufacturing operations (US NIOSH, 1980, 1984). The detection of isophorone in coal fly ash suggests that it may also be found in ambient air. 4.2 Biotransformation and environmental fate 4.2.1 Atmospheric fate By virtue of its vapour pressure of 40 Pa at 20°C, atmospheric isophorone will exist mainly in the vapour state (ECETOC, 1988). The Graphical Exposure Modelling System (GEMS) predicts that the half-life for reaction with both ambient ozone and photo-chemically generated hydroxyl radicals will be approximately 30 min. This estimate assumes a concentration of 8 × 105 molecules per cm3 and a reaction rate constant of 8.14 × 10-11 cm3 molecule-1 sec-1 at 25°C for hydroxyl radicals and 1.0 × 1012 molecules per cm3 with a reaction rate constant of 5 × 10-16 cm3 molecule-1 sec-1 at 25°C for ozone (US EPA, 1986). 4.2.2 Aquatic fate From an estimated Henry's Law constant of 5.8 × 10-6 atm m3 mole-1, based upon a water solubility of 12 g/litre at 20°C and a vapour pressure of 40 Pa at 20°C, the volatilization half-life in a model river flowing at 1 m/sec was calculated to be 7.5 days (Lyman et al., 1982). Based on a water solubility of 17.5 g/litre the volatilization half-life would be 11 days (Personal communication from Hüls AG, Marl, Germany, 1989, to the IPCS). The oxidation of isophorone by alkylperoxy radicals or singlet oxygen in water is unlikely to be significant in the environment (Mabey, 1981). Although dimerization has been reported in water irradiated at wave-lengths > 200 nm and in organic solvents at > 300 nm, such products are also considered unlikely at the levels existing in the environment (Callahan et al., 1979). Evidence that isophorone is photo-oxidized is provided by Borup & Middlebrooks (1986). Treatment with hydrogen peroxide (250 mg/litre) followed by UV radiation reduced an isophorone concentration of 62 mg/litre to < 0.05 mg/litre in 60 min. Isophorone has been shown to be converted to a compound(s) mutagenic to Salmonella typhimurium TA100 by aqueous chlorination under conditions of pH and reactant concentrations that may be relevant to wastewater and drinking-water chlorination (Cheh, 1986). 4.2.3 Terrestrial fate Loss from soil, as in the case of surface water, will be by volatilization and biodegradation. In view of the vapour pressure and Henry's Law constant, volatilization from both wet and dry soil surfaces would be slow. Based on a log Kow of 2.22 and assuming a water solubility of 12 g/litre at 20°C, a soil adsorption coefficient (Koc) of 25 has been estimated (Lyman et al., 1982). These values suggest that isophorone would be mobile in soil and that adsorption on suspended solids and sediment in water would be insignificant (Swann et al., 1983). 4.2.4 Biodegradation Tabak et al. (1981a,b) reported that isophorone (concentrations of 5 and 10 mg/litre) was rapidly degraded over 7 days by adapted microorganisms based on an aerobic-static culture procedure incorporating settled domestic wastewater as the microbial inoculum. Aerobic incubation of 100 mg/litre with activated sludge (30 mg/litre) for 2 weeks resulted in < 30% degradation (Kawasaki, 1980; Sasaki, 1980). Price et al. (1974) reported the removal (using a BOD procedure) of 9 and 42% isophorone from salt and fresh water, respectively, following incubation for 20 days with a settled non- adapted domestic wastewater inoculum. The losses of isophorone from wastewater treated using a trickling filter, activated sludge, aerated lagoon and facultative lagoon were 19, 98, 24 and 30%, respectively (Hannah et al., 1986). Intermediate degradation products of isophorone identified by Mikami et al. (1981) after incubation with Aspergillus niger were 3,5,5- trimethyl-2-cyclohexene-1,4-dione; 3,5,5-trimethylcyclo-hexane-1,4- dione; (S)-4-hydroxy-3,5,5-trimethyl-2-cyclohexene-1-one; and 3-hydroxymethyl-5,5-dimethyl-2-cyclohexene-1-one. In a Die-away test, the breakdown of isophorone after 14 days was approximately 70%, whereas 95% was broken down within 28 days (Schöberl, 1992). 4.2.5 Bioaccumulation Barrows et al. (1980) reported a measured bioconcentration factor of 7 for the bluegill sunfish (Lepomis macrochirus) exposed for 14 days to a mean concentration of 92.4 (± 10.5) µg/litre. The half-life of isophorone in the tissues of this species was 1 day. The bioconcentration factor was expressed as the quotient of the mean measured residues of the compound in fish tissues (whole body) during the equilibrium period divided by the mean measured concentration of the compound in water. The half-life of the compound in tissues was the time in days required for mean measured residue concentration in tissues to be reduced to half that which was measured during the equilibrium period in the uptake phase (Barrows et al., 1980). Thus, it is assumed that isophorone will not bioconcentrate in aquatic organisms. 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air Harrison et al. (1985) identified isophorone at a level of 490 µg/kg by GC/MS in electrostatically precipitated coal fly ash, suggesting that coal-fired power stations may be a source of emission to the atmosphere. 5.1.2 Water The available data on isophorone concentrations in water are presented in Table 3. Isophorone was detected in 1% of 795 surface water samples (Hauser & Bromberg, 1982). Concentrations of up to 3 µg/litre were reported in the Delaware river (Sheldon & Hites, 1979) and a concentration of 10 µg/litre was measured in urban run-off in Washington, DC, USA (Cole et al., 1984). The US Environmental Protection Agency has identified isophorone in finished drinking-water at concentrations ranging from 0.02 to 9.5 µg/litre (US EPA, 1980). A maximum concentration of 10 µg/litre has been reported in groundwater in the Netherlands (Zoeteman et al., 1981); the specific sources of this contamination were not identified. Isophorone was not detected in 36 water samples during the 1981 Environmental Survey of Chemicals in Japan (Japan Environment Agency, 1983). 5.1.3 Soil and sediment Isophorone was identified in sediment and soil taken from Love Canal, New York, USA (Hauser & Bromberg, 1982) and in sediment taken from Lake Pontchartrain, Louisiana, USA (McFall et al., 1985). The concentrations in the latter were 0.9-12 µg/kg dry weight. Isophorone was detected in 18 out of 36 bottom sediments, at concentrations ranging from 0.6 to 6.6 µg/kg dry weight, in the 1981 Environmental Survey of Chemicals in Japan (Japan Environment Agency, 1983). 5.1.4 Terrestrial organisms 184.108.40.206 Plants To estimate the decline of isophorone concentration in plants treated with pesticides containing isophorone as a carrier, 14C-isophorone was sprayed on bean plants and rice at a rate Table 3. Isophorone concentrations in water Media Location Number Analytical Range Samples with Reference of samples method (µg/litre) detectable residues (%) Surface water Delaware River, USA GC-MS 0.6-3 Hites (1979) Delaware River, USA GC-MS 3 Sheldon & Hites (1979) Drinking-water USA 0.02-9.5 US EPA (1980) Philadelphia, USA 12 GC-MS 17 Suffet et al. (1980) Ground water Netherlands 10 Zoeteman et al. (1981) Industrial effluent manufacturing effluent GC-MS 40 Jungclaus et al. (1976) industrial effluent GC-MS 100 Hites (1979) treated effluent GC-MS 10 Hites (1979) Urban run-off Washington, DC, USA 86 10 4 Cole et al. (1984) Leachate hazardous waste landfill 8 GC-MS 29 12.5 Ghassemi et al. (1984) equivalent to 7.5 kg/ha. Samples of the plants were taken periodically and assayed for total radioactivity. No attempt was made to characterize metabolites or degradation products. In bean plants total residues declined rapidly from 16 mg/kg one hour after application to below 0.1 mg/kg on day 42. Beans harvested on day 56 did not contain detectable radioactivity. Residues in rice plants declined from 7.3 mg/kg one hour after application to 3.1 mg/kg on day 35 and 0.12 mg/kg on day 128. Immature rice heads did not contain radioactivity on days 110 and 128. The relatively slow decay in rice plants was considered by the authors to be due to unfavourable growing conditions in this particular study (Rohm & Haas, 1972). In a similar study, sugar beet was sprayed at the 2-leaf stage with a herbicide containing 14C-isophorone. Total radioactivity found on day 30 was reported to be 10% of the initial value. On day 90, residues in the plants were below 0.01 mg/kg except in dry leaves where 0.07 mg/kg were found. The results suggested some uptake of radiolabel from the soil from day 60 onwards; this was considered likely to be due to the uptake of small carbon fragments or 14CO2 resulting from degradation of isophorone in the soil (Schering, 1974). 220.127.116.11 Animals No data are available. 5.1.5 Aquatic organisms Isophorone was found in three samples of commercial mussels (Mytilus edulis) collected near Holbaek, Denmark, but no concentrations were reported (Rasmussen et al., 1993). Isophorone was detected in fish sampled from several tributary rivers of Lake Michigan, Canada. Composite samples of several fish carcasses were analysed using GC-MS, and the following concentration ranges (mg/kg wet weight) were reported: common carp, < 0.02-3.13; smallmouth bass, 0.74-3.61; largemouth bass, 0.72; small bass, 0.74-3.61; pumpkinseed, 0.4; bowfin, < 0.02-0.76; northern pike, < 0.02-0.48; rock bass, < 0.02-1.44; and lake trout, 2.38. Isophorone was not detected in the channel catfish sample (Camanzo et al., 1987). 5.2 General population exposure No data were available. 5.3 Occupational exposure In the USA, the ACGIH (1986) have adopted a short-term (15 min) ceiling value of 28 mg/m3 (5 ppm) and the NIOSH recommended a 10-h TWA exposure limit of 23 mg/m3 (4 ppm) based primarily on unpublished reports, supplied to the TLV committee, of fatigue and malaise in workers exposed to concentrations of 28-46 mg/m3 (5-8 ppm) for 1 month. On lowering the concentration to between 5.7 and 22.8 mg/m3 (1 and 4 ppm) no further complaints were received (ACGIH, 1986). Exposure to isophorone has been determined in a screen printing plant, where several environmental conditions favoured evaporation of this solvent and where working conditions increased the risk of employee exposure. The highest exposures in screen printers were reported to be 131 ± 31 mg/m3 (23 ± 5.4 ppm) (50-90 min TWA) in the breathing zone of printing press workers. Other workers also received significant exposures: paint mixers, 102 ± 31 mg/m3 (17.8 ± 5.5 ppm); manual drying, 86 ± 23 mg/m3 (15 ± 4.1 ppm); automatic drying, 54 ± 19 mg/m3 (9.5 ± 3.3 ppm); and screen washer, 47 ± 32 mg/m3 (8.3 ± 5.6 ppm) (Samimi, 1982). A NIOSH health hazard evaluation (US NIOSH, 1980) conducted at a screen printing process in 1980 found workshift (6´ h) average exposures of printers to isophorone of 4 and 80 mg/m3 (0.7 and 14 ppm). Symptoms of respiratory tract and eye irritation reported by workers were attributed to the antistatic agent containing principally isophorone. A NIOSH evaluation (US NIOSH, 1984) conducted at another screen printing operation in 1984 found no detectable (< 2.8 mg/m3, 0.5 ppm) exposure to isophorone. Isophorone has been found in 6 out of 29 samples of printer's inks from different European manufacturers. The method of analysis was headspace gas chromatography and mass spectrometry. All of the samples in the present study were for serigraphy on electric and electronic articles (Rastogi, 1991). 6. KINETICS AND METABOLISM 6.1 Human No data are available. 6.2 Laboratory mammals Isophorone is absorbed by oral, dermal and inhalation routes. Following a single oral administration of isophorone to rats (4 g/kg) and rabbits (1 g/kg), the substance was distributed rapidly in the body and detected in the stomach, pancreas, adrenals, spleen and liver. Following inhalation (2284 mg/m3, 400 ppm for 4 h) isophorone was detected in the kidney, adrenals, liver, pancreas and brain of rats (Dutertre-Catella, 1976). Strasser et al. (1988) studied the distribution of isophorone in male rats following administration by gavage of a single dose (3.6 mmol/kg) of 14C-isophorone (in corn oil) containing 177 µCi/kg. Determination of radiolabel distribution of 14C-isophorone 24 h after dosing showed that 93% of the label had been excreted in the urine, faeces and expired air (approximate ratio 1200:1:67). The remainder of the radiolabel was concentrated in the liver, kidney and preputial glands, which contained 3.7, 1.1 and 0.7%, respectively, of the original dose. The high concentration of radiolabel in the preputial gland may have been due to the high concentrations of alpha2u-globulin to which it could bind (see also section 7.6). Following oral administration of isophorone to rats and rabbits, the substance was partly eliminated unchanged in expired air and urine; the remainder was metabolized (see Fig. 1) to: a) 5,5-dimethyl-cyclohex-1-en-3-one-1-carboxylic acid (i), derived from isophorone by methyl-oxidation; b) isophorol (3,5,5-trimethyl-cyclohex-2-en-1-ol) (ii), formed by the reduction of the ketonic group to a secondary alcohol and eliminated as a glucuronide; and c) dihydroisophorone (3,5,5-trimethyl-cyclohexanone) (iii), proceeding from the hydrogenation of the cyclohexene ring, and small quantities of cis- and trans-3,5,5-trimethyl- cyclohexanol-1 (iv), likely to have been formed from dihydroisophorone. The amounts of the identified metabolites as a proportion of the administered dose were not reported (Truhaut et al., 1970; Dutertre- Catella et al., 1978). A single oral isophorone dose of 500 mg/kg to male SD rats has been reported to cause significant depletion of hepatic, testicular and epididymal glutathione. Evidence was subsequently presented for enhanced ethyl methane sulfonate (EMS)-induced alkylation of DNA taken from epididymal spermatozoa (Gandy et al., 1990). Although individual compounds were not identified, the data suggest glutathione may play a significant role in the elimination of isophorone and its metabolites. Quantitative data regarding the excretion of isophorone are not available. In the study of Dutertre-Catella et al. (1978), unchanged isophorone, isophorol (ii), dihydroisophorone (iii), 3-carboxy-5,5- dimethyl-2-cyclohexene-1-one (i), and cis- and trans-3,5,5- trimethyl-cyclohexanols (iv) were detected in the urine of rats and rabbits 24 h after an oral dose of isophorone. The expired air contained unchanged isophorone 6 h after dosing. 7. EFFECTS ON LABORATORY MAMMALS, AND IN VITRO TEST SYSTEMS 7.1 Acute toxicity The acute LD50 values for various routes of exposure are presented in Table 4. Table 4. Acute LD50 values for technical (commercial) grade isophorone Route Species Sex LD50 Reference (mg/kg) Oral rat male and female 1500 Schering (1968) female 2104 Smyth et al. (1969) male 2700 Dutertre-Catella (1976) female 2100 Dutertre-Catella (1976) mouse male and female 2200 Dutertre-Catella (1976) rabbit male and female 2000 Dutertre-Catella (1976) female 2000 Smyth et al. (1969) Dermal rat male and female 1700 Schering (1968) rabbit male and female 1200 Dutertre-Catella (1976) 7.1.1 Oral Median lethal doses for isophorone in laboratory mammals ranging from 1500 to 2700 mg/kg body weight have been reported. The signs of toxicity were similar to those of solvents and narcotics, prostration being followed rapidly by coma. Deaths occurred within 24 h, otherwise recovery was complete. Degenerative changes in the liver were reported in animals that died (Dutertre-Catella, 1976). 18.104.22.168 ß-Isophorone An oral LD50 of 2950 mg/kg in rats has been reported for ß-isophorone (purity 97.5%, containing 2.5% alpha-isophorone). The predominant systemic effect was non-specific CNS depression shortly after dosing. Cirrhosis-like changes on the surface of the liver and severe irritation of the stomach were observed in the animals that died following dosing (Hüls, 1988d). 7.1.2 Dermal Dermal LD50 values indicate that isophorone is rapidly absorbed through the skin under occlusion. During the first 6 h of occluded application an increase in respiratory rate, followed by prostration and narcosis, was reported in rabbits exposed to 500-1000 mg/kg. Occluded skin contact for 24 h resulted in erythema, followed after several days by scarring. Skin damage was still evident after 14 days. In this study, 10-25 ml isophorone was applied to skin as compared to 0.5 ml in the skin irritation test (see section 7.2.1) (Dutertre-Catella, 1976). 7.1.3 Inhalation Rats and guinea-pigs were exposed to atmospheres containing isophorone concentrations of 1713 or 4282 mg/m3 (300 or 750 ppm) for 24 h, of 5025 mg/m3 (880 ppm) for 12 h, or of 7823 to 26 266 mg/m3 (1370 to 4600 ppm) for 8 h. In both animal species, in the order of development, eye and respiratory irritation, lachrymation, ataxia, dyspnoea, diarrhoea, light narcosis and death were observed. Postmortem examination of rats dying after exposure to isophorone showed haemorrhage in the lungs, congestion of the stomach and liver, peritoneal effusion and discoloration of the kidney and spleen (Smyth & Seaton, 1940). However, it would appear that some of the atmospheric concentrations could not have been achieved with pure isophorone, and therefore the results reported in the study are of little relevance (Rowe & Wolf, 1963). Groups of six rabbits and rats were exposed to isophorone for 5 h. The observation period was 2 weeks. At a concentration of 39.9 g/m3 (7000 ppm), 10% of the rats and 30% of the rabbits died. No LC50 value could be established from this study (Dutertre- Catella, 1976). 7.2 Skin, eye and respiratory irritation, sensitization 7.2.1 Skin irritation The skin irritation of isophorone was studied by Truhaut et al. (1972). A single application of 0.5 ml isophorone under an occlusive patch for a period of 24 h on the shaved or scarified skin of six rabbits produced a light erythema which disappeared rapidly after exposure. Microscopical examination did not show any histopathological changes. In the rabbit, occlusive and semi-occlusive contact with 0.5 ml neat isophorone for 1 or 4 h was non-irritating (Potokar et al., 1985). 22.214.171.124 ß-Isophorone A single application of 0.5 ml ß-isophorone (containing 2.5% alpha-isophorone) under a semi-occlusive patch over a period of 4 h on the shaved skin of three rabbits produced moderate erythema and swelling (Hüls, 1988e). 7.2.2 Eye irritation A single instillation of 0.1 ml isophorone in the eyes of six rabbits caused opacity in four animals, which in some instances covered the entire area of the cornea, inflammation of the conjunctiva and purulent discharge. In a supplementary experiment in which the rabbits' eyes were washed with 20 ml of warm water 2-4 seconds after the introduction of 0.1 ml isophorone, considerable recovery from these effects occurred over 7 days (Dutertre-Catella, 1976). Grant (1974) reported that application of one drop of isophorone to rabbit cornea caused mild transient injury, graded 4 on a scale of 1 to 10 after 24 h. Pronounced irritation of eyes and nose occurred in rats and guinea-pigs exposed to atmospheres containing isophorone (see section 7.1.3) (Smyth & Seaton, 1940; Smyth et al., 1942). 126.96.36.199 ß-Isophorone A single instillation of 0.1 ml in the eyes of three rabbits produced moderate conjunctival and corneal opacities. Iridial changes were also reported in two animals. Although the corneal and iridial changes had resolved by 7 days, minor conjunctival irritation was still in evidence (Hüls, 1988f). 7.2.3 Respiratory irritation In a study of sensory irritation, De Ceaurriz et al. (1981) estimated the concentration of various chemicals causing a 50% decrease in respiratory rate (measured using individual plethysmographs) in mice (RD50). The RD50 for isophorone was 158.7 mg/m3 (27.8 ppm) for a 5-min exposure. For comparison, the RD50 values for toluene diisocyanate and acetone were 0.24 and 23 480 ppm, respectively. Exposure of rats to airborne concentrations of 383-514 mg/m3 (67 or 90 ppm) for a single 4-h period produced statistically significant reductions in circulating leukocytes. This leukopenia was considered to be due to the stress-induced release of cortico-steroids resulting from sensory irritation (Brondeau et al., 1990). 7.2.4 Sensitization Isophorone, administered at a concentration of 10% intradermally (in maize germ oil) and 100% topically to female guinea-pigs in the Magnusson-Kligman test, showed no sensitizing potential (Hüls, 1988a). 7.3 Subchronic toxicity 7.3.1 Inhalation The effects of repeated whole body exposure to isophorone vapour were reported by Smyth et al. (1942). Groups of male Wistar rats and guinea-pigs of both sexes, 16-20 animals per group, were exposed to isophorone concentrations up to approximately 2855 mg/m3 (500 ppm) 8 h/day, 5 days/week, for 6 weeks. As in the Smyth & Seaton (1940) study (section 7.1.3), the air concentrations of isophorone could not have been attained under conditions employed by Smyth et al. (1942). In the presentation of these results no distinction was made between the two species and no control data were presented. Growth retardation was noted in all animals exposed to higher concentrations. Postmortem examination of animals dying after exposure revealed severely injured kidneys and lungs. The kidneys of surviving animals were congested with dilation of Bowman's capsules and cloudy swelling of tubular cells. The lungs and liver were also reported to be congested with desquamation of the bronchial epithelium in the lungs and cloudy swelling in the liver cells. Dutertre-Catella (1976) exposed groups of 10 male and 10 female rats to atmospheres reported to contain 2855 mg/m3 8 h/day, 5 days/week for 6 and 4 months, respectively. Two males and one female exposed to isophorone died; there were no deaths in the control animals. The only reported effects were irritation of the eyes and nose. Groups of 10 male and 10 female young adult Charles River CD rats were exposed to isophorone at air concentrations of 0 or 250 mg/m3, 6 h/day, 5 days/week, for 4 weeks. Results of daily spectroscopic determinations indicated that the average daily exposure was 208 mg/m3. Body weight measurements and haematological studies were made before exposure and after 4 weeks. The rats were killed and gross necropsy was performed. Organ weights were determined for lungs, liver, kidneys, adrenals and spleen. Histological examination of those tissues were performed in three males and three females per group. The following effects were observed: transient nasal bleeding, increased percentages of lymphocytes, decreased percentages of neutrophils and increased haemoglobin concentration in males and females and significantly lower terminal body weights and significantly decreased absolute and relative liver weights of exposed males, compared with controls (Littlefield, 1968). 7.3.2 Oral Four groups of 20 male and 20 female albino rats were fed diets containing 0, 750, 1500 or 3000 mg isophorone/kg (0, 37.5, 75 or 150 mg/kg body weight) for 90 days. Isophorone in corn oil (ratio 1:2) was blended with the diet; fresh diets were prepared each week. Haematology, serum chemistry and urine analyses were carried out on five animals of each sex from each group at week 4 and at termination. Comprehensive histopathological examination was confined to five animals of each sex from the control and high dose groups. The liver and kidney from five animals of the intermediate dose levels were also examined histopathologically. Under the conditions of this study, no effects on the general appearance of the test animals, on their behaviour, on body weight gain or on food consumption were observed at a dietary level of 1500 mg/kg or less. Isophorone did not alter the composition of the formed elements of the blood, nor did it interfere with the general metabolism or with liver and kidney function. No detectable gross or microscopic pathological changes were noted in any of the animals examined after 28 or 90 days of feeding. Organ/body weight ratios for vital organs were not changed. The only adverse finding reported was a reduction in body weight gain in the male rats receiving the highest dose (Affiliated Medical Enterprises, 1972a). As part of a preliminary investigation prior to an NTP carcinogenicity study, five rats (F-344) and five mice (B6C3F1) of each sex were given by gavage 12 oral doses of up to 2000 mg isophorone/kg body weight administered in corn oil over a 16-day period (US NTP, 1986). Lethargy was reported in all rats following dosing while in mice uncertain locomotion was reported among animals receiving 1000 mg/kg. All mice and 50% of the rats receiving 2000 mg/kg died. The weight gain of animals surviving 2000 mg/kg and all animals receiving 1000 mg/kg was reduced. As part of the same programme, 10 rats and mice of each sex were given by gavage daily doses of 0, 62.5, 125, 250, 500 or 1000 mg isophorone/kg body weight for 90 days (US NTP, 1986). The rats were drowsy and lethargic following administration. At the highest dose level, one female rat and three female mice died. No macroscopic or microscopic changes were observed in the organs examined from either study. A subsequent histopathological review of the kidney, which included re-sectioning and additional staining, also failed to reveal any treatment-related effects. The no-observed-effect level in this study was considered to be 500 mg/kg body weight per day. Groups of four male and four female beagle dogs were given 90 daily oral doses of isophorone in gelatin capsules at dosage rates of 35, 75 or 150 mg/kg per day. Comprehensive haematology, clinical chemistry and urine analyses were carried out initially and at 1, 2 and 3 months. Apart from a mild intermittent incidence of soft stools in animals of the high-dose group, there was no observable effect as demonstrated by the data on general appearance and conditions as well as those from haematological or biochemical investigations. At autopsy, no changes in the organ/body weight ratios and no histopathological changes were observed. No toxic effect was found at any of the dose levels used. It was concluded that the no-observed- effect level for isophorone in the dog was 150 mg/kg per day (Affiliated Medical Enterprises, 1972b). 7.3.3 Dermal The daily occluded application on shaved and abraded skin of 0.1 or 0.2 ml of isophorone to rats for 8 weeks produced erythema and scabs at the site of application (Dutertre-Catella, 1976). The only apparent systemic effect reported was an 8% reduction in mean weight gain in the females compared with controls; the dose levels at which this occurred were not given. 7.4 Mutagenicity The results of mutagenicity tests are summarized in Table 5. 7.4.1 Gene mutation in bacteria (Ames tests) The mutagenic potential of isophorone was examined using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, following a preincubation protocol. The test substance concentrations used were between 10 and 5000 µg per plate. There was no increase in the number of revertants at any of the concentrations tested with and without rat liver S9 fraction (Hüls, 1988b). Two further studies were conducted on Salmonella typhimurium TA1535, TA1537 and TA1538 strains (1 to 1000 µg/plate) (Atochem, 1978a) and TA98, TA100, TA1535 and TA1537 strains (100 to 10 000 µg/plate) (US NTP, 1986). No evidence of mutagenic potential was provided in the presence or absence of rat or hamster liver S9 fractions. Salmonella strains TA1535, TA1537, TA97, TA98 and TA100 were used to test isophorone (up to 10 000 µg/plate) with and without Aroclor 1254-induced rat and hamster metabolic activation systems (male Sprague-Dawley rats and male Syrian hamsters). When negative results were obtained in the initial assay, the chemicals were retested in all strains with and without activation. Isophorone showed no mutagenic activity (Mortelmans et al., 1986). 188.8.131.52 Dihydroisophorone The mutagenic potential of dihydroisophorone (iii in Fig. 1, see section 6.2), a metabolite of isophorone, was examined using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 at concentrations of 25 to 2500 µg per plate. There was no increase in the number of revertants at the concentrations tested, either with or without rat liver S9 fraction (BP, 1988a). Table 5. Isophorone genotoxicity tests Test system Results Reference Gene Ames test (with and negative Atochem (1978a); US NTP mutation without S9) (1986); Mortelmans et al. (1986); Hüls (1988b) mouse lymphoma assay positive US NTP (1986) (without S9) (with and without S9) negative Microbiological Associates (1984a) (with and without S9) positive McGregor et al. (1988) (with and without S9) negative O'Donoghue et al. (1988) positive Mitchell (1993) sex-linked recessive negative Foureman et al. (1994) lethal in Drosophila melanogaster Chromosome CHO cells (in vitro) negative US NTP (1986) aberrations (with and without S9) negative Gulati et al. (1989) Micronucleus mouse (in vivo) negative Atochem (1978b); Microbio- test logical Associates (1984b); O'Donoghue et al. (1988) Sister CHO cells (in vitro) chromatid (without S9) positive US NTP (1986) exchanges (with S9) negative US NTP (1986) (without S9) positive Gulati et al. (1989) Direct DNA unscheduled DNA negative Microbiological Associates damage synthesis (primary rat (1984c); O'Donoghue et al. hepatocyte culture) (1988) DNA binding liver and kidney DNA negative Thier et al. (1990); in vivo (mouse and rat) Thier & Xu (1990) Morphological BALB/c-3T3 cells positive Matthews et al. (1993) transformation (without S9) 7.4.2 Gene mutation in mammalian cells Isophorone was tested in the L5178Y TK +/- mouse lymphoma mutagenesis assay (MLA) in the presence and absence of rat liver S9 fraction. The experiment was performed only once and all doses were tested in duplicate. In the absence of S9, isophorone concentrations of 0.13 to 1.3 ml/litre produced total growth of 111% to 12% compared to the control. In the presence of S9, isophorone concentrations of 0.067 to 0.89 ml/litre produced total growth of 86 to 9% compared to the control. None of these cultures exhibited mutation frequencies which were significantly greater than the mean mutation frequency of the solvent control (Microbiological Associates, 1984a; O'Donoghue et al., 1988). The authors pointed out that while the results of the MLA without metabolic activation were negative in the present study, these assays were positive in the NTP study and thus were not reproducible between laboratories. In contrast to O'Donoghue et al. (1988), McGregor et al. (1988) reported isophorone to be positive in the MLA both with and without rat liver S9 mix (from male Fischer-344 rats). Isophorone was toxic to the cultures only at doses of 600 ml/litre or more. The authors viewed the experiment with reservations since the cloning efficiency was low. Another MLA study employing concentrations of 400 to 1200 mg/litre was carried out in the absence of rat liver S9 fraction. Duplicate experiments produced gradations of total growth relative to control values of 112 or 118% for the low concentrations to 7 or 14% for the high concentrations. Dose-related increases in mutation frequencies compared with control were observed; at the highest dose level the increase was 4-fold (US NTP, 1986). Mitchell (1993) compared the induction of mutation frequencies by the in situ variant (ISV) approach with the standard MLA. Isophorone was one of the compounds tested, and showed a higher induced mutation frequency with the ISV approach than in the standard MLA. Isophorone was tested for its ability to induce sex-linked recessive lethal mutations in post-meiotic and meiotic germ cells of male Drosophila melanogaster. No induction was observed at 2000 ppm by feeding or at 15 000 ppm by injection (Foureman et al., 1994). 7.4.3 Chromosome aberrations and sister chromatid exchange 184.108.40.206 Chromosome aberrations Isophorone was tested in an in vitro cytogenetic assay using Chinese hamster ovary (CHO) cell cultures. Treatments were performed both in the absence and presence of rat liver S9 fraction. Under the conditions of this test, isophorone did not induce chromosomal aberrations (ABS) at concentrations up to 1600 or 1500 mg/litre, respectively (US NTP, 1986). Twenty-seven chemicals (including isophorone), previously tested in rodent carcinogenicity assays were tested for induction of ABS in CHO cells as part of a more extensive analysis of the correlation between results of in vitro genetic toxicity assays and carcinogenicity bioassays. Chemicals were tested up to toxic doses both with and without exogenous metabolic activation. A liver fraction (S9) prepared from Aroclor 1254-induced male Sprague-Dawley rats was used to provide exogenous metabolic activation. No ABS were observed in CHO cells following incubation with isophorone in either the presence or the absence of S9 (Gulati et al., 1989). 220.127.116.11 Sister chromatid exchange The ability of isophorone to induce sister chromatid exchange (SCE) was studied in CHO cells, in the presence and absence of rat liver S9 fraction at concentrations up to 1000 mg isophorone/litre. Without S9, isophorone induced a small but dose-related increase in the frequencies of SCE. No effect was observed in the presence of S9 (US NTP, 1986). A significant increase in SCE frequency with isophorone was observed only in the absence of S9, at doses of 500-1000 mg/litre (Gulati et al., 1989 - see also section 18.104.22.168). At these high dose levels, isophorone is extremely cytostatic; therefore, the increase in SCE frequency was observed only after delayed harvest (6-13 h additional culture time). 7.4.4 Micronucleus test Isophorone doses of 450, 900 and 1800 mg/kg body weight were administered by gavage to CFLP mice of both sexes in two equal doses separated by an interval of 24 h. Six hours after the last treatment the mean micronucleated cell counts and the bone marrow cytotoxicity were similar in all test groups and controls (Atochem, 1978b). Male and female CD-1 mice were treated by intraperitoneal injection of isophorone (0.54 ml/kg body weight). There was no evidence of micronuclei or cytotoxicity in bone marrow samples collected 12, 24 and 48 h after administration (Microbiological Associates, 1984b; O'Donoghue et al., 1988). 7.4.5 Primary DNA damage 22.214.171.124 Bacterial tests In the Bacillus subtilis (strain H17) rec-assay, isophorone showed DNA-damaging potential without metabolic activation and a reverse effect with activation (Matsui et al., 1989). Isophorone did not induce the SOS function responses in the umu test using Salmonella typhimurium strain TA1535/pSK1002 (Ono et al., 1991). 126.96.36.199 Unscheduled DNA synthesis Isophorone was tested at dose levels ranging from 0.005 to 0.40 ml/litre using primary rat cultures of hepatocytes. There was no increase in the mean nuclear grain count compared to the controls or in the incidence of cells undergoing repair at any dose level (Microbiological Associates, 1984c; O'Donoghue et al., 1988). 188.8.131.52 DNA binding A DNA-binding study was performed with radiolabelled 1,3,5- 14C-isophorone (Thier et al., 1990; Thier & Xu, 1990). Male and female F-344 rats and male and female B6C3F1 mice received doses (500 mg/kg body weight) of unlabelled isophorone containing 0.4 mCi (per rat) and 0.8 mCi (per mouse) labelled isophorone in neutral oil by gavage. No binding of the radioactivity to liver or kidney DNA was observed in either species. 7.4.6 Morphological transformation Isophorone was positive in a standard transformation assay using BALB/c-3T3 cells without exogenous activation at concentrations of 1.07 g/litre (7.76 mmol/litre) (Matthews et al., 1993). 7.5 Chronic toxicity and carcinogenicity In 18-month inhalation studies, groups of 10 rats and 2 rabbits of each sex were exposed to atmospheres containing 1413 mg isophorone/m3 (250 ppm) for 6 h/day, 5 days/week. Slight conjunctivitis and irritation of the nasal mucosa with a bloody discharge were observed. In the lungs of the animals, frequent haemorrhages were found with oedema in the alveoli. Vacuolization was found in the liver of the treated animals (Dutertre-Catella, 1976). Toxicological and carcinogenesis studies of isophorone (more than 94% pure) were conducted by administering 0, 250 or 500 mg isophorone/kg body weight per day by gavage in corn oil to groups of 50 F-344/N rats and 50 B6C3F1 mice of each sex, 5 days per week for 103 weeks. Throughout the 2-year study, the mean body weights of the high-dose male rats were on average 5% less than those of the vehicle controls. During the second year, the mean body weights of the female high-dose rats were on average 8% less than those of the vehicle controls, and the high-dose female mice were 5% lower. The survival of high-dose male rats was significantly lower than that of the vehicle controls after week 96 (final survival: vehicle control, 33/50; low dose, 33/50; high dose, 14/50). The survival of dosed female rats was poor (30/50; 23/50; 20/50), due in part to 20 gavage- related accidental deaths of dosed animals. The survival of male mice was also low (16/50; 16/50; 19/50), but there was a significant trend toward increased survival of dosed female mice relative to that of the vehicle controls (26/50; 35/50; 34/50). Dosed male rats showed a variety of proliferative lesions of the kidney (tubular cell hyperplasia: 0/50; 1/50; 4/50; tubular cell adenoma: 0/50; 0/50; 2/50; tubular cell adenocarcinoma: 0/50; 3/50; 1/50; epithelial hyperplasia of the renal pelvis: 0/50; 5/50; 5/50). Dosed male rats also exhibited increased mineralization of the medullary collecting ducts (1/50; 31/50; 20/50), and low-dose male rats showed a more severe nephropathy than is commonly seen in ageing F-344/N rats. Carcinomas of the preputial gland were increased in high-dose male rats (0/50; 0/50; 5/50). With the exception of a moderate increase in nephropathy (21/50; 39/50; 32/50), female rats did not show chemically related increased incidences of neoplastic or non-neoplastic lesions. In high-dose male mice, isophorone exposure was associated with increased incidences of hepatocellular adenomas and carcinomas (18/48; 13/50; 29/50) and of mesenchymal tumours of the integumentary system (fibroma, fibrosarcoma, neurofibrosarcoma or sarcoma: 6/48; 8/50; 14/50). An increased incidence of lymphomas or leukaemias was noted in low-dose male mice (8/48; 18/50; 5/50). Coagulative necrosis (3/48; 10/50; 11/50) and hepatocytomegaly (23/48; 39/50; 37/50) were observed more frequently in the livers of dosed male mice than in vehicle controls. No compound-related neoplastic or non-neoplastic lesions associated with isophorone exposure were seen in female mice. Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenicity of isophorone in male F-344/N rats as shown by the occurrence of renal tubular cell adenomas and adenocarcinomas in animals given 250 or 500 mg/kg per day; carcinomas of the preputial gland were also observed at increased incidence in male rats given 500 mg/kg. There was no evidence of carcinogenicity in female F-344/N rats given 250 or 500 mg/kg per day. For male B6C3F1 mice, there was equivocal evidence of carcinogenicity of isophorone as shown by an increased incidence of hepatocellular adenomas or carcinomas (combined) and of mesenchymal tumours in the integumentary system in animals given 500 mg/kg per day and by an increase in malignant lymphomas in animals given 250 mg/kg per day. There was no evidence of carcinogenicity of isophorone in female B6C3F1 mice given 250 or 500 mg/kg per day (US NTP, 1986). In the above NTP study, preputial gland carcinomas were observed in five high-dose male rats. The apparent absence of this tumour in vehicle controls or in the low-dose group, and the very low incidence (12/1094, i.e. 1%) in corn oil vehicle controls in previous 2-year studies, suggest that this may be a chemical-related effect. No preputial gland tumours were observed in male mice, but two histogenetically related clitoral gland adenomas were seen in low-dose female rats, providing some support for an association of isophorone exposure with this tumour type (Bucher et al., 1986). 7.6 Mechanisms of toxicity Swenberg et al. (1992) and US EPA (1991a,b) reviewed the mechanisms involved in alpha2u-globulin nephropathy and renal carcinogenesis of several chemicals in various animal, biochemical and molecular modelling systems. All of the data are consistent with the hypothesis that reversible binding of chemicals or their metabolites to this abundant protein is causally related to the induction of disease. Alpha2u-globulin, found mainly in male rats, is synthesized by the liver and subsequently transported to the kidney. It is normally present in the cytoplasm of the proximal convoluted tubules of untreated animals in the form of hyaline droplets, visible by light microscopy. Xenobiotics, or their metabolites, are bound to the alpha2u-globulin in the liver of animals; this conjugate is even more difficult to hydrolyse than the alpha2u-globulin itself and induces the formation of the hyaline droplets which accumulate in the tubules (Swenberg et al., 1989). Accumulation of the chemical/ alpha2u-globulin complex causes lysomal protein overload and necrosis of the cells with subsequent cellular regeneration. Thus cellular proliferation may contribute to the development of renal tumours. In order to determine whether isophorone and other compounds that cause alpha2u-globulin to accumulate have the same binding characteristics, binding studies were conducted with kidney cytosol preparations from male Fischer-344 rats. The inhibition constant value (Ki) for isophorone was in the range of 10-6 to 10-7 mol/litre, while those for d-limonene, 1,4-dichlorobenzene and 2,5-dichlorophenol were higher (around 10-4 mol/litre). This suggests that other factors besides binding are involved in the accumulation of alpha2u-globulin. Results so far indicate that binding is dependent on both hydrophobic interactions and hydrogen bonding (Borghoff et al., 1991). Investigations (Charbonneau et al., 1988; Strasser et al., 1988) have shown that isophorone, isophorol and dihydroisophorone bind to alpha2u-globulin, resulting in an increased accumulation of hyaline droplets in renal tubular cells. These findings suggest the same sequence of events may be responsible for the small increase in the incidence of renal tubular neoplasias seen in male rats. alpha2u-Globulin was not detected at significant levels in plasma and urine of female rats or either sex of mice and humans (Swenberg et al., 1989; Olson et al., 1990). In addition, other laboratory rodents, dogs and primates do not develop hydrocarbon-related nephropathy (Swenberg et al., 1989). For these reasons, it was concluded that the low increase in the incidence of tubular adenomas and adenocarcinomas observed in male rats following isophorone administration was attributable to the accumulation of alpha2u-globulin and was sex- and species-specific (Charbonneau & Swenberg, 1988; Strasser et al., 1988; Swenberg et al., 1989). Further evidence to support this view was gained by a study of Dietrich & Swenberg (1991) using male animals of the NCI Black Reiter (NBR) strain, which does not synthesize alpha2u-globulin. It was shown that an oral dose (gavage) of 1000 mg isophorone/kg on four consecutive days failed to produce the early features of the alpha2u-globulin-related nephropathy syndrome (i.e. hyaline droplets or alpha2u-globulin) in this strain. In terms of human risk assessment, renal tubule tumours produced in male rats in association with chemicals inducing alpha2u-globulin accumulation should be distinguished from renal tubule tumours of other origin (Borghoff et al., 1990; Swenberg, 1991; US EPA, 1991a,b). It has been recognized that the induction of such tumours does not necessarily indicate a potential carcinogenic hazard to humans. The significance of this information with respect to the etiology of pathological changes is uncertain at present. Studies have shown that high levels of alpha2u-globulin and its messenger RNA (mRNA) are present in the preputial gland of both male and female rats. The preputial gland in both sexes contained about 3 times more alpha2u-globulin mRNA and about 300 times more alpha2u-globulin than the male rat liver (Murty et al., 1987). It was claimed that this high content of alpha2u-globulin was primarily due to the cellular and ductal accumulation of the protein in the preputial gland but did not reflect a difference in the rate of transcription of the alpha2u-globulin gene. The high amount of radiolabel derived from 14C-isophorone found in the preputial gland of the male rats following single gavage dosing with 5 ml/kg (Strasser et al., 1988) may therefore be related to its high content of alpha2u-globulin. 7.7 Appraisal for mutagenicity/carcinogenicity Isophorone does not induce gene mutations in bacteria, chromosomal aberrations in vitro, bone-marrow micronuclei in mice or DNA repair in primary cultures of rat hepatocytes. No DNA binding in vivo was observed in a DNA binding study using 1,3,5- 14C-isophorone. In one study in the absence and in another study in the absence and presence of metabolic activation, weak mutagenic responses were obtained in three out of five L5178Y TK +/- mouse lymphoma mutagenesis assays and a small increase of sister chromatid exchange (SCE) was found only without metabolic activation in CHO cells. Another study with SCE showed no increases in chromosomal aberrations (for details see section 7.4). Isophorone was negative in a test for sex-linked recessive lethal mutations in Drosophila. It induced morphological transformation in mouse cells without activation. The weight of the evidence of all mutagenicity data supports the contention that isophorone is not a potent DNA reactive compound. There was no DNA binding in the liver and kidneys (sites affected in the carcinogenicity bioassays). The available data on in vitro mutagenicity and related end- points do not suggest that isophorone is genotoxic. When tested in a two-year carcinogenicity bioassay, isophorone produced a variety of lesions of the kidneys, such as severe nephropathy and tubular cell hyperplasia. A low but statistically significant increase in the frequency of renal tubular cell adenomas and adenocarcinomas was found in male F-344 rats dosed at levels of 250 and 500 mg/kg per day (see section 7.5). An increase in the number of carcinomas of the preputial gland was found only in male rats given 500 mg/kg per day. In high-dose male B6C3F1 mice hepatocellular adenomas and carcinomas were observed. No compound related neoplastic lesions were seen in female rats (for details, see section 7.5). The induction of these two types of tumours in male rats may be associated with nephropathy, epithelial hyperplasia and alpha2u-globulin formation and accumulation of the isophorone/ alpha2u-globulin complex, causing lysomal protein overload and necrosis of the kidney cells with subsequent cellular regeneration (for details see section 7.6). 7.8 Reproduction, embryotoxicity, teratogenicity The teratogenicity of isophorone to rats and mice was studied by Traul et al. (1984). Groups of 22 confirmed mated females of each species were exposed 6 h/day on days 6-15 of gestation to atmospheres containing 0, 143, 285 or 656 mg/m3 (0, 25, 50 or 115 ppm) isophorone. At the highest atmospheric concentration there was evidence of maternal toxicity which showed as reduced food consumption, alopecia and cervical or anogenital staining in the rats and reduced body weights in the mice. Comprehensive uterine and fetal examinations did not show any significant differences between animals exposed to isophorone and their respective controls. From the results of this study, it can be concluded that no teratogenic or fetotoxic effect was observed with 656 mg/m3 in F-344 rats and CD-1 mice. In a study by Dutertre-Catella (1976), groups of 10 rats of each sex, exposed 6 h/day, 5 days/week to 2855 mg/m3 (500 ppm) isophorone for 3 months, were mated to exposed animals or to controls. All females were reported to have delivered 7-10 pups. Anatomo- pathological examination did not show any abnormalities. However, only one isophorone concentration was used, the group size was small, and no information was provided on reproductive success and maternal survival. 7.9 Neurotoxicity Central nervous system depression is a characteristic feature of isophorone intoxication in experimental animals (Smyth & Seaton, 1940; Dutertre-Catella, 1976; De Ceaurriz et al., 1981). Isophorone and a number of aliphatic ketones have been studied using the mouse behavioural despair swimming test (De Ceaurriz et al., 1984). This test, which was developed for screening antidepressant drugs, is based on the duration of the periods of immobility exhibited by mice when placed in water. Mice were exposed to isophorone at atmospheric concentrations of 508-782 mg/m3 (89-137 ppm) for 4 h. The ID50 (an estimated concentration producing a 50% reduction in the immobility time) for animals thus exposed was 628 mg/m3 (110 ppm). 7.10 Other special studies One of the current research approaches for assessing cytotoxicity is to monitor the respiratory activity of the mitochondrion, a sensitive, nonspecific subcellular target site. Detected changes in mitochondrial function after the addition of a test chemical could be correlated to toxic effects. In this case, rat (male, Charles River) liver mitochondria were used, and isophorone was shown to induce no observable effects on rat liver mitochondria respiratory activity over the tested concentration range of 0.273-113.8 mg/litre (1.98-825 µmol/litre) (Haubenstricker et al., 1990a). Isophorone was tested for its ability to perturb glutathione (GSH) levels in the testes and epididymides, as well as liver, following single acute dosages to rats. Groups of four mature male Sprague-Dawley rats were administered isophorone intraperitoneally (2 ml/kg). Concurrent control groups were maintained for all time points examined to account for possible circadian fluctuation of GSH throughout the day. Animals were sacrificed at 1, 2, 4, 8 or 16 h after administration of the test compound. Liver, testes and epididymides were excised for determination of total GSH content by spectrophotometry. Isophorone (500 mg/kg) significantly reduced GSH in the liver and in both reproductive organs examined. The ability of isophorone to enhance the covalent binding of tritiated ethyl methanesulfonate (3H-EMS) to spermatocytes was assessed. Perturbation of reproductive tract GSH by isophorone treatment significantly enhanced the extent of 3H-EMS-induced binding to sperm heads. The authors postulate that chemical-induced lowering of GSH in the male reproductive tract may be a mechanism for potentiation of chemical-induced germ-cell mutations (Gandy et al., 1990). 8. EFFECTS ON HUMANS 8.1 Acute Groups of 11 or 12 subjects exposed for a few minutes to atmospheric isophorone concentrations of 228 mg/m3 (40 ppm) or more experienced irritation of the eyes, nose and throat. A few complaints of nausea, headache, dizziness, faintness, inebriation and a feeling of suffocation occurred at concentrations 1142 mg/m3 (> 200 ppm). The symptoms of irritation and narcosis were said to be less intensive following exposure to concentrations below 1142 mg/m3 (Smyth & Seaton 1940). However, this study has been criticized because of uncertain actual concentrations and the use of impure substances (Rowe & Wolf, 1963). 8.2 Sub-chronic Complaints of fatigue and malaise were reported among workers exposed for 1 month to atmospheres containing 28.5 to 48 mg isophorone/m3 (5 to 8 ppm) (Communication from Ware, GD, to Chairman, TLV Committee, 1973). No further complaints were received following a reduction of the concentration to between 5.7 and 22.8 mg/m3 (1 and 4 ppm). On the basis of these data, the American Conference of Governmental Industrial Hygienists (ACGIH, 1986) recommended a ceiling limit (15 min) of 29 mg/m3 (5 ppm) for isophorone. 8.3 Irritation and sensitization No report on skin irritation or skin sensitization has been published. 8.3.1 Eye and respiratory irritation Smyth & Seaton (1940) reported eye, nose and throat irritation following exposure to isophorone levels of 228 mg/m3 (40 ppm) or more. Silverman et al. (1946) estimated the sensory threshold of a number of ketones including isophorone. An average of 12 subjects of both sexes were used for each solvent exposure. The time of exposure was 15 min. Irritation of the eyes, nose and throat was experienced at 142.7 mg/m3 (25 ppm) isophorone; the highest concentration considered acceptable by the majority of subjects for an 8-h exposure was 57.1 mg/m3 (10 ppm). A NIOSH health hazard evaluation (US NIOSH, 1980) conducted at a screen printing process in 1980 found that the workshift (6´ h) average exposures of printers to isophorone were 4 and 80 mg/m3 (0.7 and 14 ppm). Symptoms of respiratory tract and eye irritation reported by workers were attributed to the antistatic agent containing principally isophorone. Amoore & Hautala (1983) reported an air odour threshold for isophorone of 1.14 mg/m3 (0.2 ppm). The "odour safety factor", which was defined as the threshold limit value 28.5 mg/m3 (5 ppm) divided by the odour threshold, was 25. From the magnitude of this value the authors predicted that 50% of exposed persons would perceive sensory warning of the TLV (28.5 mg/m3, 5 ppm). 8.4 Chronic toxicity and carcinogenicity No long-term surveys have been carried out on occupationally exposed workers or other potentially exposed populations. 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1 Microorganisms Yoshioka et al. (1985) studied the acute toxicity of isophorone in Tetrahymena pyriformis. The EC50 after a 24-h exposure was 420 mg/litre. Yoshioka et al. (1986) found that the EC50 in the Activated Sludge Respiration Inhibition Test was 100 mg/litre. In a 2-day laboratory test with the marine red alga Champia parvula, 38.3 mg isophorone/litre caused 50% reduction in cystocarp formation. Above 138.5 mg/litre no cystocarps were formed, indicating absence of sexual reproduction. In a 2-week laboratory test, 107.3 mg/litre completely inhibited cystocarp formation (Thursby & Steele, 1986). Isophorone was tested in air-saturated media with Saccharomyces cerevisiae at concentrations of 0.272 to 113.7 mg/litre (1.97-824 µmol/litre). These produced no detectable effect on the rate of yeast respiration as measured with a dissolved oxygen electrode (Haubenstricker et al., 1990b). 9.2 Aquatic organisms The acute toxicity of isophorone to fish, crustaceans and algae is summarized in Table 6. With the exception of Mysidopsis bahia (US EPA, 1978) all acute LC50 values were above 100 mg/litre, indicating a relatively low aquatic toxicity. This was consistent with the results of a subacute (14 day) study with the marine red alga Champia parvula. The toxicity of isophorone was determined by means of various biological end-points, namely vegetative growth, formation of tetrasporangia (asexual reproduction) and production of cystocarps (sexual reproduction). Depending on the toxicological end-point, the lowest concentration resulting in a significant difference from controls ranged between 50 and 138 mg/litre (Thursby et al., 1985). In addition to the results of acute toxicity tests shown in Table 6, an early life stage toxicity test was conducted with the freshwater fathead minnow Pimephales promelas (Cairns & Nebeker, 1982) using concentrations of 11, 19, 30, 56 and 112 mg isophorone/litre. Survival was affected at a concentration of 112 mg/litre, but not at 56 mg/litre or less; fork length was affected at 30 mg/litre but not at 19 mg/litre or less; body weight gain was decreased at 19 mg/litre or more but not at 11 mg/litre. The authors calculated a no-observed- effect concentration of 14 mg/litre. Using the same test with the same species, Lemke et al. (1983) found a no-observed-effect level of 19.5 mg/litre, and in an interlaboratory (6 laboratories) comparison no-observed-effect levels of up to 45.4 mg/litre were found (Lemke, 1983). 9.3 Terrestrial organisms No data are available concerning the effects of isophorone on terrestrial organisms. Table 6. Acute toxicity of technical isophorone to aquatic species Test species Parameter Duration Static/flowa Resultsb Reference (h) (mg/litre) Fish Bluegill sunfish LC50 96 static 220 (n) US EPA (1978) (Lepomis macrochirus) LC50 24 static 240 (n) Buccafusco et al. (1981) Fathead minnow LC50 96 flow 145-255 (m) Cairns & Nebeker (1982) (Pimephales promelas) LC50 96 228 Geiger et al. (1990) Golden orfe LC50 72 204 Scheubel & Scholz (1989) (Leuciscus idus melanotus) Sheepshead minnow LC50 96 140 Ward et al. (1981) (Cyprinodon variegatus) LC50 96 static > 170, < 300 (n) Heitmuller et al. (1981) Invertebrates Brine shrimp LC50 24 430 Price et al. (1974) (Artemia salina) Mysid shrimp LC50 96 static 12.9 US EPA (1978) (Mysidopsis bahia) Water flea immobilization: EC50 24 static 430 (n) LeBlanc (1980) (Daphnia magna) Immobilization: EC50 24 static 254-277 Scholz (1988a) immobilization: EC50 48 static 120 (n) LeBlanc (1980) immobilization: EC50 48 static 117 US EPA (1978) Algae Selenastrum capricornutum cell count: EC50 96 static 122 US EPA (1978) chlorophyll: EC50 96 static 126 US EPA (1978) Table 6 (cont'd) Test species Parameter Duration Static/flowa Resultsb Reference (h) (mg/litre) Skeletonema costatum cell count: EC50 96 static 105 US EPA (1978) chlorophyll: EC50 96 static 110 US EPA (1978) Scenedesmus subspicatus cell count: EC50 72 475-476 Scholz (1988b) chlorophyll: EC50 72 524-527 Scholz (1988c) Red alga reproduction EC50 24 38.3 Thursby & Steele (1986) (Champia parvula) a static = static exposure; flow = flow-through exposure b n = nominal exposure concentration; m = measured exposure concentration REFERENCES ACGIH (1986) Documentation on the threshold limit values and biological exposure indices, 5th ed. 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Yoshioka Y, Nagase H, Ose Y, & Sato T (1986) Evaluation of the test method "activated sludge, respiration inhibition test" proposed by the OECD. Ecotoxicol Environ Saf, 12(3): 206-212. Zoeteman BCJ, De Greef E, & Brinkmann FJJ (1981) Persistency of organic contaminants in groundwater; lessons from soil pollution incidents in The Netherlands. Sci Total Environ, 21: 187-202. RESUME ET EVALUATION, CONCLUSIONS ET RECOMMANDATIONS 1. Résumé et évaluation 1.1 Propriétés physiques et chimiques L'isophorone est un liquide incolore d'odeur mentholée. Elle est soluble dans l'eau (12 g/litre) et miscible à la plupart des solvants organiques. Son point de congélation est de -8,1°C et son point d'ébullition de 215°C. San tension de vapeur à 20°C est de l'ordre 40 Pa et sa densité de vapeur par rapport à l'air est de 4,7. C'est une substance stable. L'isophorone de qualité technique vendue dans le commerce contient 1 à 3% d'isomère ß-(3,5,5-triméthyl-3-cyclohexène-1-one); la somme des isomères alpha et ß-dépasse 99%. 1.2 Production et usage L'isophorone est largement utilisée comme solvant pour un grand nombre de résines et de polymères de synthèse ainsi que dans certaines peintures spéciales et certaines encres d'imprimerie. Elle joue le rôle de produit intermédiaire en synthèse et on l'utilise comme solvant dans certaines formulations de pesticides. On estime qu'en 1988, la production mondiale d'isophorone était de l'ordre de 92 000 tonnes par an. 1.3 Transport, distribution et transformation dans l'environnement L'isophorone peut pénétrer dans l'environnement du fait de son utilisation par de nombreuses industries, lors du rejet de déchets ou d'eaux usées et par suite de son utilisation comme solvant, notamment dans les formulations de pesticides. Une fois libérée dans l'eau ou le sol, sa volatilisation et sa biodégradation entraînent une baisse de la concentration. L'isophorone présente dans l'atmosphère en est éliminée par des processus photochimiques avec une demi-vie estimative d'environ 30 minutes (selon un modèle mathématique). On a constaté que la biodécomposition de l'isophorone atteignait environ 70% dans les 14 jours et 95% dans les 28 jours. Les résultats des études de biodécomposition sont variables et limités. D'après la solubilité dans l'eau, les coefficients d'adsorption au sol et la polarité de ce composé, il paraît improbable qu'il soit adsorbé en quantité notable sur les matières solides en suspension et les sédiments. Bien qu'on ait trouvé de l'isophorone dans des tissus pisciaires, les données concernant ce composé et notamment ses propriétés physiques et chimiques, incitent à penser qu'il a peu de chances de subir une bioconcentration. On a mesuré une demi-vie d'un jour chez une espèce de poisson. 1.4 Concentrations dans l'environnement et exposition humaine On n'a pas procédé au dosage de l'isophorone dans l'air ambiant. On a fait état d'une concentration d'isophorone dans des cendres volantes de houille égale à 490 µg/kg. On a également mis en évidence la présence d'isophorone dans des eaux de surface (0,6 à 3 µg/litre), des eaux souterraines (10 µg/litre), des eaux de ruissellement urbaines (10 µg/litre) et des eaux de lessivage de décharge (29 µg/litre). De l'isophorone a été trouvée à la concentration de 100 µg/litre dans des eaux usées industrielles. Après un traitement secondaire classique, la concentration d'isophorone dans l'effluent était tombée à 10 µg/litre. La présence d'isophorone a été observée dans des sédiments lacustres (0,6 à 12 µg/kg de poids sec) ainsi que dans les tissus de plusieurs espèces de poisson à des concentrations allant jusqu'à 3,61 µg/kg de poids frais. On n'a pas constaté la présence d'isophorone dans les parties comestibles de plants de haricots, de riz ou dans des betteraves à sucre, après application sous la forme de véhicule pour pesticide. 1.5 Cinétique et métabolisme chez les animaux de laboratoire et l'homme Des études de distribution effectuées sur des rats au moyen de 14C-isophorone ont montré que 93% de la radioactivité administrée par voie orale, réapparaissaient principalement dans les urines et l'air expiré au bout de 24 heures. Les organes dans lesquels subsistait au bout de cette période la plus forte concentration d'isophorone étaient le foie, les reins et les glandes préputiales. Après administration par voie orale d'isophorone à des lapins, on a retrouvé dans l'urine de ces derniers des métabolites résultant d'une oxydation du groupe méthyle en position 3, de la réduction du groupe carbonyle et de l'hydrogénation de la double liaison du cycle cyclohexénique; ces métabolites ont été éliminés tels quels ou sous forme de glucuronides dans le cas des alcools. Les valeurs de la DL50 percutanée indiquent que l'isophorone passe rapidement à travers la peau. 1.6 Effets sur les mammifères de laboratoire et les systèmes d'épreuves in vitro La toxicité aiguë de l'isophorone est faible, les valeurs de DL50 par voie orale étant > 1500 mg/kg chez le rat, à > 2200 mg/kg chez la souris et à > 2000 mg/kg chez le lapin. On a obtenu pour la DL50 par voie percutanée des valeurs de 1700 mg/kg chez le rat et de > 1200 mg/kg chez le lapin. Au niveau cutané les effets aigus d'une exposition vont, chez le rat et le lapin, d'un léger érythème à la formation de croûtes. On a signalé une conjonctivite et des lésions cornéennes après instillation directe dans l'oeil ou exposition à de fortes concentrations d'isophorone. Le test de Magnusson-Kligman pratiqué sur des cobayes n'a pas permis de mettre en évidence un effet de sensibilisation cutanée. Les études de toxicité aiguë et celles au cours desquelles on a administré de l'isophorone par voie orale pendant de brèves périodes à des rongeurs ont montré qu'à fortes doses (> 1000 mg/kg), ce composé provoquait des effets dégénératifs au niveau du foie ainsi qu'une dépression du système nerveux central et une certaine mortalité. Lors d'études de 90 jours, on a évalué à 500 mg/kg de poids corporel, la dose quotidienne sans effets observables pour le rat et la souris. Une autre étude de 90 jours, au cours de laquelle on a administré de l'isophorone par voie orale à des chiens beagle (en nombre limité), n'a révélé aucun effet à des doses quotidiennes allant jusqu'à 150 mg/kg de poids corporel. Lors d'études de toxicité aiguë au cours desquelles on a fait inhaler pendant de brèves périodes de l'isophorone aux animaux de laboratoire, on a observé une irritation oculaire et respiratoire, des effets hématologiques et une diminution du poids corporel. Etant donné que la conception de ces études laissait à désirer, il n'a pas été possible de déterminer la dose sans effets observables et on ne peut en tirer aucune conclusion en ce qui concerne la santé humaine. L'isophorone ne provoque pas de mutations géniques chez les bactéries ni d'aberrations chromosomiques in vitro; on n'observe pas non plus de réparation de l'ADN dans des cultures primaires d'hépatocytes de rat, ni la présence de micro-noyaux dans des cellules de moelle osseuse de souris. Des effets positifs ont été observés, lors d'essais de mutagénèse, sur des cellules de lymphomes murins L5178Y TK +/-, mais uniquement en l'absence d'un système métabolique exogène. Le même phénomène a été observé en ce qui concerne les échanges de chromatides soeurs. L'isophorone a produit une transformation morphologique in vitro, mais là encore, en l'absence de système métabolique exogène. En revanche, elle n'a pas produit de mutations létales récessives liées au sexe chez la drosophile. Les données de mutagénicité ont un poids expérimental suffisant pour qu'on puisse soutenir que l'isophorone n'est pas un composé qui réagit énergiquement avec l'ADN. D'ailleurs, lors d'une épreuve in vivo, on n'a pas observé de lésion de l'ADN dans des cellules hépatiques et rénales (alors que ces organes sont ceux où l'on observe des lésions dans les épreuves de cancérogénicité). Les études toxicologiques au cours desquelles on a administré pendant une longue période de l'isophorone par voie orale à des souris et à des rats ont révélé la présence de plusieurs lésions rénales prolifératives chez les rats males, comprenant une néphropathie, une hyperplasie tubulaire et une faible incidence des adénomes affectant les tubules et des adénocarcinomes. Il est admis que l'accumulation d'alpha2u-globuline joue un rôle dans l'étiologie de ces lésions. Etant donné que l'on n'a pas décelé chez l'homme la présence d'alpha2u-globuline en quantités appréciables, il ne semble pas que ce type de cancérogénèse soit à envisager dans l'espèce humaine. Chez cinq rats males soumis à des fortes doses d'isophorone, on a observé des carcinomes de la glande préputiale, et deux adénomes de la glande clitoridienne ont été observés chez les rattes soumises à de faibles doses d'isophorone. Là encore, il est possible que l'accumulation d'alpha2u-globuline soit en cause. On a également attribué à l'isophorone la présence d'un certain nombre de lésions néoplasiques du foie, des téguments et du système lymphoréticulaire, observées chez les rats males, de même d'ailleurs que des lésions bénignes du foie et du cortex surrénalien, lésions qui n'ont cependant pas été observées chez les souris femelles. La seule étude d'inhalation à long terme dont on connaisse les résultats chez le rat et le lapin, a permis d'observer une irritation de la muqueuse oculaire et de la muqueuse nasale et des altérations au niveau pulmonaire et hépatique, à une dose de approx.1427 mg/m3 (environ 250 ppm). Toutefois ces résultats peuvent s'expliquer par le fait que les études en question n'offraient pas toutes les garanties de rigueur. Des études très limitées effectuées sur des rats et des souris indiquent que l'isophorone n'affecte pas la fécondité ni le développement chez les animaux de laboratoire. Le fait qu'une dépression du système nerveux central se produise chez les animaux de laboratoire par suite d'exposition à de l'isophorone, pourrait être le signe d'un effet neurotoxique. Lors d'une épreuve comportementale de nage désespérée, l'isophorone a également produit un effet positif. 1.7 Effets sur l'homme On peut déceler une odeur d'isophorone à une concentration ne dépassant pas 1,14 mg/m3 (0,2 ppm). A des concentrations inférieures à 28,55 mg/m3 (5 ppm), on a signalé une irritation des yeux, du nez et de la gorge; au-delà de 1142 mg/m3 (200 ppm) on a fait état de nausées, de maux de tête, d'étourdissements, de faiblesse et de sensation ébrieuse. 1.8 Effets sur les autres êtres vivants au laboratoire et dans leur milieu naturel En ce qui concerne les effets aigus sur un certain nombre d'espèces marines ou dulçaquicoles, on possède plusieurs valeurs de la CL50. Les valeurs de CE50 à 96 heures (basées sur la numération cellulaire et la chlorophylle) s'échelonnent de 105 à 126 mg/litre. Pour Daphnia magna, les valeurs de la CL50 à 48 heures vont de 177 à 120 mg/litre et pour les poissons d'eau douce; celles de la CL50 à 96 heures s'étagent de 145 à 255 mg/litre. Les valeurs de la CL50 à 96 heurs pour les invertébrés marins vont de 12,9 à 430 mg/litre et pour une espèce de poisson de mer, cette valeur se situe entre 170 et 300 mg/litre. Les données fournies par les études au cours desquelles on a utilisé des concentrations mesurées ne diffèrent pas de celles où ce sont les concentrations nominales dont on s'est servi. Les épreuves effectuées par différents laboratoire sur Pimephales promelas ont fait ressortir, pour la dose sans effets nocifs observables, des valeurs allant de 14 à 45,4 mg/litre. Les données disponibles incitent à penser que l'isophorone n'est que faiblement toxique pour les organismes aquatiques. 2. Conclusions 2.1 Population générale L'isophorone est utilisée comme solvant pour les résines, les polymères et certaines formulations de pesticides. Il peut y avoir exposition par voie cutanée ou respiratoire, mais il y a de grandes chances pour qu'elle reste minime. Les données disponibles montrent que l'isophorone peut être présente à des concentrations de l'ordre du µg/litre (ou par kg) dans l'eau de boisson et dans le poisson. Les études expérimentales ayant montré que ce composé était faiblement toxique et du fait que l'exposition aux sources d'isophorone présentes dans l'environnement est peu importante, on peut considérer que le risque pour la population générale est minime. 2.2 Exposition professionnelle Faute de contrôles techniques suffisants et de mesures d'hygiène industrielle convenables, il est possible que l'exposition professionnelle à l'isophorone dépasse les limites acceptables et provoque une irritation oculaire, cutanée ou respiratoire. A plus fortes concentrations, d'autres effets nocifs peuvent se produire. Le groupe de travail ne disposait pas d'études sur les effets à long terme de ce composé chez les ouvriers. 2.3 Environnement Il est possible que l'isophorone soit libérée dans l'environnement lorsqu'on l'utilise comme véhicule de pesticides et du fait de son emploi généralisé comme solvant. On en a trouvé de faibles concentrations dans plusieurs compartiments du milieu, mais sa persistance est faible par suite des processus de biodécomposition, volatilisation et oxydation photochimique qu'elle subit. D'après les données disponibles, il semble que l'isophorone soit peu toxique pour les organismes aquatiques. 3. Recommandations 3.1 Protection de la santé humaine et de l'environnement Des précautions sont à prendre pour éviter la pollution des eaux souterraines et de l'air. Les travailleurs qui sont employés à la production d'isophorone doivent se prémunir contre l'exposition à ce composé grâce à des mesures de contrôle technique suffisantes et à des précautions d'hygiène industrielle appropriées. L'exposition professionnelle doit rester dans des limites acceptables et être régulièrement contrôlée. 3.2 Recherches futures a) Surveillance médicale des travailleurs exposés. b) Détermination de la concentration effective d'isophorone dans les eaux à l'entour des zones industrielles. c) Etudes d'inhalation satisfaisantes à court et à long terme sur des animaux de laboratoire afin de déterminer les limites de sécurité pour l'exposition professionnelle. d) Nécessité d'obtenir des données sur la biodécomposition anaérobie de l'isophorone, notamment du fait qu'on en a observé la présence dans les lixiviats de décharges. RESUMEN Y EVALUACION, CONCLUSIONES Y RECOMENDACIONES 1. Resumen y evaluación 1.1 Propiedades físicas y químicas La isoforona es un líquido incoloro con un olor parecido al de la menta. Es soluble en agua (12 g/litro) y se mezcla con la mayoría de disolventes orgánicos. Su punto de congelación es -8,1°C y su punto de ebullición 215°C. Su presión en vapor a 20°C es del orden de 40 Pa, y su densidad en vapor (aire = 1) es 4,7. Es una sustancia estable. Las muestras comerciales de isoforona de calidad técnica contienen 1-3% del isómero ß-isoforona (3,5,5-trimetil-3-ciclo- hexeno-1-1); la suma de isómeros alpha y supera el 99%. 1.2 Producción y utilización La isoforona se utiliza mucho como disolvente para cierto número de resinas y polímeros sintéticos, así como en pinturas y tintas de imprenta para aplicaciones especiales. Es también un producto químico intermedio y un disolvente en determinadas formulaciones de plaguicidas. Se ha estimado que en 1988 su producción mundial fue del orden de 92 000 toneladas. 1.3 Transporte, distribución y transformación en el medio ambiente La isoforona puede introducirse en el medio ambiente teniendo como procedencia numerosas industrias, la evacuación de desechos y de aguas residuales y a raíz de su utilización como disolvente y como portador de plaguicida. Tras su descarga en el agua o el suelo, la concentración ambiental disminuye a consecuencia de la volatilización y la biodegradación. La isoforona de la atmósfera se elimina por procesos fotoquímicos con una semivida estimada de unos 30 minutos (sobre la base de un modelo matemático). En una prueba de estimación, la isoforona se biodegradó hasta aproximadamente un 70% en 14 días y un 95% en 28 días. Los resultados de los estudios de biodegradación son variables y limitados. Los coeficientes de solubilidad en agua y de adsorción en el suelo y la polaridad indican que es improbable que tenga lugar una adsorción significativa por sólidos en suspensión y sedimentos. Aunque se ha hallado isoforona en tejidos de peces, los datos y las propiedades fisicas y químicas parecen indicar que es improbable una bioconcentración significativa. Se ha medido una semivida de un día en una única especie de peces. 1.4 Niveles ambientales y exposición humana No se ha medido isoforona en el aire ambiente. Se ha notificado una concentración de isoforona en ceniza volátil de carbón de 490 µg/kg. Se ha identificado isoforona en aguas superficiales (0,6 a 3 µg/litro), en aguas subterráneas (10 µg/litro), en aguas de escorrentía urbanas (10 µg/litro) y en lixiviado de terraplenados (29 µg/litros). Se ha hallado isoforona en aguas residuales industriales en una concentración de 100 µg/litro. Tras un tratamiento secundario clásico, la concentración de isoforona en el efluente fue de 10 µg/litro. Se ha identificado isoforona en sedimentos lacustres (0,6 a 12 µg/kg de peso en seco) y en los tejidos de varias especies de peces en concentraciones de hasta 3,61 mg/kg de peso en húmedo. No se detectó isoforona en las partes comestibles de las plantas del frijol, del arroz o de la remolacha azucarera tras la aplicación de un portador de plaguicida. 1.5 Cinética y metabolismo en animales de laboratorio y en el ser humano Los estudios de distribución en ratas utilizando 14C-isoforona mostraron que el 93% de la radiactividad administrada por vía oral aparecía principalmente en la orina y el aire espirado en 24 horas. Los tejidos que retuvieron la mayor concentración tras ese periodo fueron el hígado, los riñones y las glándulas prepuciales. Los metabolitos tras administración oral de isoforona identificados en la orina de conejos fueron resultado de la oxidación del grupo 3-metilo, la reducción del grupo keto y la hidrogenación del enlace doble del anillo de ciclohexeno, y se eliminaron como tales o como derivados de glucuronida en el caso de los alcoholes. Los valores de la DL50 percutáneas indican que la isoforona se absorbe rápidamente a través de la piel. 1.6 Efectos en mamíferos de laboratorio y en sistemas de prueba in vitro La toxicidad aguda de la isoforona es baja, con valores de DL50 oral > 1500 mg/kg en la rata, > de 2200 mg/kg en el ratón y > 2000 mg/kg en el conejo. Los valores de la DL50 cutáneas fueron 1700 mg/kg en la rata y > 1200 mg/kg en el conejo. Los efectos agudos por exposición cutánea en ratas y conejos oscilaron entre eritema leve y escaras. Se han notificado casos de conjuntivitis y lesión corneana tras la aplicación directa al ojo o la exposición a concentraciones elevadas de isoforona, pero ningún caso de sensibilización de la piel en cobayos utilizando la prueba Magnusson- Kligman. En estudios de administración por vía oral sobre efectos agudos y a corto plazo a roedores en dosis altas (> 1000 mg/kg) se observaron efectos degenerativos en el hígado, así como depresión del sistema nervioso central y algunas defunciones. En estudios de 90 días, se determinó un NOEL en ratas y ratones de 500 mg/kg de peso corporal por día. En un estudio de administración por vía oral de 90 días a perros pachón (en número limitado) no se apreciaron efectos en dosis de hasta 150 mg/kg de peso corporal por día. En los experimentos examinados de inhalación aguda y a corto plazo, se observaron irritación ocular y respiratoria, efectos hematológicos y reducción del peso corporal. Como el diseño de los estudios era inadecuado, no pudo determinarse NOEL y no puede hacerse ninguna deducción con respecto a la salud humana. La isoforona no induce mutaciones genéticas en bacterias, aberraciones cromosómicas in vitro, reparación del ADN en los hepatocitos primarios de la rata, ni micronúcleos de médula ósea en los ratones. Se observaron efectos positivos únicamente en ausencia de un sistema metabólico exógeno en valoraciones L5178Y TK+/- de los mutagénesis de linfoma del ratón, así como en una valoración de intercambio de cromátides hermanos. La isoforona indujo transformación morfológica in vitro en ausencia de un sistema metabólico exógeno. No indujo mutaciones recesivas letales ligadas al sexo en Drosophila. El peso probatorio de conjunto de datos sobre mutagenicidad avala la tesis de que la isoforona no es un potente compuesto ADN-reactivo. En una valoración in vivo no se observó enlace con el ADN en el hígado y los riñones (órganos afectados en las biovaloraciones de carcinogenicidad). En estudios de toxicidad por administración oral a largo plazo en ratones y ratas, las ratas macho mostraron varias lesiones del riñón, incluidas nefropatía, hiperplasia de las células tubulares y una baja incidencia de adenomas y adenocarcinomas de ese tipo de células. Se ha reconocido el papel que desempeña la acumulación de alpha2u-globulina en la etiología de estas lesiones. Como no se han detectado cantidades significativas de alpha2u-globulina en el hombre, este mecanismo de carcinogénesis no parece ser importante en la especie humana. Se observaron carcinomas de la glándula prepucial en cinco ratas macho sometidas a dosis elevadas y dos adenomas de la glándula clitorídea en ratas hembra expuestas a bajas dosis de isoforona. Estos tumores pueden estar relacionados también con la acumulación de alpha2u-globulina. La exposición a la isoforona se asoció con algunas lesiones neoplásicas del hígado, y de los sistemas integumentario y linforreticular de los ratones macho, así como con lesiones no neoplásicas del hígado y de la corteza suprarrenal, pero esto no se observó en los ratones hembra a los que se administraron estas dosis. En el único estudio a largo plazo disponible sobre inhalación en ratas y conejos, se observaron irritación de los ojos y de la mucosa nasal, así como cambios pulmonares y hepáticos, a dosis de approx.1427 mg/m3 (approx. 250 ppm). Sin embargo, estos resultados pueden haberse debido a las limitaciones del estudio. Estudios muy limitados en ratas y ratones indican que la isoforona no afecta a la fecundidad ni produce toxicidad para el desarrollo en los animales de experimentación. El hecho de que en los animales de experimentación se produzca depresión del sistema nervioso central podría indicar un posible efecto neurotóxico. La isoforona también produjo un efecto positivo en la prueba de comportamiento de natación desesperada. 1.7 Efectos en el ser humano El olor de la isoforona puede detectarse a concentraciones de sólo 1,14 mg/m3 (0,2 ppm). Se ha observado irritación de los ojos, la nariz y la garganta a concentraciones inferiores a 28,55 mg/m3 (5 ppm); y por encima de 1142 mg/m3 (200 ppm), náuseas, cefalea, vértigo, desmayo y embriaguez. 1.8 Efectos en otros organismos en laboratorio y sobre el terreno No se dispuso de datos sobre animales terrestres. Se dispone de valores de LC50 agudos en varias especies de agua dulce y marinas. Los valores EC50 en 96 horas (basados en recuento celular y clorofila) oscilan entre 105 y 126 mg/litro. Los valores LC50 en 48 horas para Daphnia magna oscilan entre 117 y 120 mg/litro, y los valores LC50 en 96 horas para peces de agua dulce, entre 145 y 255 mg/litro. Los valores de la LC50 en 96 horas para animales invertebrados marinos oscilan entre 12,9 y 430 mg/litro, y el valor de la LC50 en 96 horas para una única especie de pez marino, entre 170 y 300 mg/litro. Los datos de estudios a concentraciones de exposición medida no fueron diferentes de los obtenidos en estudios a concentraciones nominales. Los valores NOEL para Pimephales promelas sometidos a prueba en diferentes laboratorios oscilaron entre 14 y 45,4 mg/litro. Los datos disponibles parecen indicar que la isoforona tiene una baja toxicidad para los organismos acuáticos. 2. Conclusiones 2.1 Población general La isoforona se utiliza como disolvente para formulaciones de resinas, polímeros y plaguicidas. Puede producirse una exposición cutánea y por inhalación, pero lo más probable es que sea insignificante. Los datos muestran que la isoforona puede aparecer en concentraciones de µg/litro (kg) en el agua de bebida y en los peces. En vista de la baja toxicidad en los estudios experimentales y de los bajos niveles de exposición a partir de fuentes ambientales, parece ser mínimo el riesgo para la población general. 2.2 Exposición ocupacional A falta de controles técnicos adecuados y de medidas de higiene industrial, la exposición ocupacional a la isoforona puede superar los niveles aceptables y producir irritación ocular, cutánea y respiratoria. En concentraciones superiores pueden producirse otros efectos sobre la salud. El Grupo Especial no dispuso de estudios sobre los efectos a largo plazo en la salud de los trabajadores. 2.3 El medio ambiente La isoforona puede pasar al medio ambiente tras su utilización como portador de plaguicida y su uso generalizado como disolvente. Se han identificado concentraciones bajas en varios compartimientos ambientales, aunque tiene una baja persistencia ambiental a causa de los procesos de biodegradación, volatilización y oxidación fotoquímica. Los datos disponibles parecen indicar que la isoforona tiene una baja toxicidad para los organismos acuáticos. 3. Recomendaciones 3.1 Protección de la salud humana y del medio ambiente Hay que tener cuidado en prevenir la contaminación de las aguas subterráneas y del aire. Los trabajadores que fabrican o utilizan isoforona deben protegerse de la exposición a ésta por medio de controles técnicos y medidas de higiene industrial adecuados. Su exposición ocupacional debe mantenerse dentro de niveles aceptables y vigilarse de forma regular. 3.2 Investigaciones ulteriores a) Debe procederse a la vigilancia sanitaria de los trabajadores expuestos. b) Deben determinarse los niveles efectivos de isoforona en las aguas que rodean a las zonas industriales. c) Deben realizarse estudios adecuados a corto y largo plazo sobre inhalación en animales de experimentación para determinar los niveles inocuos de exposición ocupacional. d) Es necesaria información sobre la biodegradación anaerobia de la isoforona, especialmente por haberse identificado en la lixiviación de terraplenados.
See Also: Isophorone (CHEMINFO) Isophorone (ICSC)