INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 148 BENOMYL 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. Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization First draft prepared by Dr L.W. Hershberger and Dr G.T. Arce, E.I. Du Pont de Nemours and Company, Wilmington, Delaware, USA World Health Orgnization Geneva, 1993 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 Benomyl. (Environmental health criteria ; 148) 1.Benomyl - adverse effects 2.Benomyl - toxicity 3.Environmental exposure I.Series ISBN 92 4 157148 9 (LC Classification: SB 951.3) 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 1993 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 BENOMYL 1. SUMMARY AND CONCLUSIONS 1.1. Summary 1.1.1. Identity, physical and chemical properties, and analytical methods 1.1.2. Sources of human and environmental exposure 1.1.3. Environmental transport, distribution and transformation 1.1.4. Environmental levels and human exposure 1.1.5. Kinetics and metabolism 1.1.6. Effects on laboratory mammals; in vitro test systems 1.1.7. Effects on humans 1.1.8. Effects on other organisms in the laboratory and field 1.2. Conclusions 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1. Chemical identity 2.1.1. Primary constituent 2.1.2. Technical product 2.2. Physical and chemical properties 2.3. Analytical methods 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Anthropogenic sources 3.2.1. Uses 3.2.2. Worldwide sales 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Transport and distribution between media 4.1.1. Air 4.1.2. Water 4.1.3. Soil 4.1.4. Leaching 4.1.5. Crop uptake 4.2. Transformation 4.2.1. Biodegradation 188.8.131.52 Water 184.108.40.206 Soil 220.127.116.11 Crops 4.2.2. Abiotic degradation 4.2.3. Bioaccumulation 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air, water and soil 5.1.2. Food and feed 5.1.3. Terrestrial and aquatic organisms 5.2. General population exposure 5.2.1. USA 5.2.2. Sweden 5.2.3. Maximum residue limits 5.3. Occupational exposure during manufacture, formulation or use 5.3.1. Use 6. KINETICS AND METABOLISM 6.1. Absorption 6.2. Distribution and accumulation 6.3. Metabolic transformation 6.4. Elimination and excretion 6.5. Reaction with body components 7. EFFECTS ON LABORATORY MAMMALS; IN VITRO TEST SYSTEMS 7.1. Single exposure 7.2. Short-term exposure 7.2.1. Gavage 7.2.2. Feeding 18.104.22.168 Rat 22.214.171.124 Dog 7.2.3. Dermal 7.2.4. Inhalation 7.3. Skin and eye irritation; sensitization 7.3.1. Dermal 7.3.2. Eye 7.3.3. Sensitization 7.4. Long-term exposure 7.4.1. Rat 7.4.2. Mouse 7.5. Reproduction, embryotoxicity and teratogenicity 7.5.1. Reproduction 126.96.36.199 Rat feeding studies 188.8.131.52 Rat gavage studies 184.108.40.206 Dog inhalation studies 7.5.2. Teratogenicity and embryotoxicity 220.127.116.11 Mouse gavage studies 18.104.22.168 Rat gavage studies 22.214.171.124 Rat feeding studies 126.96.36.199 Rabbit feeding studies 7.6. Mutagenicity and related end-points 7.7. Carcinogenicity 7.7.1. Rat 7.7.2. Mouse 7.8. Special studies 7.8.1. Neurotoxicity 7.8.2. Effects in tissue culture 7.9. Factors modifying toxicity; toxicity of metabolites 7.10. Mechanisms of toxicity - mode of action 8. EFFECTS ON HUMANS 8.1. General population exposure 8.2. Occupational exposure 8.2.1. Acute toxicity 8.2.2. Effects of short- and long-term exposure 9. EFFECTS ON ORGANISMS IN THE LABORATORY AND FIELD 9.1. Microorganisms 9.2. Aquatic organisms 9.3. Terrestrial organisms 9.4. Population and ecosystem effects 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Evaluation of human health risks 10.2. Evaluation of effects on the environment 10.3. Conclusions 11. FURTHER RESEARCH 12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES REFERENCES RESUME ET CONCLUSIONS RESUMEN Y CONCLUSIONES WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR BENOMYL AND CARBENDAZIM Members Dr G. Burin, Office of Pesticide Programmes, US Environmental Protection Agency, Washington, D.C., USA Dr R. Cooper, Reproductive Toxicology Branch, US Environmental Protection Agency, Research Triangle Park, North Carolina, USA Dr I. Desi, Department of Public Health, Albert Szent-Györgyi University Medical School, Szeged, Hungary Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon, United Kingdom Dr A. Helweg, Department for Pesticide Analysis and Ecotoxicology, Danish Research Service for Plant and Soil Science, Flakkebjerg, Slagelse, Denmark Dr M. Lotti, Institute of Occupational Medicine, University of Padua, Padua, Italy ( Chairman) Dr K. Maita, Toxicology Division, Institute of Environmental Toxicology, Kodaira-Shi, Tokyo, Japan Dr F. Matsumura, Department of Environmental Toxicology, Institute of Toxicology and Environmental Health, University of California, Davis, California, USA Dr T.K. Pandita, Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore, Indiaa Dr C. Sonich-Mullin, Environmental Criteria and Assessment Office, US Environmental Protection Agency, Cincinnati, Ohio, USA Dr P.P. Yao, Institute of Occupational Medicine, Chinese Academy of Preventive Medicine, Beijing, China a Invited but unable to attend the meeting Secretariat Dr B.H. Chen, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland ( Secretary) Dr L.W. Hershberger, Dupont Agricultural Products, Walker's Mill, Barley Mill Plaza, Wilmington, Delaware, USA ( Rapporteur) Mr P. Howe, Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntington, United Kingdom NOTE TO READERS OF THE CRITERIA MONOGRAPHS Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are kindly requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda. * * * A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Palais des Nations, 1211 Geneva 10, Switzerland (Telephone No. 7988400 or 7985850). ENVIRONMENTAL HEALTH CRITERIA FOR BENOMYL A WHO Task Group on Environmental Health Criteria for Benomyl and Carbendazim, sponsored by the US Environmental Protection Agency, met in Cincinnati, USA, from 14 to 19 September 1992. On behalf of the host agency, Dr T. Harvey opened the meeting and welcomed the participants. Dr B.H. Chen of the International Programme on Chemical Safety (IPCS) welcomed the participants on behalf of the Director, IPCS, and the three IPCS cooperating organizations (UNEP/ILO/WHO). The Task Group reviewed and revised the draft criteria monograph and made an evaluation of the risks for human health and the environment from exposure to benomyl. The first draft of this monograph was prepared by Dr L.W. Hershberger and Dr G.T. Arce of E.I. Du Pont de Nemours and Company, Wilmington, Delaware, USA. The second draft was prepared by Dr L.W. Hershberger incorporating comments received following the circulation of the first draft to the IPCS Contact Points for Environmental Health Criteria monographs. Dr M. Lotti (Institute of Occupational Medicine, University of Padua, Italy) made a considerable contribution to the preparation of the final text. Dr B.H. Chen and Dr P.G. Jenkins, both members of the IPCS Central Unit, were responsible for the overall scientific content and technical editing, respectively. The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged. Financial support for the meeting was provided by the US Environmental Protection Agency, Cincinnati, USA. ABBREVIATIONS ADI acceptable daily intake a.i. active ingredient BIC butyl isocyanate BUB 2-(3-butylureido)benzimidazole EEC European Economic Community HPLC high performance liquid chromatography Koc Distribution coefficient between pesticide adsorbed to soil organic carbon and pesticide in solution Kom Distribution coefficient between pesticide adsorbed to soil organic matter and pesticide in solution MRL maximum residue limits NOEL no-observed-effect level OECD Organisation for Economic Co-operation and Development STB 3-butyl-1,3,5-triazino[1,2a]-benzimidazol-2,4(1H,3H)dione 2-AB 2-aminobenzimidazole 5-HBC methyl (5-hydroxy-1H-benzimidazol-2-yl)-carbamate 1. SUMMARY AND CONCLUSIONS 1.1 Summary 1.1.1 Identity, physical and chemical properties, and analytical methods Benomyl, a tan crystalline solid, is a systemic fungicide belonging to the benzimidazole family. It decomposes just above its melting point of 140 °C and has a vapour pressure of < 5 x 10-6 Pa (< 3.7 x 10-8 mmHg) at 25 °C. Benomyl is virtually insoluble in water at pH 5 and 25 °C, the solubility being 3.6 mg/litre. It is stable under normal storage conditions but decomposes to carbendazim in water. Residual and environmental analyses are performed by extraction with an organic solvent, purification of the extract by a liquid-liquid partitioning procedure, and conversion of the residue to carbendazim. Measurement of residues may be determined by high performance liquid chromatography or immunoassay. 1.1.2 Sources of human and environmental exposure In 1988, the estimated worldwide use of benomyl was approximately 1700 tonnes. It is a widely used fungicide registered for use on over 70 crops in 50 countries. Benomyl is formulated as a wettable powder. 1.1.3 Environmental transport, distribution and transformation Benomyl is rapidly converted to carbendazim in the environment with half-lives of 2 and 19 h in water and in soil, respectively. Data from studies on both benomyl and carbendazim are therefore relevant for the evaluation of environmental effects. Carbendazim decomposes in the environment with half-lives of 6 to 12 months on bare soil, 3 to 6 months on turf, and 2 and 25 months in water under aerobic and anaerobic conditions, respectively. Carbendazim is mainly decomposed by microorganisms. 2-Aminobenzimidazole (2-AB) is a major degradation product and is further decomposed by microbial activity. When phenyl-14C-labelled benomyl was decomposed, only 9% of the 14C was evolved as CO2 during 1 year of incubation. The remaining 14C was recovered mainly as carbendazim and bound residues. The fate of a possible degradation product (1,2-diaminobenzene) may further clarify the degradation pathway of benzimidazole fungicides in the environment. Field and column studies have shown that carbendazim remains in the soil surface layer. There is no available determination of carbendazim adsorption in soil, but it is expected to be as strongly adsorbed to soil as benomyl, with Koc values ranging from 1000 to 3600. The log Kow values for benomyl and carbendazim are 1.36 and 1.49, respectively. No risk of leaching was apparent when this was evaluated in a screening model based on adsorption and persistence data. This statement is supported by analyses of well-water in the USA where benomyl was not found in any of 495 wells and carbendazim not in any of 212 wells (limit of detection not available). Surface run-off of benomyl and carbendazim is expected to consist only of fungicide adsorbed to soil particles, and these compounds are expected to be strongly adsorbed to sediments in the aqueous environment. Benomyl in solutions, plants and soil degrades to carbendazim (methyl-1H-benzimidazol-2-carbamate) and to 2-AB, STB (3-butyl-1,3,5-triazino[1,2a]-benzimidazol-2,4(1H,3H)dione) and BBU (1-(2-benzimidazolyl)-3- n-butylurea). There is little or no photolysis of benomyl. In animal systems, benomyl is metabolized to carbendazim and other polar metabolites, which are rapidly excreted. Neither benomyl nor carbendazim has been observed to accumulate in any biological system. 1.1.4 Environmental levels and human exposure No environmental monitoring data for benomyl appear to be available. However, the following can be summarized from environmental fate studies. Since benomyl and carbendazim remain stable for several weeks on plant material, they may become accessible to organisms feeding on leaf litter. Soil and sediments may contain residues of carbendazim for up to 3 years. However, the strong adsorption of carbendazim to soil and sediment particles reduces the exposure of terrestrial and aquatic organisms. The main source of exposure for the general human population is residues of benomyl and carbendazim in food crops. Dietary exposure analysis in the USA (combined benomyl and carbendazim) and the Netherlands (carbendazim) yielded an expected mean intake of about one-tenth of the recommended Acceptable Daily Intake (ADI) for benomyl of 0.02 mg/kg body weight and for carbendazim of 0.01 mg/kg body weight. Occupational exposures during the manufacturing process are below Threshold Limit Values. Agricultural workers engaged in pesticide mixing and loading or re-entering benomyl-treated fields are expected to be exposed dermally to a few mg of benomyl per hour. This type of exposure could be reduced by the use of protective devices. Furthermore, since dermal absorption is expected to be low, the probability of benomyl having systemic toxic effects on human populations through this route is very low. 1.1.5 Kinetics and metabolism Benomyl is readily absorbed in animal experiments after oral and inhalation exposure, but much less so following dermal exposure. Absorbed benomyl is rapidly metabolized and excreted in the urine and faeces. In rats fed 14C-labelled benomyl, its metabolites carbendazim and methyl(5-hydroxy-1H-benzimidazol-2-yl)-carbamate (5-HBC) were found in the blood and in small amounts in the testes, kidneys and livers. The tissue distribution showed no bioconcentration. In urine the primary metabolite was 5-HBC, some carbendazim also being present. By 72 h after administration, 98% of the given amount had been excreted. In cows dosed by capsule for 5 days with radiolabelled benomyl at a dose equivalent to 50 mg/kg diet, there was a benomyl equivalent level of 4 mg/kg in the liver, 0.25 mg/kg in the kidney and no significant levels in other tissues or fat. During feeding, 65% of the radiolabel was excreted in the urine, 21% in the faeces and 0.4% in the milk. The major metabolite in the milk was 5-HBC. Similar metabolism and elimination patterns were found in other animals. Benomyl does not inhibit acetyl cholinesterase in vitro. It has been shown to induce liver epoxyhydrolase, gamma-glutamyl transpeptidase and glutathione- S-transferase in in vivo studies on mice and rats. 1.1.6 Effects on laboratory mammals; in vitro test systems 188.8.131.52 Single exposure Benomyl has low acute toxicity with an oral LD50 in the rat of > 10 000 mg/kg and an inhalation 4-h LC50 of > 4 mg/litre. Carbendazim, like its parent compound benomyl, has an LD50 in rats of > 10 000 mg/kg. Dogs, exposed via inhalation for 4 h at 1.65 mg/litre and examined 28 days after exposure, showed decreased liver weight. A single dose of benomyl to rats by gavage showed reproductive effects at 70 days after exposure (see section 184.108.40.206). 220.127.116.11 Short-term exposure Short-term gavage, dietary or dermal administration of benomyl for up to 90 days slightly increased liver weights in the rat (125 mg/kg per day, dietary) and produced effects on male reproductive organs (decreased testis and epididymal weights, decreased sperm production) in the rat (45 mg/kg per day, gavage; no-observed-effect level (NOEL) = 15 mg/kg), rabbit (1000 mg/kg per day, oral; 500 mg/kg body weight per day, dermal) and beagle dog (62.5 mg/kg; NOEL = 18.4 mg/kg per day, dietary). Liver and testicular effects were not observed in rats exposed via inhalation to benomyl concentrations of up to 200 mg/m3 for 90 days. 18.104.22.168 Skin and eye irritation and sensitization Application to the skin of the rabbit and guinea-pig produced either mild or no irritation and moderate skin sensitization. Application to the eyes of rats produced temporary mild conjunctival irritation. 22.214.171.124 Long-term exposure A long-term feeding study in rats did not demonstrate any compound-related effects at dose levels up to and including 2500 mg/kg diet (125 mg/kg body weight per day). This study was not considered adequate to evaluate reproductive effects. In the CD-1 mouse, liver weights were increased at dose levels of 1500 mg/kg diet or more. Male mice had decreased absolute testes weights and thymic atrophy at a level of 5000 mg/kg diet. 126.96.36.199 Reproduction, embryotoxicity, and teratogenicity Benomyl causes a decrease in testis and epididymis weight, a reduction in caudal sperm reserves, a decrease in sperm production, and a lowering of male fertility rates. At higher doses, there is hypospermatogenesis with generalized disruption of all stages of spermatogenesis. Benomyl does not effect copulatory behaviour, seminal vesicles, sperm mobility or related reproductive hormones. The lowest benomyl concentration shown to induce a statistically significant spermatogenic effect in male rats was 45 mg/kg per day. The NOEL for these effects was 15 mg/kg per day. A single dose of benomyl (100 mg/kg or more) administered to rats by gavage showed effects, at 70 days aftr exposure, which included decreased testis weight and seminiferous tubular atrophy. When administered via gavage from days 7 to 16 of gestation to ChD-CD rats and Wistar rats, benomyl was found to be teratogenic at 62.5 mg/kg for both strains, but not at 30 mg/kg for ChR-CD rats and not at 31.2 mg/kg for Wistar rats. When Sprague-Dawley rats were administered by gavage on days 7 to 21 of gestation, benomyl was found to be teratogenic at 31.2 mg/kg. The effects were microphthalmia, hydrocephaly, and encephaloceles. Postnatal development of rats was adversely affected at dose levels greater than 15.6 mg/kg. In mice, gavage dosing at a concentration of 50 mg/kg or more induced supernumery ribs and other skeletal and visceral anomalies. A NOEL was not established in the mouse because no doses lower than 50 mg/kg were tested. Except for a marginal increase in supernumery ribs in rabbits, no teratogenic effects were observed at dose levels as high as 500 mg/kg diet. 188.8.131.52 Mutagenicity and related end-points Studies in somatic and germ cells show that benomyl does not cause gene mutations or structural chromosomal damage (aberrations) and it does not interact directly with DNA (causing DNA damage and repair). This has been demonstrated in both mammalian and non-mammalian systems. Benomyl does, however, cause numerical chromosome aberrations (aneuploidy and/or polyploidy) in experimental systems in vitro and in vivo. 184.108.40.206 Carcinogenicity Benomyl or carbendazim caused liver tumours in two strains of mice (CD-1 and Swiss (SPF)) that have a high spontaneous rate of liver tumours. In contrast, carbendazim was not carcinogenic in NMRKf mice, which have a low spontaneous rate of such tumours. The first carcinogenicity study using CD-1 mice showed a statistically significant dose-related increase of hepatocellular neoplasia in females, and a statistically significant response was also observed in the mid-dose (1500 mg/kg) males but not in the high-dose males because of the high mortality rate. A second carcinogenicity study of carbendazim in a genetically related mouse strain, SPF mice (Swiss random strain), at doses of 0, 150, 300 and 1000 mg/kg (increased to 5000 mg/kg during the study) showed an increase in the incidence of combined hepatocellular adenomas and carcinomas. A third study carried out in NMRKf mice at doses of 0, 50, 150, 300 and 1000 mg/kg (increased to 5000 mg/kg during the study) showed no carcinogenic effects. Carcinogenicity studies with both benomyl and carbendazim were negative in rats. 220.127.116.11 Mechanism of toxicity - mode of action The biological effects of benomyl and carbendazim are thought to be the result of their interaction with cell microtubules. These structures are involved in vital functions such as cell division, which is inhibited by benomyl and carbendazim. Benomyl and carbendazim toxicity in mammals is linked with microtubular dysfunction. Benomyl and carbendazim, like other benzimidazole compounds, display selective toxicity for species. This selectivity is, at least in part, explained by the different binding of benomyl and carbendazim to tubulins of target and non-target species. 1.1.7 Effects on humans Benomyl causes contact dermatitis and dermal sensitization. No other effects have been reported. 1.1.8 Effects on other organisms in the laboratory and field Benomyl has little effect on soil microbial activity at recommended application rates. Some adverse effects have been reported for groups of fungi. The 72-h EC50, based on total growth, for the green alga Selenastrum capricornutum was calculated to be 2.0 mg/litre; the no-observed-effect concentration (NOEC) was 0.5 mg/litre. The toxicity of benomyl to aquatic invertebrates and fish varies widely, 96-h LC50 values ranging from 0.006 mg/litre for the channel catfish (yolk-sac fry) to > 100 mg/litre for the crayfish. Benomyl is toxic to earthworms in laboratory experiments at realistic exposure concentrations and as a result of recommended usage in the field. It is of low toxicity to birds and its degradation product carbendazim is "relatively non-toxic" to honey-bees. 1.2 Conclusions Benomyl causes dermal sensitization in humans. Both benomyl and carbendazim represent a very low risk for acute poisoning in humans. Given the current exposures and the low rate of dermal absorption of these two compounds, it is unlikely that they would cause systemic toxicity effects either in the general population or in occupationally exposed subjects. These conclusions are drawn from animal data and the limited human data available, and are supported by the understanding of the mode of action of carbendazim and benomyl in both target and non-target species. Further elucidation of the mechanism of toxicity of benomyl and carbendazim in mammals will perhaps permit a better definition of no-observed-effect levels. Binding studies on tubulins of target cells (testis and embryonic tissues) will facilitate inter-species comparisons. Carbendazim is strongly adsorbed to soil organic matter and remains in the soil for up to 3 years. Carbendazim persists on leaf surfaces and, therefore, in leaf litter. Earthworms have been shown to be adversely affected (population and reproductive effects) at recommended application rates. There is no information on other soil or litter arthropods that would be similarly exposed. The high toxicity to aquatic organisms in laboratory tests is unlikely to be seen in the field because of the low bioavailability of sediment-bound residues of carbendazim. However, no information is available on sediment-living species, which would receive the highest exposure. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Chemical identity 2.1.1 Primary constituent Chemical structure: Molecular formula: C14H18N4O3 Common name: Benomyl CAS chemical name: Carbamic acid, [1-(butylamino)carbonyl]-1H- benzimidazol-2-yl]-, methyl ester IUPAC chemical name: Methyl 1-[(butylamino)carbonyl]-1H- benzimidazol-2-ylcarbamate CAS registry number: 17804-35-2 Relative molecular mass: 290.3 Synonym: Methyl 1-(butylcarbamoyl)-2-benzimida- zolecarbamate 2.1.2 Technical product Major trade names: Benlate, Tersan, Fungicide 1991, Fundazol Purity: > 95% (FAO specifications) 2.2 Physical and chemical properties Table 1. Some physical and chemical properties of Benomyl Physical state Crystalline solid Colour Tan Odour Negligible Melting point/boiling point/ Decomposes just after flash point melting at 140 °C Explosion limits LEL = 0.05 g/litre in air Vapour pressure < 5.0 x 10-6 Pa (< 3.7 x 10-8 mmHg) at 25 °Ca Density 0.38 g/cm3 Log n-octanol/water partition coefficient 1.36 Solubility in water 3.6 mg/litre (at pH 5 and 25 °C) Solubility in organic solvents Chloroform 9.4 (g/100 g solvent at 25 °C) Dimethylformamide 5.3 Acetone 1.8 Xylene 1.0 Ethanol 0.4 Heptane 40 Henry's constant < 4.2 x 10-9 atm-m3/mol at pH 5 and 25 °C Soil/water partition coefficient 1090 mg/g (Kom); 1860 mg/g (Koc)b a Barefoot (1988) b Koc = Distribution coefficient between pesticide adsorbed to soil organic carbon and pesticide in solution. Kom = Distribution coefficient between pesticide adsorbed to soil organic matter and pesticide in solution. 2.3 Analytical methods Most methods for determining benomyl and its by-product residues in plant and animal tissue and in soil involve isolation of the residue by extraction with an organic solvent, purification of the extract by a liquid-liquid partitioning procedure, and conversion of the residue to carbendazim. Residues may be measured by procedures using high-speed cation-exchange liquid chromatography, reversed phase HPLC, and immunoassay. One method for analysis of water samples can distinguish between benomyl and carbendazim. Recoveries of benomyl, carbendazim and 2-AB (2-aminobenzimidazole) from various types of soils average 92, 88 and 71%, respectively. The lower limit of sensitivity of the method is 0.05 ppm for each of these components. The recoveries and sensitivities for plant tissues are similar. Table 2 outlines various analytical methods for soil, water, plant and animal tissue. Table 2. Analytical Methods for Benomyl Analytical method Medium Detection limit Comments Reference Strong cation exchange/HPLC soil 0.05 mg/kg acidic methanol extraction converts Kirkland et al. (1973) residual benomyl to carbendazim Strong cation exchange/HPLC plant 0.05 mg/kg acidic methanol extraction converts Kirkland et al. (1973) residual benomyl to carbendazim Strong cation exchange/HPLC animal 0.01 mg/kg (milk) acidic aqueous hydrolysis followed Kirkland (1973) 0.05 mg/kg by organic extraction converts (tissue) benomyl to carbendazim and frees metabolites from conjugates Reversed phase HPLC water 9.0 x 10-6 g/litre on-line HPLC with preconcentration; Marvin et al. (1991) benomyl and carbendazim determined separately Reversed phase blueberries 0.03 mg/kg acidic methanol extraction converts Bushway et al. (1991) HPLC/fluorescence detection residual benomyl to carbendazim Radioimmunoassay plant 0.05-1.0 mg/kg ethyl acetate extraction converts Newsome & Shields (dependent on crop) residual benomyl to carbendazim (1981) Enzyme-linked immunosorbent plant 0.50 mg/kg ethyl acetate extraction converts Newsome & Collins assay (ELISA) residual benomyl to carbendazim (1987) 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence Benomyl does not occur naturally. 3.2 Anthropogenic sources 3.2.1 Uses Benomyl is one of the most widely used members of a family of fungicides known as benzimidazoles. It is registered in more than 50 countries for use on more than 70 crops, including cereals, cotton, grapes, bananas and other fruits, ornamentals, plantation crops, sugar beet, soybeans, tobacco, turf, vegetables, mushrooms and many other crops, and is used under most climatic conditions. Registered benomyl usage specifies rates from 0.1 to 2.0 kg a.i./ha and applications from once per year to spray intervals ranging from 7 to 14 days (FAO/WHO, 1985a; 1988a). Benomyl is effective at low usage rates against more than 190 different fungal diseases such as leaf spots, blotches and blights; fruit spots and rots; sooty moulds; scabs; bulb, corn and tuber decays; blossom blights; powdery mildews; certain rusts; and common soilborne crown and root rots. A key limitation to the use of benomyl and other benzimidazoles is the development of fungal resistance. Resistance management can be achieved by using benomyl in combination with a non-benzimidazole companion fungicide as a tank mix or it may be used alternately with a non-benzimidazole fungicide (Delp, 1980; Staub & Sozzi, 1984). Benomyl is formulated as a wettable powder and dry flowable or dispersible granules. In some countries the latter formulation is no longer available. 3.2.2 Worldwide sales In 1991, the estimated worldwide sales of benomyl was US$ 290 million. This was about 50% of the worldwide market for benzimidazole products. Carbendazim (20%) and thiophanatemethyl (20%) account for most of the rest of the benzimidazole market (County NatWest WoodMac). 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1 Transport and distribution between media 4.1.1 Air Benomyl has a vapour pressure of < 5.0 x 10-6 Pa (< 3.7 x 10-8 mmHg) and a solubility in water of 3.6 mg/litre at pH 5 and 25 °C. As a result, it has a Henry's constant of < 4.2 x 10-9 atm-m3/mol. Benomyl is essentially non-volatile from water surfaces. 4.1.2 Water The half-life of benomyl in surface water and sediment under aerobic conditions has been shown to be approximately 2 h. Its metabolite carbendazim had a half-life of 61 days under non-sterile conditions. After 30 days, 22% of the applied radioactivity was bound to sediments and < 1% of the applied radioactivity was evolved as carbon dioxide (Arthur et al., 1989a). 4.1.3 Soil Radiolabelled benomyl was found to be strongly adsorbed (Ka = 6.1 and 13 µg/g) to two different sandy loam soils and very strongly adsorbed (Ka = 50 and 90 µg/g) to two different silt loam soils. Adsorption was not significantly affected by the benomyl concentration over the range 0.2-2.3 ppm. Adsorbed radioactivity was not readily desorbed from any of the test soils. The Ka, corrected for the organic matter content of the soils, was 2-4 times higher on the silt loam than on the sandy loam soil. This difference suggests that variables other than percentage organic matter (i.e. cation exchange capacity, particle size or compound degradation) influence adsorption. The ease of desorption appears to be inversely related to the organic content of the soils (Priester, 1985). The structure of benomyl and its soil degradation products, i.e. carbendazim (methyl 1H-benzimidazol-2-ylcarbamate), 2-AB (2-aminobenzimidazole), STB (3-butyl-1,3,5-triazino[1,2a]-benzimidazol-2,4(1H,3H)dione), and BBU (1-(2-benzimidazolyl)-3-n-butylurea), which is also known as 2-(3-butylureido)-benzimidazole (BUB), are given in Fig. 1. The major proportion of each of the metabolites was found in the uppermost (0-12.7 cm) soil layer. The extent of mobility correlated with the type and characteristics of the soil to which benomyl was applied. The 14C label was less mobile in soils of lower sand content and higher silt or clay content. It was also found to be less mobile on soils of higher organic content and lower pH (Chang, 1985). In a soil column leaching experiment in rice paddy soil, benomyl did not leach significantly. Approximately 94% was found in the top 5 cm, 9% in the next 5-10 cm, and less than 1% was detected in any lower segments (Ryan, 1989). These data indicate that benomyl, carbendazim, BUB and STB are highly immobile. Similar mobility results have been observed in the field. Benomyl and its degradates were studied on bare soil and turf in four areas of the USA. Carbendazim and 2-AB were the major and minor degradates, respectively. After 1 and 2 years of outdoor exposure, the half-life of total benzimidazole-containing residues was about 3 to 6 months on turf and about 6 to 12 months on bare soil (Baude et al., 1974). Under these conditions, benomyl, carbendazim and 2-AB showed little or no downward movement. 4.1.4 Leaching To evaluate the risk of pollution of ground and drainage water, screening models based on adsorption and persistence can be used, together with existing analyses of groundwater samples. Gustafson (1989) proposed the use of the equation GUS = log T´ (4 - log Koc); GUS values < 1.8 = "improbable leachers", GUS values of 1.8-2.8 = "transition" and GUS values > 2.8 = "probable leachers". For benomyl, Kom values of 550, 620, 2100 and 1100 (mean 1093) were found in four different soils (Priester, 1985). A Kom of 1093 is equal to a Koc of 1857 since Koc = Kom x 1.7. The half-life of 320 days given by Marsh & Arthur (1989) seems in good agreement with field half-lives of 6 to 12 months (Baude et al., 1974). When the calculation of the GUS value is based on a Koc of 1857 and a T0.5 of 320 days, a value of 1.83 is obtained. According to this value, benomyl/carbendazim lies between the "improbable leachers" and "transition", and, therefore, would not be expected to occur in ground water. The adsorption of benomyl and of carbendazim is expected to be of the same order of magnitude since the Kow values are almost identical (log Kow = 1.49 and 1.36 for carbendazim and benomyl, respectively). In ground water studies in the USA (Parsons & Witt, 1988), benomyl was not found in any of 495 wells tested and carbendazim not in any of 212 wells (detection limit not reported). In an EEC survey (Fielding, 1992), the presence of carbendazim in groundwater in the Netherlands and in Italy was investigated. Carbendazim was found in one of two samples from the Netherlands (0.1 µg/litre), and the level was above 0.1 µg/litre in 23 of 70 samples in Italy. Detection of the non-polar DDT and lindane in many wells in the Italian study may indicate macropore transport or artifacts such as direct pollution of wells. 4.1.5 Crop uptake Various greenhouse and outdoor tests, in which benomyl was applied to several crops (apples, bananas, cucumbers, grapes and oranges), indicate that benomyl and carbendazim remain on plant surfaces as major components of the total residue (Baude et al., 1973). Benomyl is primarily converted to carbendazim once inside plant tissues. Although benomyl is systemic when applied directly to plant foliage, crop uptake of soil residues is extremely low, even when the crop is planted in the same growing season as the benomyl treatment. In a greenhouse crop-rotation study, [2-14C]- carbendazim, the more persistent benomyl metabolite, was applied to a loamy sand soil. Aging periods of 30, 120 or 145 days were used and the crops studied were beets, barley and cabbage. Radioactivity did not accumulate in these crops grown to maturity in a loamy sand soil treated 30 days earlier with 1 kg a.i./ha or 120 to 145 days earlier with 3 kg a.i./ha. Accumulation factors, calculated as the ratio of radioactivity in the crop to that in the corresponding soil, were very low in beet foliage (0.04) and beet roots (0.03), low in cabbage and barley grain (0.2), and ranged from 0.9 to 1.2 in barley straw (Rhodes, 1987). 4.2 Transformation Numerous field studies to determine the fate and behaviour of benomyl in soil have shown the instability of benomyl under various conditions. In solutions, plants, and soil, it degrades to carbendazim. The conversion of benomyl under alkaline conditions to STB and BBU has also been reported (section 4.1). The environmental fate of benomyl has been thoroughly reviewed by Zbozinek (1984). 4.2.1 Biodegradation 18.104.22.168 Water Anaerobic aquatic degradation studies in pond water and sediment showed a half-life of 2 h for benomyl and 743 days for its degradation product carbendazim. Some (1-8%) transformation to STB occurred. After one year 36% of the applied radioactivity was bound to the sediment (Arthur et al., 1989b). 22.214.171.124 Soil In a study by Marsh & Arthur (1989), non-sterile and sterile samples of Keyport silt loam soil were treated with [phenyl(U)- 14C]benomyl at a concentration of approximately 7.0 mg/kg. This is equivalent to the expected soil residues in the surface 10 cm of topsoil when benomyl is applied at 8 kg a.i./ha. Distilled water was added to each sample until it reached 75% of its moisture-holding capacity at 0.33 bar. The soils were incubated in the dark at approximately 25 °C. The non-sterile soil flasks were sampled after 0.1, 0.2, 1, 3, 7, 14, 30, 60, 120, 270 and 365 days. Samples of sterilized soil were taken after 14, 30, 120, 270 and 365 days. The half-life of benomyl in non-sterile silt loam was 19 h, but this was not determined in the sterilized soil. Benomyl was rapidly converted to carbendazim. The carbendazim had a half-life of 320 days under non-sterile aerobic conditions (Marsh & Arthur, 1989). This is in close agreement with reported half-lives of 6-12 months for benzimidazoles applied to bare soil (Baude et al., 1974). After 365 days of incubation, 9% of the 14C was evolved as 14CO2, 34% could still be recovered as carbendazim, and 36% was not extractable. The total recovery of 14C was 88%. In the sterilized soil, the half-life of carbendazim was approximately 1000 days (Marsh & Arthur, 1989). When the degradation of 2-14C-carbendazim (20 mg/kg) was determined, 33% of the 14C label added was evolved as 14CO2 during 270 days. Identical or even faster 14C evolution was observed from 2-14C-labelled 2-AB (Helweg, 1977). The relatively low 14C evolution from phenyl-14C-labelled benomyl/carbendazim may be caused by the formation of strongly adsorbed degradation products or compounds that are readily incorporated into soil organic matter. Thus, most of the remaining radioactivity was accounted for in the organic fraction of the soil. To elucidate the reason for the low 14C evolution from phenyl-14C-labelled fungicide, the fate of a possible degradation product, 1,2-diaminobenzene, needs to be determined. 126.96.36.199 Crops Metabolism studies in various crops (soybeans, rice, sugar beet and peaches) using [phenyl(U)-14C]benomyl have shown that the only species of significance in plant tissues are benomyl, carbendazim and 2-AB. Soybeans were treated twice with 1 kg a.i./ha and harvested 35 days later. Rice was treated twice with 2 kg a.i./ha and harvested at 21 days, sugar beet was treated with 0.5 kg a.i./ha five times and harvested at 21 days, and peaches were treated twice at 1 kg a.i./ha and harvested 20 min after spraying. Soybeans, rice and sugar beet were treated at twice the recommended application rate. The concentration of radiolabelled compounds in mature soybeans was 0.7 mg/kg and consisted of 0.42 mg 2-AB/kg, 0.05 mg benomyl/kg and 0.14 mg carbendazim per kg (Bolton et al., 1986a). Levels in the rice grain were 2.7 and 7.3 mg/kg for benomyl and carbendazim, respectively (Bolton et al., 1986b). Sugar beet tops retained 99% of the total recovered radioactivity, 6.8 mg/kg being present as carbendazim and 0.4 mg/kg as benomyl. The roots retained only 0.01 mg carbendazim per kg (Tolle, 1988). After the first application to peaches, benomyl was present at 0.65 mg/kg and carbendazim at 0.72 mg/kg. The second application resulted in 0.33 mg benomyl/kg and 0.92 mg carbendazim/kg. No other radioactive metabolites were found in peaches (Stevenson, 1985). Chiba & Veres (1981) applied benomyl to apple trees as Benlate 50% WP at a rate of 1.7 kg/ha. Three successive applications were made in 1977 and a single spray was applied in 1979. Between 3 and 7 days after application there was a marked reduction of about 50% in benomyl residues from an initial level of about 110 mg/kg. This fall in benomyl was accompanied by a doubling in the level of carbendazim residues over the same period due to benomyl degradation to carbendazim. Within 46 days of the single application in 1979, benomyl residues fell to 0.63 mg/kg foliage and carbendazim was present at 1.2 mg/kg. Following the three sprayings in 1977 (at 0, 13 and 27 days after the initial application), residue levels were 2.6 and 17.1 mg/kg foliage for benomyl and carbendazim 83 days after the first spraying. Both experiments showed an exponential fall in benomyl residues but the rate of decline was much slower in the case of the more persistent metabolite. 4.2.2 Abiotic degradation In a study by Wheeler (1985), the hydrolysis of benomyl was studied in sterilized aqueous solutions maintained at 25 °C in the dark for 30 days at pH 5, 7 and 9. In pH 5 buffer, the major product was carbendazim, whereas at pH 7 and 9 carbendazim and STB were the major products. STB represented approximately 25% of the total radioactivity at pH 7 and approximately 80% at pH 9. The half-lives of benomyl in the pH 5, 7 and 9 solutions were approximately 3.5, 1.5 and less than 1 h, respectively. There was no further degradation of carbendazim at pH 5 and 7 over 30 days. At pH 9, however, carbendazim was slowly hydrolysed to 2-AB with a half-life of 54 days (Priester, 1984). Aqueous photolysis studies conducted in natural sunlight have shown that benomyl is mainly degraded by hydrolysis rather than photolysis (Powley, 1985). 4.2.3 Bioaccumulation Although only low concentrations of benomyl or its metabolites would be expected in natural waters, studies have evaluated the metabolism and bioaccumulation in fish. Bluegill sunfish ( Lepomis macrochirus) were exposed to radiolabelled carbendazim concentrations of 0.018 or 0.17 mg/litre for 4 weeks in a dynamic study designed to measure the bioaccumulation of 14C residues in edible tissue, viscera, remaining carcass and whole fish. A two-week depuration phase followed the exposure phase. Results were similar at the two exposure concentrations, the peak whole fish bioconcentration factors (BCFs) being 27 and 23 at the low and high exposure levels, respectively. The radioactivity was concentrated more in the viscera than in other tissues, the peak viscera BCFs being 460 and 380 for the low and high exposure levels, respectively. Very little bioconcentration occurred in the muscle tissue (BCF = < 4) or the remaining carcass (BCF = < 7). During the 14-day depuration phase, > 94% of the peak level of radioactivity was lost from the whole fish, viscera and muscle. The rate of loss from the carcass tissue was lower (77% and 82% loss for the low and high exposure levels, respectively) (Hutton et al., 1984). When rainbow trout ( Oncorhynchus mykiss), channel catfish ( Ictalurus punctatus) and bluegill sunfish ( Lepomis macrochirus) were injected intraperitoneally with carbendazim, branchial and biliary excretion were the major pathways for the elimination (Palawski & Knowles, 1986). In a separate experiment, the three fish species were exposed to 45 µg carbendazim/litre for 96 h, except in the case of catfish, which were exposed for 48 h. This was followed by a 96-h depuration phase. Rainbow trout had the highest uptake rate constant (1.78 per h) and bioconcentration factor (159) of the three species. Much less carbendazim was accumulated by channel catfish than by the other two species, but this residue level (0.44 µg/g) appeared to be lethal after 48 h of exposure. The elimination rate constant and the biological half-life of carbendazim were similar for rainbow trout and bluegill sunfish. However, the elimination rate constant was greater and the biological half-life shorter in channel catfish (13 h) than in the other two species. 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air, water and soil The environmental levels in air, water and soil are discussed in detail in section 4. 5.1.2 Food and feed Levels of benomyl in food and feed are indicated in section 5.2. 5.1.3 Terrestrial and aquatic organisms Benomyl levels in terrestrial and aquatic organisms are discussed in detail in sections 4 and 6. 5.2 General population exposure The principal exposure of the general population to benomyl is through dietary exposure. It was recommended by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) (FAO/WHO, 1988b) that all maximum residue limits (MRLs) for benomyl, thiophanate-methyl and carbendazim be listed as carbendazim (see Table 5). 5.2.1 USA A system called the Dietary Risk Evaluation System (DRES), which was developed by the US Environmental Protection Agency, was used to quantify the intake of residues occurring in various commodities. The system assumes a diet consistent with the 1977-1978 USDA Nationwide Food Consumption Survey. This survey was a stratified probability survey in which 3-day dietary records of approximately 30 000 individuals were collected. The dietary intake of residues resulting from registered food crop uses of benomyl was then estimated using mean residue levels found in controlled field trials and adjusting for the effects of food processing, e.g., washing and cooking, on residues of benomyl and its metabolites. Based on this analysis, the total dietary exposure was determined for the general population and for a number of population subgroups. The exposure of the average person to residues resulting from benomyl use was estimated to be 0.218 µg/kg body weight per day. The highest exposure was found in the population subgroup entitled "non-hispanic other than black or white", the estimated exposure being 1.479 µg/kg body weight per day. The lowest exposure was found in the > 20-year-old males where the estimated exposure was 0.144 µg/kg body weight per day (Eickhoff et al., 1989). These estimates are below the benomyl ADI allocated by JMPR (0-0.02 mg/kg body weight per day) (FAO/WHO, 1985a,b). 5.2.2 Sweden Residue monitoring data for benzimidazole fungicides, i.e. benomyl, carbendazim and thiophanate-methyl, on food crops from Sweden is shown in Table 3 (FAO/WHO, 1988b). No further analysis to determine dietary intake was performed. 5.2.3 Maximum residue limits National MRLs for certain commodities are listed in Table 4 (FAO/WHO, 1988a). A complete list of MRLs for carbendazim, including new proposals and an indication of the source of the data (application of benomyl, carbendazim, or thiophanate-methyl) on which the MRL is based, is given in Table 5 (FAO/WHO, 1988b). 5.3 Occupational exposure during manufacture, formulation or use The levels of inhalation exposure to benomyl and carbendazim experienced by workers in a major manufacturing facility (DuPont) were reviewed from 1986 to 1989. The average levels of benomyl and carbendazim were less than 0.2 mg/m3 and 0.3 mg/m3, respectively. Table 6 lists established inhalation exposure limits for benomyl and carbendazim. 5.3.1 Use Potential dermal and respiratory exposure to benomyl wettable powder formulation under actual use situations has been determined for: a) tank loading and mixing for aerial application; b) re-entry into treated fields; and c) home use (garden, ornamental and greenhouse). For crop treatments, approximately 17 kg benomyl (formulation) was handled per cycle. Maximum exposure occurred in the loading and mixing operation for aerial application, where dermal exposure was 26 mg benomyl per mixing cycle, primarily to hands and forearms (90%) and respiratory exposure averaged 0.08 mg benomyl. Re-entry data revealed dermal and respiratory exposures of 5.9 mg/h and < 0.002 mg/h, respectively. Home-use situations (application of 7 to 8 litres of benomyl in hand-held compressed air sprayers) produced exposures of 1 mg and 0.003 mg per application cycle for dermal and respiratory routes, respectively (Everhart & Holt, 1982). Similar average dermal exposure levels (5.39 mg/h) for strawberry harvesters were reported by Zweig et al (1983). Table 3. Benomyl/carbendazim/thiophanate-methyl residues in food in Swedena Samples Swedish/imported No. of samples Samples with residues Residue level Median value >0.20 mg/kg (mg/kg) (mg/kg) 1986 Pineapples imported 3 1 0.69 Grapes imported 20 3 0.17-0.35 0.26 Strawberries imported 7 1 0.29 Mangoes imported 17 4 0.20-1.82 0.70 Papayas imported 5 2 0.25-0.45 Pears Swedish 17 3 0.32-0.62 0.43 imported 45 7 0.20-0.45 0.34 Apples Swedish 78 17 0.20-0.72 0.40 imported 91 30 0.21-0.74 0.39 1987 Grapes imported 28 3 0.52-0.87 0.60 Strawberries imported 7 0.23 Mangoes imported 14 5 0.29-1.30 0.66 Papayas imported 4 2 0.86-1.14 Pears Swedish 14 1 0.52 imported 62 13 0.21-0.45 0.29 Apples Swedish 61 25 0.20-1.17 0.45 imported 94 12 0.21-0.82 0.36 a From: FAO/WHO (1988b) Table 4. National Maximum Residue Limits (mg/kg) for certain commoditiesa banana cereal cherries citrus bean cucumber peach pome fruit strawberries grapes Australia 1 0.05 5 10 3 3 5 5 6 2 Austria 0.2 0.5 7 1 0.5 2 1.5 3 Belgium 2 0.5 2 2 0.5 2 5 5 2 Brazil 1 0.5 10 10 2 0.5 10 5 5 10 Bulgaria 0.5 10 5 5 10 Canada 5 10 1 0.5 10 5 5 5 Denmark 2 0.1 2 5 2 2 2 2 5 5 France 1 1.5 6 Finland 0.2 1 2 0.5 0.5 1 1 1 Germany 0.2 0.5 2 7 1 0.5 2 2 3 Hungary 2 1 Israel 10 10 10 5 10 Italy 0.5 0.5 1 1 Mexico 10 2 1 15 7 5 10 Netherlands 3 0.1 3 4 3 3 3 3 3 3 New Zealand 5 1 5 5 2 2 5 5 5 5 Spain (guidelines) 1 0.5 5 7 2 2 5 5 1 5 Switzerland 1 0.2 3 7 0.2 0.1 3 3 3 3 United Kingdom 1 0.5 10 0.5 10 5 5 10 (proposed) USA 1 0.2 15 10 2 1 15 7 5 10 USSR 1 0.5 10 10 2 0.5 10 5 5 5 Yugoslavia 0.1 7 0.5 0.1 2 0.5 2 a From: FAO/WHO (1988a) Table 5. Proposed Maximum Residue Limits for carbendazim from any sourcea Commodity MRL (mg/kg) Applicationb Apricot 10c B,C Asparagus 0.1d B,T Avocado 0.5 B Banana 1c B,C,T Barley straw and fodder, dry 2 B Bean fodder 50 C Beans, dry 2 B Berries and other small fruit 5 B,C,T Brussel sprouts 0.5 B Broad bean 2 T Carrot 5c C,T Cattle meat 0.1d B Celery 2 B,C Cereal grains 0.5 B,C,T Cherries 10c B,C,T Citrus fruits 10c B,C,T Coffee beans 0.1d C Common beane 2 C Cucumber 0.5 B,C,T Eggs (poultry) 0.1d B,T Egg plant 0.5 C Gherkin 2 C,T Hops, dry 50 C Lettuce, head 5 B,C,T Mango 2 B Melons, except watermelons 2c B,C Milk 0.1d B Mushrooms 1 B,C,T Nectarine 2 B Onion, bulb 2 C,T Peach 10c B,C,T Peanut 0.1d B,C Peanut fodder 5 B,C Peppers 5 C Pineapple 20c B Plums (including prunes) 2c B,C,T Pome fruit 5c B,C,T Potato 3c,f B,C Poultry meat 0.1d B,T Rape seed 0.05d C Rice straw and fodder, dry 15 B,C,T Sheep meat 0.1d B Soya bean, dry 0.2 C Soya bean fodder 0.1d C Squash, summer 0.5 B Table 5 (contd). Commodity MRL (mg/kg) Applicationb Sugar beat 0.1d B,C,T Sugar beat leaves on tops 10 B,C,T Swedeg 0.1d C Sweet potato 1 B Taro 0.1d B Tomato 5 B,C,T Tree nuts 0.1d B Wheat straw and fodder, dry 5 B Winter squash 0.5 B a From: FAO/WHO (1988b) b B = benomyl; C = carbendazim; T = thiophanate-methyl c MRL based on post-harvest use d At or about the limit of detection e JMPR recommended 2 mg/kg for dry, dwarf, lima and snap beans. These are all covered by "VP 0526, Common bean" and "VP 0071, Beans, dry" in the new classification f washed before analysis g Described as rutabagas in 1983 recommendation Table 6. Established inhalation exposure limitsa Country and agency Compound TWAb STELc (mg/m3) (mg/m3) Australia benomyl 10 - Belgium benomyl 10 - Denmark benomyl 5 - Finland benomyl 10 30 France benomyl 10 - Switzerland benomyl 10 - United Kingdom benomyl 10 15 USA: ACGIHd benomyl 10 - USA: NIOSHe/OSHAf benomyl 10 - (inhalable dust) USA: NIOSH/OSHA benomyl 5 - (respirable dust) USSR carbendazim - 0.1 a From: ILO (1991) b Time-weighted average c Short-term exposure limit d American Conference of Governmental Industrial Hygienists e National Institute of Occupational Safety and Health f Occupational Safety and Health Administration Air concentrations of benomyl ranged from 0.0074 to 0.053 mg/m3 (average 0.027 mg/m3) during its application in greenhouses. Spraying tall plants (over 1.5 m) caused three times higher concentrations in air than spraying low plants. No detectable amounts of benomyl or its metabolites (carbendazim, 4-HBC and 5-HBC) were found in the urine of applicators during the 48 h following the application. However, information describing protective clothing, ventilation, and other hygienic factors was not reported (Liesivuori & Jääskeläinen, 1984). 6. KINETICS AND METABOLISM Benomyl is extensively metabolized by animals, as described in detail in section 6.3. Metabolite names and structures are given in Table 6 and Figures 2 and 3. 6.1 Absorption Absorption in ChR-CD male rats was monitored after dermal application of 0.1, 1, 10, and 100 mg benomyl (as 2-14C-Benlate 50 WP) at 0.5, 1, 2, 4 and 10 h intervals. Four rats were used for each treatment and time interval. Benomyl was slowly absorbed across an area of skin (16% of the animal), appearing in the blood and urine within 30 min after treatment and reaching a maximum between 2 and 4 h after dosing (Belasco, 1979b). The concentration of benomyl and its metabolites in the blood peaked at 0.05 mg/litre (2 h sample) in the low-dose group (0.1 mg) and at 0.10 mg/litre (4 h sample) in the high-dose group (100 mg). This represented a 20-fold increase in blood concentration after a 1000-fold dose increase. Thus, absorption into the bloodstream was non-linear with respect to dose. An in vitro study on the penetration of formulated benomyl (Benlate 50 WP) through human skin showed that benomyl penetrates human skin poorly when it is applied as a recommended spray strength solution. Much less penetration was detected when dry concentrated benomyl was applied (Ward & Scott, 1992). In a rat gavage study, the absorption of carbendazim given in the form of a corn oil suspension was estimated to be approximately 80% (Monson, 1990). 6.2 Distribution and accumulation Blood levels of benomyl and its metabolites in male rats were measured 6 and 18 h after exposure in male rats. The rats were exposed to time-weighted averages of 0.32 and 3.3 mg/litre of air for 0.5, 1, 2 and 6 h. The methodology did not distinguish between benomyl and carbendazim. At both exposure levels, the blood concentrations of benomyl/carbendazim were greater than that of 5-HBC 6 h after exposure; the levels were 0.39-2.3 mg/litre and 0.25-1.2 mg/litre, respectively. At 18 h after exposure, only 5-HBC was detected in the blood (1.1 mg/litre) and this only at the highest dose. Urinary metabolites consisted primarily of 5-HBC, and limited amounts of benomyl/carbendazim were also detected (Turney, 1979). Table 7. Chemical names of benomyl and its metabolites in animalsa Common or abbreviated Chemical name name Benomyl Carbamic acid, [1-(butylamino)carbonyl]- 1H-benzimidazol-2-yl]-, methyl ester Carbendazim (MBC) methyl (1-H-benzimidazol-2-yl)carbamate 5-HBC methyl (5-hydroxy-1H-benzimidazol-2-yl)- carbamate 4-HBC methyl (4-hydroxy-1H-benzimidazol-2-yl)- carbamate 5-HBC-Sb 2-[(methoxycarbonyl)amino]-1H-benzimidazol-5- yl hydrogen sulfate 5-HBC-Gc [2-[(methoxycarbonyl)amino]-1H-benzimidazol- 5-yl] ß-D-glucopyranosiduronic acid MBC-4,5-epoxide MBC-5,6-epoxide MBC-4,5-dihydrodiol (4,5-dihydro-4,5-dihydroxy-1H-benzimidazol- 2-yl) carbamate MBC-5,6-dihydrodiol (5,6-dihydro-5,6-dihydroxy-1H-benzimidazol- 2-yl) carbamate MBC-4,5-diol MBC-5,6-diol 5-OH-6-GS-MBCd S-[5,6-dihydro-5-hydroxy-2-(methoxycarbonyl amino)-1H-benzimidazol-4-yl]glutathione 5-OH-4-GS-MBC S-[4,5-dihydro-5-hydroxy-2-(methoxycarbonyl amino)-1H-benzimidazol-4-yl]glutathione 5,6-HOBC-N-oxide methyl (6-hydroxy-5-oxo-5H-benzimidazol-2- yl)-carbamate-N-oxide Table 7 (contd). Common or abbreviated Chemical name name 5,6-HOBC-N-oxide-G [2-[(methoxycarbonyl)amino]-6-oxo-6H- benzimidazol-5-yl] ß-D-glucopyranosiduronic acid-N-oxide 5,6-DHBC methyl (5,6-dihydroxy-1H-benzimidazol-2-yl) carbamate 5,6-DHBC-G [6-hydroxy-2-[(methoxycarbonyl)amino]-1H- benzimidazol-5-yl] ß-D-glucopyranosiduronic acid 5,6-DHBC-S 6-hydroxy-2-[(methoxycarbonyl)amino]-1H- benzimidazol-5-yl 5-(hydrogen sulfate) 2-AB 2-aminobenzimidazole 2-AB dihydrodiol 2-amino-4,5-dihydro-4,5-dihydroxy-1H- benzimidazol 5-HAB 5-hydroxy-2-aminobenzimidazole a From: Krechniak & Klosowska (1986); Monson (1986a,b); Monson (1990) b S = conjugate with sulfuric acid c G = conjugate with glucuronic acid d GS = conjugate with glutathione In a study by Han (1979), ten male ChR-CD rats were given 1 and 10 µg benomyl intravenously (as 14C-Benlate 50% WP). Radioactivity was found in the urine as 5-HBC at 6, 12 and 24 h after dosing, and there was little radioactivity in the blood or faeces at these sampling times. No radioactivity (< 0.1%) was found in any tissue after 24 h except in blood, which contained trace quantities of 14C residues. In a further study, three groups of five rats of each sex were gavaged with [phenyl(U)-14C] carbendazim. One group received a single dose of 14C-carbendazim (50 mg/kg). The second group received a single dose of 14C-carbendazim (50 mg/kg) following 14 days of pre-conditioning with non-radiolabelled carbendazim (50 mg/kg per day). The third group received a single dose of 14C-carbendazim (1000 mg/kg). For all groups, > 98% of the recovered radioactivity was excreted in the urine or faeces by the time of sacrifice (72 h after 14C dosing). The 14C remaining in tissues was < 1% of the applied dose (Monson, 1990). In a study by Belasco et al. (1969), 14C-benomyl was administered to male ChR-CD rats and the blood and testes were analysed. The fungicide was given by gavage as: (a) a single dose of 1000 mg/kg to five rats, which were sacrificed either 1, 2, 4, 7 or 24 h later; (b) 10 repeated doses of 200 mg/kg per day to two rats, which were sacrificed either 1 or 24 h after the last dose. In addition, blood and testes from rats fed 2500 mg/kg diet for one year were analysed. In rats given 1000 mg/kg, results show that: (a) the total 14C radioactivity (calculated as benomyl) ranged from 3 to 13 ppm in the blood and from 2 to 4 ppm in the testes; (b) 5-HBC appeared in the blood and testes as early as 1 h after dosing; and (c) the concentration of benomyl and/or carbendazim decreased with time and there was a corresponding increase in the concentration of 5-HBC in the blood and testes. Analyses of blood and testes from rats given 10 repeated oral doses of 200 mg/kg per day showed that one hour after the last dose no benomyl/carbendazim (< 0.1 ppm) was detected and only low levels of 5-HBC were found (1.5 ppm blood and 0.3 ppm in testes). No benomyl/carbendazim or 5-HBC (< 0.1 ppm) was found 24 h after the last dose. With rats fed 2500 mg benomyl/kg diet for one year, no benomyl/carbendazim (< 0.1 ppm) was detected in blood or testes. Only a minimal amount of 5-HBC was found in blood (0.2 ppm) and none was found in the testes (< 0.1 ppm) (Belasco et al., 1969). In a series of metabolic studies, benomyl and/or Benlate (50% benomyl formulation) were administered either by gavage or in the diet to pregnant ChR-CD rats to determine the concentrations of benomyl, carbendazim and two carbendazim metabolites (4-and 5-HBC) in maternal blood and embryonic tissue (Culik, 1981a,b). Dosing took place on days 7 to 16 of gestation at levels of 125 mg/kg body weight per day via gavage or 5000-10 000 mg/kg diet (approximately 400-800 mg/kg body weight). Blood samples from the dams and tissue samples from their embryos were examined on the first, sixth and tenth days of dietary administration and on days 12 and 16 of gavage administration. Embryos and maternal blood were analysed 1, 2, 4, 8 and 24 h after gavage. The levels of benomyl/carbendazim in maternal blood and embryonic tissues, 24 h following each dose, markedly decreased with the number of treatments. The level of benomyl (one hour after treatment) ranged from 0.98 to 8.4 mg/kg with a mean value of 5.0 mg/kg on the first day of treatment. After 10 treatments, the levels of benomyl/carbendazim ranged from < 0.12 to 0.39 mg/kg (one hour after last treatment). In the embryo there was 0.13 mg/kg benomyl/carbendazim after the tenth treatment compared with a mean of 1.9 mg/kg after the first treatment. The half-life of benomyl in maternal blood was approximately 45 min and was less in the embryos. The level of 5-HBC (0.84-2.9 mg/kg) 2 h following the last gavage increased with the number of exposures, the half-life in the blood being 2-3 h in the dam and 4-8 h in the embryo. 4-HBC was not detected. In the dietary studies, the levels of benomyl, carbendazim and 4-HBC were too low to be measured in the embryonic tissue. 4-HBC could not be detected in the dams. Irrespective of the dose level (5000 and 10 000 mg/kg diet active ingredient) of benomyl or Benlate, the level of benomyl/carbendazim in maternal blood was very low. In three separate groups of animals, the mean highest blood concentrations of benomyl/carbendazim were 0.35, 0.61 and 0.23 mg/kg in each group of dams. The mean highest value of 5-HBC (5000 mg benomyl/kg diet) was 0.44 mg/kg in the blood and 0.33 mg/kg in the embryos. Animals fed benomyl or Benlate at a level of 10 000 mg/kg diet had 5-HBC levels an order of magnitude higher (Culik, 1981a,b). A lactating Holstein cow was dosed by capsule twice daily (515 mg [2-14C]-benomyl each dose), equivalent to 50 mg/kg in the average total daily feed, for 5 consecutive days, and samples of urine, faeces and milk were collected at each dosing. Approxi mately 17 h after the tenth dose, the cow was sacrificed and organ, tissue and blood samples were subsequently collected. 14C residue levels in the milk averaged 0.2 mg/kg (calculated as benomyl), 49% of the radioactive metabolites being extractable in ethyl acetate, 36% soluble in water, and 8% isolated as solids. Small amounts of radioactivity were detected in the liver (4.12 mg/kg) and kidney (0.25 mg/kg), most of which was bound. No significant levels of radioactivity (0.06 mg/kg) were detected in other tissues or fat (Monson, 1985). Lactating and non-lactating goats were given daily capsule doses of [2-14C]-benomyl, equivalent to 36 and 88 mg/kg, respectively, in the total daily diet, for five days. Milk residues accounted for approximately 2% of the total dose. Approximately 25% of the milk radioactivity was incorporated into the natural milk components casein and whey protein. There were no detectable residues in muscle tissue and fat (< 0.01 mg/kg). However, radioactivity detected in liver and kidney amounted to 3.8 and 0.09 mg/kg (calculated as benomyl equivalents), respectively (Han, 1980). In a study by Johnson (1988), the total 14C residue and metabolic fate of carbendazim in the liver was examined in non-lactating female goats. Twelve goats were administered a feed-rate-equivalent dose of [phenyl(U)-14C]-carbendazim (> 50 mg/kg), once a day, for up to 30 days. Within 2 weeks of dose initiation, a plateau of 14C residues in the liver was achieved at a level of 9.48 mg/kg (group mean of the total radiolabelled liver residues for goats sacrificed 2, 3, and 4 weeks after initiation of dosing). The total 14C residue levels in the liver decreased to 5.17, 3.55 and 1.67 mg/kg (calculated as carbendazim equivalents) 1, 2 and 3 weeks, respectively, after dosing ceased. The elimination half-life for total 14C residues from the liver, based on this depuration data, was calculated to be approximately 9 days. The half-life for removal of carbendazim from the general circulation, based on 14C-carbendazim equivalent whole blood levels, was approximately 10 h. The level of bound, non-extractable 14C residues in the liver of goats sacrificed after 28 days was 1.0 mg/kg. The results of this study suggest that levels of carbendazim- derived residues do not accumulate beyond 2 weeks when goats are exposed to a constant feed level of 50 mg carbendazim/kg. Furthermore, discontinuation of exposure results in a clearing of residues from the liver (Johnson, 1988). The metabolism of benomyl was studied in laying hens by Monson (1986a). Two hens were individually dosed daily for three consecutive days with 3.5 mg [2-14C]-benomyl at a rate equivalent to 29 mg/kg in the daily feed, and two hens were individually dosed with 3.29 mg [phenyl(U)-14C]-benomyl at rates equivalent to 27 mg/kg in the daily feed. Faeces and eggs from the previous 24 h were collected just before each dosing. Twenty-two hours after the third dose, the hens were killed and samples of muscle (breast and thigh), liver, kidney and fat were analysed. The concentration of radioactivity (calculated as benomyl equivalents) in the tissues and day-3 eggs of the [2-14C]-benomyl- and [phenyl(U)-14C]-benomyl- dosed hens, respectively, was as follows: liver (0.54 and 0.41 mg/kg), kidney (0.28 and 0.16 mg/kg), thigh and breast muscle (both 0.01 mg/kg), fat (0.05 and 0.02 mg/kg) and eggs (0.08 and 0.05 mg/kg). The distribution of benomyl in this study was comparable to that in a 20-hen [2-14C]-carbendazim metabolism study. The concentrations of radioactivity, calculated as mg carbendazim/kg, in the high-dose laying hens (dose equivalent 120 mg/kg carbendazim in the diet) were liver (2.63), kidney (1.74), thigh muscle (0.06), breast muscle (0.05), fat (0.03), day-6 eggs (0.63) (Monson, 1986b). This study is discussed in detail in the Environmental Health Criteria monograph on Carbendazim (WHO, 1993). When bluegill sunfish were exposed to benomyl, carbendazim and 2-AB at nominal concentrations of 0.05 mg/litre (measured concentrations of 0.01 to 0.04 mg/litre) and 5.0 mg/litre (measured concentrations of 2 to 5 mg/litre), no residues were found in the tissues of fish exposed to low levels of these three compounds. Detectable residues were found in the tissues of fish exposed to the high levels, but there was no build-up or bioconcentration with time (DuPont, 1972). 6.3 Metabolic transformation Benomyl is extensively metabolized by rats to carbendazim, which is then further metabolized. Studies with rats administered benomyl intravenously (Han, 1979), dermally (Belasco, 1979b) or by inhalation (FAO/WHO, 1985a) showed that 5-HBC is the main urinary metabolite, some carbendazim also being present. In a rat gavage study (Monson, 1990; see section 6.2), carbendazim was extensively metabolized. Three dosing regimens (five rats of each sex per group) were used: a single oral dose of 50 mg/kg (low dose); a single oral dose of 50 mg/kg following pre-conditioning gavage with non-radiolabelled carbendazim at 50 mg/kg for 14 days (pre-conditioned low dose); and a single oral dose of 1000 mg/kg (high dose). The 48-h urine from the low-dose and the high-dose rats and the 14-day urine from the pre-conditioned low-dose group were collected. The total recovery from urine was 61.5 and 61.7% of given doses for the low-dose and pre-conditioned low-dose male groups, 53.2 and 59.3% for the low-dose and pre-conditioned low-dose female groups, and 39 and 41% for both male and female high-dose groups, respectively. 5-HBC-S (21-43% of given dose) was identified as the main metabolite, except in the case of the pre-conditioned low-dose and high-dose female rat groups (5.5-10%), while in all female rat groups 5,6-HOBC-N-oxide-G (10-19%) was predominant. Both 5,6-DHBC-S and 5,6-DHBC-G were identified as minor metabolites. In the same study, the faeces were collected at the same periods as the urine. The total recovery from faeces was about 24% for the low-dose and pre-conditioned low-dose male groups, 33-38% for the low-dose and pre-conditioned low-dose female groups, and higher (> 60%) for both male and female high-dose groups. Unchanged carbendazim was about 10-15% of the given dose in the faeces of high-dose rats (Monson, 1990). The proposed metabolic pathway for benomyl in rats is given in Fig. 2. When a lactating Holstein cow was dosed by capsule twice daily (515 mg per dose), equivalent to 50 mg/kg diet, for 5 consecutive days with [2-14C]-benomyl, the major metabolites of whole milk were 5-HBC (0.06 mg/litre), 4-HBC (0.03 mg/litre) and MBC-4,5- dihydrodiol (< 0.07 mg/litre). The proportions of radioactive residues in the urine were 46% 5-HBC, 3% 4-HBC, and 50% polar aqueous-soluble metabolites, which included MBC-4,5-dihydrodiol, 2-AB-dihydrodiol and 5-OH-4-GS-MBC (Monson, 1985). Lactating and non-lactating goats were given five consecutive daily doses of 2-14C-benomyl by capsule at rates equivalent to 36 and 88 mg/kg, respectively, in the total daily diet. The main metabolite in milk was 5-HBC, and there were minor amounts of 4-HBC and 5-HAB. The principal metabolites in urine and faeces were 5-HBC and 4-HBC. The main identified metabolite in the kidney and liver was 5-HBC (about 6% of the residue). Much of the liver residue was incorporated into glycogen, protein, fatty acids and cholesterol, and accounted for approximately 35% of the liver residues. Further characterization of the bound liver tissue residues following enzymatic and trifluoroacetic anhydride hydrolysis identified 5-hydroxy-benzimidazole moieties as the principal (at least 77%) 14C residue in goat liver. No free benomyl, carbendazim or 5-HBC was detected in the liver (Han, 1980; Hardesty, 1982). In a further study, the total 14C residue and metabolic fate of carbendazim in the liver were examined in non-lactating female goats. Twelve goats were administered a dose equivalent to 50 mg/kg feed once a day for up to 30 days. Extraction of composite liver homogenate from goats sacrificed 4 weeks after initiation of dosing ("plateau level") indicated that the major ethyl acetate extractable and identifiable radiolabelled residues in the liver were 5-HBC (2 to 3 mg/kg) and carbendazim (approximately 0.2 mg/kg) (Johnson, 1988). The metabolism of [2-14C]-benomyl and [phenyl(U)-14C]- benomyl has been studied in laying hens (see section 6.2 for a detailed description of the study). Benomyl was extensively metabolized to carbendazim, 5-HBC, MBC-4,5-dihydrodiol and a metabolite tentatively identified as 5-OH-4-GS-MBC. The metabolic profile observed in hens indicates that the benzimidazole ring is not broken during metabolism (Monson, 1986a). The proposed metabolic pathway for benomyl in the laying hen is given in Fig. 3. Monson (1991) analysed the release and characterization of bound benomyl and carbendazim metabolites in diary cow, goat, hen and rat liver after treatment with 14C-benomyl or 14C-carbendazim via Raney nickel desulfurization and acid dehydration. Using this technique, he was able to show that bound 14C residue was released from the liver of cows (76% bound before desulfurization and 36% bound after desulfurization) and hens (58% bound before desulfurization and 19% bound after desulfurization). The major part of the reduced residue was identified as 5-HBC, 5,6-HOBC or carbendazim, suggesting that the bound liver residue consisted of conjugates of benzimidazole-related products and not natural products resulting from breakdown and incorporation. In fish, benomyl and carbendazim are metabolized to 5-HBC (Dupont, 1972). 6.4 Elimination and excretion Absorbed benomyl and carbendazim are rapidly excreted in the urine and faeces. In a study where rats were administered 1 or 10 µg formulated 14C-benomyl (50% wettable powder) in a single intravenous dose by tail injection, more than 80% of the dose was excreted in the urine and faeces within 6 h after injection and the total urine and faeces recovery was > 95% in 24 h (Han, 1979). [Phenyl(U)-14C]-carbendazim was administered by gavage to Sprague-Dawley rats using three dosing regimens: a single oral dose of 50 mg/kg (low dose); a single oral dose of 50 mg/kg following pre-conditioning gavage with unlabelled carbendazim of 50 mg/kg for 14 days (pre-conditioned low dose); and a single oral dose of 1000 mg/kg (high dose). Each dosing group consisted of five animals of each sex. A preliminary study conducted with two rats of each sex, each rat having received a single oral dose of 50 mg/kg, demonstrated that 95% of the radioactivity excreted in the urine and faeces was recovered within 72 h after dosing and that < 1% of the dose was expired as volatile metabolites. In the full study, > 98% of the recovered radioactivity was excreted by the time of sacrifice (i.e. 72 h after dosing) for each dosing group. Urinary excretion accounted for 62% to 66% of the dose in males and 54% to 62% of the dose in low-dose and pre-conditioned low-dose female groups. In the high-dose group, this pathway accounted for 41% of the dose in all animals. Elimination of radiolabel in faeces accounted for virtually all of the remaining radiolabel. There were no apparent differences between male and female rats with respect to the extent of absorption and extent and rate of elimination of 14C-carbendazim equivalents within a given treatment group (Monson, 1990). In a study by Han (1978), two male ChR-CD-1 mice were fed a diet of non-radiolabelled benomyl (2500 mg/kg) for 21 days and were then gavaged with 2.5 mg [2-14C]-benomyl in corn oil. An identical experiment was performed with one male ChR-CD hamster. More than 90% of the radioactivity was eliminated in the urine and faeces within 72 h (Han, 1978). A lactating Holstein cow was dosed by capsule twice daily (515 mg [2-14C]-benomyl each dose), equivalent to 50 mg/kg in the average total daily feed, for 5 consecutive days, and samples of urine, faeces and milk were collected at each dosing. Approximately 17 h after the tenth dose, the cow was sacrificed and organs, tissues and blood were collected for analysis. At sacrifice, 65% of the radiolabel had been excreted in the urine, 21% in the faeces and 0.4% in the milk. Carbon-14 residue levels in the milk averaged 0.2 mg/litre (calculated as benomyl equivalents) with 49% of the radioactive metabolites being extractable in ethyl acetate, 36% soluble in water, and 8% isolated as solids (Monson, 1985). Lactating and non-lactating goats were given 5 consecutive daily doses of [2-14C]-benomyl by capsule at rates equivalent to 36 and 88 mg/kg, respectively, in the total daily diet. Most of the radioactivity (96%) had been eliminated in the urine and faeces by the time of sacrifice (Han, 1980). The excretion of benomyl was studied in laying hens dosed daily for three consecutive days with 3.5 mg [2-14C]-benomyl or 3.29 mg [phenyl(U)-14C]-benomyl. At sacrifice (22 h after the last dose), an average of 107% and 95% of the dose had been excreted for the [2-14C]-benomyl- and [phenyl(U)-14C]-benomyl-dosed birds, respectively (Monson, 1986a). In a similar study on 14C-carbendazim, groups of laying hens were fed at a rate equivalent to 5 and 120 mg/kg diet. At sacrifice, 24 h after the sixth daily dose, an average of 95% of the dose had been excreted by the low-dose birds and 92% by the high-dose birds (Monson, 1986b). 6.5 Reaction with body components An in vitro study using acetyl cholinesterase from bovine erythrocytes showed that benomyl did not inhibit this enzyme. The acetyl cholinesterase inhibition constant (KI) for benomyl was greater than 1 x 10-3 mol/litre (Belasco, 1970). Another in vitro study by Krupka (1974) verified that benomyl did not inhibit either acetyl cholinesterase or butyryl cholinesterase. In a study by Guengerich (1981), the effects of benomyl and carbendazim on hepatic enzymes were studied in male and female Crl-CD rats and CD-1 mice. The treatment groups included animals fed for 28 days with diets that contained benomyl or carbendazim at concentrations of 0, 10, 30, 100, 300, 1000 or 3000 mg/kg. In these studies, microsomal epoxide hydrolase and cytosolic glutathione- S- transferase were monitored in subcellular fractions isolated from the livers of animals in each treatment group. Liver weights were also recorded. Elevated mean absolute liver weights were observed at 1000 and 3000 mg carbendazim/kg in both male and female rats and at 300 mg carbendazim/kg in female rats. However, the only significantly elevated liver weight was found in females after a dose of 3000 mg benomyl/kg. No apparent liver toxicity or effect on body weight was observed. Both benomyl and carbendazim induced epoxide hydrolase in both sexes of rats and mice at 1000 and 3000 mg/kg. Induction of glutathione- S-transferase was observed at 3000 mg/kg in the case of both benomyl and carbendazim. In general, the level of induction seemed to be slightly greater in females than males. There did not appear to be any substantial difference in enzyme induction between rats and mice. In a study by Shukla et al. (1989), levels of gamma-glutamyl transpeptidase (GGT) were evaluated after benomyl exposure. Female albino rats (eight per group) and female Swiss albino mice (eight per group) were given 1000 and 4000 mg benomyl/kg feed for 15 days, and blood and liver GGT levels were analysed. Benomyl exposure increased the activity of both blood and liver GGT in both rats and mice, and the degree of induction was dose related (Shukla et al., 1989). 7. EFFECTS ON LABORATORY MAMMALS; IN VITRO TEST SYSTEMS 7.1 Single exposure The acute toxicity of benomyl in several animal species is summarized in Table 8. Benomyl has an oral LD50 in the rat of > 10 000 mg/kg and an inhalation 4-h LC50 > 4 mg/litre. Several other minor metabolites were evaluated and the approximate lethal doses were 3400 mg/kg for 2-AB, 7500 mg/kg for 5-HBC, 17 000 mg/kg for BUB and 17 000 mg/kg for STB. In a study by Littlefield & Busey (1969), three groups of male dogs (around 10 dogs/group) were exposed to benomyl at air concentrations of 0, 0.65 and 1.65 mg/litre. One half of the dogs in each group were killed on day 14 and the remainder on day 28. The liver weight of the high-dose dogs was significantly decreased on day 28. For further discussion of single dose toxic effects, see section 7.5.1. 7.2 Short-term exposure 7.2.1 Gavage In a 14-day rat study, benomyl (200 and 3400 mg/kg in peanut oil) was given by gavage five times a week for two weeks to six male ChR-CD rats per group. Four out of six rats died after 5, 7, 8 and 9 doses, respectively, of 3400 mg/kg. No clinical signs of toxicity were observed in the group treated with 200 mg/kg per day. Degeneration of germinal epithelium, multinucleated giant cells and reduction or absence of sperm were observed in the testes after multiple doses of 3400 mg/kg per day. Less than 10% of the testicular tubules were affected in only two out of six animals dosed with 200 mg/kg. At the high dose level, there was erosion and thickening of the squamous mucosa of the stomach with submucosal inflammation and a decrease in the large globular-shaped vacuoles located centrolobularly in the liver (Sherman & Krauss, 1966). 7.2.2 Feeding 188.8.131.52 Rat In a 90-day study by Sherman et al. (1967), groups of rats (4-week-old ChR-CD rats, 16 rats of each sex per group) were fed Benlate 70 WP (72% benomyl) in the diet at levels of 0, 100, 500 and 2500 mg benomyl/kg. The animals were observed daily for behavioural changes and body weights, and food consumption was recorded at weekly intervals. Haematological examinations were conducted on six male and six female rats in each group at 30, 60 and 90 days. Routine urine and plasma alkaline phosphatase and glutamic pyruvic transaminase activity analyses were performed on the same animals. After 96-103 days of continuous feeding, 10 male and 10 female rats in each group were killed, and selected organs were weighed and examined microscopically. The remaining six male and female animals in each group after the terminal sacrifice were used in a one-generation reproductive study. No effect was observed with respect to reproduction or lactation in the delivery or rearing of the F1A litters. There were no compound-related effects on weight gain, food consumption, food efficiency, clinical signs, or on haematology, biochemistry or urinalysis determinations. The liver-to-body weight ratio in females was slightly increased at 2500 mg/kg, compared with control rats. Gross and microscopic examinations of tissues and organs showed no significant effects attributable to the presence of benomyl in the diet at levels up to and including 2500 mg/kg. 184.108.40.206 Dog Groups of beagle dogs (four males and four females per group; 7-9 months old) were administered benomyl 50% wettable powder in the diet at dosage levels of 0, 100, 500 and 2500 mg/kg diet (based on active ingredient) for three months (this corresponded to treatment levels of 0, 3.8, 18.4 and 84 mg/kg body weight). Food consumption and body weight data were recorded weekly, and clinical laboratory examinations (including haematology, biochemistry and urinalysis) were performed pre-test and after 1, 2 and 3 months of feeding. At the conclusion of the study, selected organs were weighed and subjected to gross and microscopic examinations. No mortality or adverse clinical effects were observed over the course of the study, and growth and food consumption were not effected by the treatment. Urine parameters showed no differences from the control, and there were no dose-related effects on the haematological values. Alkaline phosphatase and glutamic pyruvic transaminase activities were increased in high-dose males and females. There were statistically significant decreases in the albumin/globulin ratio in either males or females fed 2500 mg/kg diet. Organ-to-body weight ratio changes were observed in the high-dose males and females for the thymus (decreased) and thyroid (increased). One of the four females fed 2500 mg/kg diet had an enlarged spleen at the end of the exposure period, as well as a decreased erythrocyte count, haemoglobin concentration and haematocrit value. Histopathological examination revealed myeloid hyperplasia of the spleen and bone marrow and erythroid hyperplasia of bone marrow. This did not appear to be compound related since group mean values were not significantly different. Three out of four males fed 2500 mg/kg diet had reduced relative prostate weights when compared with controls. Microscopic examinations of tissues and organs did not indicate changes in dogs fed benomyl for 90 days. The no-observed-effect level (NOEL) was 500 mg/kg diet (Sherman, 1968). Table 8. Acute toxicity of benomyl and its metabolites for laboratory mammals Chemical Species Sex Number of Route Vehicle Concentrationa Reference animals (mg/kg body weight) Benomyl ratb M/F 10/dose oral peanut oil LD50 > 10 000 Sherman (1969a) rabbitc M 1/dose oral 50% wettable powder ALD > 3400 Fritz (1969) dogd M 1 oral evaporated milk and ALD > 1000 Sherman (1969b) water (1:1) Benlate OD (50% rat M 10/dose oral corn oil LD50 > 12 000 Hostetler (1977) benomyl) Fungicide 1991 rabbit M/F 4/dose dermal (4 h) 50% wettable powder LD50 > 10 000 Busey (1968a) (50% benomyl) rat M 6/dose inhalation (4 h) 50% wettable powder LC50 > 4.01 mg/litre Busey (1968b) (analytical) dog M 10/dose inhalation 50% wettable powder LC50 > 1.65 mg/litre Littlefield & Busey (analytical) (1969) Benlate fungicide rat M/F 10/dose oral aqueous suspension LD50 > 10 000 Sherman (1969a) (52-53% benomyl) rat M 5/dose inhalation 50% wettable powder LC50 > 0.82 mg/litre Hornberger (1969) Benlate PNW (50% rabbit M/F 10/dose dermal 50% wettable powder LD50 > 2000 Gargus & Zoetis benomyl) (1983c) Benlate 50 DF (50% rat M/F 5/dose oral aqueous suspension LD50 > 5000 Sarver (1987) benomyl) rabbit M/F 5/dose dermal 50% dry flowable LD50 > 2000 Brock (1987) Table 8 (contd). Chemical Species Sex Number of Route Vehicle Concentrationa Reference animals (mg/kg body weight) Benomyl metabolites 2-Benzimidazole carbamic rat M/F 10/dose oral corn oil LD50 > 10 000 Goodman (1975) acid, methyl ester 5-Hydroxy-2-benzimidazole- rat M 1/dose oral corn oil ALD > 7500 Snee (1969) carbamic acid, methyl ester 2-Aminobenzimidazole rat M 1/dose oral peanut oil ALD > 3400 Fritz & Sherman (1969) Benzimidazole 2- rat M 1/dose oral corn oil ALD > 17 000 Dashiell (1972) (3-butylureido) S-Triazine, 3-butyl- rat M 1/dose oral corn oil ALD > 17 000 Barbo & Carroll benzimidazole (1,2a), (1972) -2,4(1H,3H)-dione a Based on active ingredient; ALD = approximate lethal dose b ChR-CD or Crl:CD rats c New Zealand white rabbits d Beagle dogs 7.2.3 Dermal In a study on groups of five male and five female New Zealand albino rabbits, weighing 2 to 2.4 kg, 15 dermal applications of a 50% benomyl formulation (equivalent to 1000 mg/kg) were made on both abraded and intact abdominal skin sites. The animals were exposed for 6 h/day, 5 days/week for 3 weeks. After each daily application, the abdomen was washed with tap water. Observations were made daily for mortality and toxic effects and weekly for body weight changes. Gross necropsy and microscopic examinations were performed. Slight erythema, oedema and atonia were observed at both abraded and intact skin sites. Slight to moderate desquamation occurred throughout the exposure period. No apparent compound-related body weight or organ weight changes were reported. Microscopic examination of the males demonstrated that benomyl produced degeneration of the spermatogenic elements of the seminiferous tubules of the testes, the changes including vacuolated and multi-nucleated spermatocytes (Busey, 1968d). In a separate repeated-dose dermal study, groups of five male and five female New Zealand albino rabbits, weighing 3 kg, were exposed to doses of benomyl equivalent to 0, 50, 250, 500, 1000 and 5000 mg/kg applied to non-occluded abraded dorsal skin sites 6 h a day, five days a week, for three weeks. Test material was removed by washing the skin site and drying with a towel. There were decreased body weight gains for both males and females at the two highest dose levels. Mild to moderate skin irritation was reported for all groups but was most notable at the highest dose level. Diarrhoea, oliguria and haematuria were observed in males and females at 1000 and 5000 mg/kg. Decreased average testicular weights and testes-to-body weight ratios were observed at 1000 mg/kg only. There were no histopathological changes reported (Hood, 1969). 7.2.4 Inhalation In an inhalation study, groups of 20 male and 20 female CD rats were exposed, nose-only, 6 h a day for 90 days, to 0, 10, 50 and 200 mg benomyl/m3. At 45 and 90 days, blood and urine samples were collected from 10 rats of each sex per group for clinical analysis and then killed for pathological examination. After approximately 45 days of exposure, test-compound-related degeneration of the olfactory epithelium was observed in all males and in eight of the ten females exposed to 200 mg/m3. Two male rats exposed to 50 mg/m3 had similar but less severe olfactory degeneration. After approximately 90 days of exposure, all of the animals showed olfactory degeneration at 200 mg/m3, along with three males exposed to 50 mg/m3. No other compound-related pathological effects were observed. Male rats exposed to 200 mg/m3 had depressed mean body weights compared to controls and this correlated with a reduction in food consumption (Warheit et al., 1989). 7.3 Skin and eye irritation; sensitization 7.3.1 Dermal A 50% wettable powder applied to the clipped intact and abraded abdomen of albino rabbits produced moderate to marked erythema, slight oedema and slight desquamation. Exposure was for 24 h to occluded skin sites at doses > 0.5 g/animal. Albino guinea-pigs similarly exposed to 10, 25 and 40% dilutions of technical grade benomyl in dimethyl phthalate presented only mild irritation of both intact and abraded skin sites (Majut, 1966; Busey, 1968a; Colburn, 1969; Frank, 1969). When "Benlate" 50 DF (50% benomyl, 0.5 g Benlate 50 DF) was evaluated for primary dermal irritation potential in six male New Zealand white rabbits, no dermal irritation was observed at 4 or 24 h after application. By 48 h, slight to mild erythema was observed in two rabbits and was still evident at 72 h. The primary irritation scores ranged from 0-1 (not an irritant) (Vick & Brock, 1987). In a study by Desi (1979), benomyl (98% purity) was applied to a shaved area of the back of four albino rabbits at 5 mg/cm2. Draize scores (Draize et al., 1944) were assessed 4 and 72 h after application. Lesions produced by this method were classified as "mild irritation". 7.3.2 Eye The eye irritation properties of benomyl were examined in albino rabbits in several tests using technical grade benomyl, 50% wettable powder and a suspension in mineral oil. Mild conjunctival irritation and minor transitory corneal opacity were reported after 48 to 96 h in all tests (Reinke, 1966; Frank, 1972). Similar results were obtained with Benlate PNW (a 50% wettable powder) (Gargus & Zoetis, 1983a,b). Another eye irritation experiment was performed with 5 mg pure benomyl using four albino rabbits (Desi, 1979). Results assessed according to Draize (Draize et al., 1944) indicated that benomyl is a mild eye irritant. 7.3.3 Sensitization Albino guinea-pigs exposed to benomyl, either technical material or a 50% sucrose formulation, produced mild to moderate skin erythema during the challenge phase following both intradermal injections or repeat applications to abraded skin (Majut, 1966; Colburn, 1969; Frank, 1969). In another sensitization study, Benlate PNW (50% benomyl prepared as a 0.1% solution in saline) was injected weekly (four injections) into ten albino guinea-pigs (Hartley strain). Ten control animals were injected with saline. Fourteen days after the final injection, 8% or 80% Benlate PNW saline solutions were applied to the backs of the induced animals and saline was applied to the backs of the control animals. No significant increase in score occurred in any of the control animals at either challenge concentration. Benlate PNW produced an unequivocal and significant (two-step) increase at two of ten sites challenged with an 8% suspension, and at seven of ten sites challenged with an 80% suspension (Gargus & Zoetis, 1984). Technical benomyl produced sensitization in all ten animals tested in a guinea-pig maximization test (Matsushita et al., 1977). 7.4 Long-term exposure 7.4.1 Rat Groups of weanling rats (36 male and 36 female Charles River albino rats/group) were fed benomyl (50-70% a.i.) in the diet for 104 weeks at levels of 0, 100, 500 and 2500 mg/kg. Growth, as observed by body weight changes and food consumption data, was recorded weekly for the first year and twice a month thereafter. Daily observations were made of clinical effects and mortality. At periodic intervals during the study, haematological, urinalysis and selected clinical chemistry examinations were performed. After one year each group was reduced to 30 males and 30 females by interim sacrifice for gross and microscopic evaluations. At the conclusion of the study, all surviving animals were sacrificed and gross examinations of tissues and organs were made. Initially, microscopic examinations of tissues and organs from the control and 2500 mg/kg groups were conducted, as were liver, kidney and testes examinations of animals in the 100 and 500 mg/kg dose groups. In follow-up pathological evaluations, all of the tissues and organs of the control and low-, intermediate- and high-dose groups were examined microscopically. There was no mortality attributable to benomyl in the diet. Survival decreased to approximately 50% during the second year, but was comparable among all groups. Body weight, food consumption and food efficiency were unaffected by treatment. The average daily dose for the 2500 mg/kg group was 330 mg/kg body weight per day initially, 91-106 mg/kg body weight per day at one year and 70-85 mg/kg body weight per day at two years. There were no compound-related clinical manifestations of toxicity. Haematological, urine and liver function tests were unaffected by treatment. There were no differences in organ weight or organ-to- body-weight ratios between control and treated groups (Sherman, 1969c; Lee, 1977). 7.4.2 Mouse In a study by Weichman et al. (1982), male and female CD-1 mice (80 males and 80 females per group) were administered benomyl (99% a.i.) in the diet at levels of 0, 500, 1500 and 5000 mg/kg (the highest levels was reduced from 7500 mg/kg after 37 weeks) for two years. The mice were 6-7 weeks old at the start of the study. Median survival time was unaffected by treatment. Male and female mice fed 1500 or 5000 mg/kg exhibited dose-related body weight decreases. Food consumption was variable throughout the study, although high-dose females appeared to consume less food. The average daily intake of benomyl for males was 1079 mg/kg body weight per day initially, 878 mg/kg body weight per day for 1 year and 679 mg/kg body weight per day for 2 years; for females it was 1442 mg/kg body weight per day initially, 1192 mg/kg body weight per day for 1 year, and 959 mg/kg body weight per day for 2 years. There were no apparent differences between treatment and control groups with respect to palpable mass, number of mice affected or latency period of discovery. Haematology examinations revealed no abnormalities except for slightly decreased erythrocyte counts in the case of males at 1500 mg/kg and females at 5000 mg/kg. Haemoglobin and haematocrit values were also slightly depressed in 1500-mg/kg males. Significant compound-related changes were seen in the absolute and relative liver weights for males at 1500 and 5000 mg/kg for females at 5000 mg/kg. Male mice also presented decreased absolute testes weights at the highest dose level. Non-neoplastic organ changes in males (5000 mg/kg) were confined to the liver (degeneration, pigment, cytomegaly), thymus (atrophy), testes, epididymis (degeneration of seminiferous tubules, atrophy, aspermatogenesis, distended acini) and prostate. In female mice, splenic haemosiderosis was significantly increased at 5000 mg/kg, as was submucosal lymphocytic infiltration of the trachea at 1500 mg/kg (Weichman et al., 1982). 7.5 Reproduction, embryotoxicity and teratogenicity 7.5.1 Reproduction A number of reproduction studies have been conducted on carbendazim, the main metabolite of benomyl. A description of these studies can be found in the Environmental Health Criteria 149: Carbendazim (WHO, 1993). 220.127.116.11 Rat feeding studies A 2-generation reproduction study was conducted using Crl:CD rats (Mebus, 1990). Throughout the study, animals were fed diets containing 0, 100, 500, 3000 or 10 000 mg benomyl/kg. P1 parental rats received the test diets for 71 days (mean daily intake 0, approx. 6, approx. 30, approx. 190 or approx. 350 mg/kg per day, respectively) before being bred to animals from the same dietary concentration group for production of the F1 parental rats. F1 rats were mated after being maintained on their respective diets (mean daily intake 0, approx. 8, approx. 20, approx. 250, or approx. 1000 mg/kg per day, respectively) for at least 105 days after weaning for production of the F2A litter. F1 dams were mated again, to different non-sibling males, at least 1 week after weaning the F2A litter, to produce the F2B litters. The following indices of reproductive function were calculated for the P1 and F1 adults: mating, fertility, gestation, viability, lactation, percent of pups born alive and percent litter survival. In addition, mean body weights, body weight gains, food consumption and food efficiency were measured, and clinical observations were recorded. After litter production, all parental generation rats were sacrificed for gross and histopathological (gross lesions and target organs only) examination. Complete histopathological examination was conducted on control and high-dose animals. Twenty F2A and 20 F2B weanlings were also given a gross pathological examination. There were no compound-related effects on parental mortality at any benomyl concentration. Mean body weights, body weight gains and overall food consumption of P1 and F1 male and female rats were significantly lower in the animals receiving 10 000 mg/kg than they were in controls. At 10 000 mg/kg, there was a significant compound-related decrease in the number of F2A and F2B offspring alive prior to culling (on day 4). In addition, male and female offspring of rats fed 10 000 mg/kg weighed consistently less at birth than did the offspring of control rats. With the exception of the 14-day male pups in the F2B generation, F2A and F2B offspring in the 3000 mg/kg group also had compound-related significantly depressed body weights on days 14 and 21 of lactation. Testicular sperm counts in P1 and F1 rats were decreased in the 3000 and 10 000 mg/kg groups. This was accompanied by decreased testicular weight and histopathological changes in the testes at 10 000 mg/kg. Microscopic observations included atrophy and degeneration of the seminiferous tubules in the testes of rats in the 3000 and 10 000 mg/kg groups and oligospermia in the epididymides of the high-dose P1 generation and the 3000 and 10 000 mg/kg F1 generation. However, there were no compound-related differences in mating indices, fertility indices or gestation length which could be attributed to benomyl feeding (Mebus, 1990). In a feeding study by Barnes et al. (1983), adult male Wistar rats (27 animals/group) were fed 0, 1, 6.3 or 203 mg/kg for 70 days (13 animals/group) and allowed to recover for 70 days (14 animals/group). Ejaculated sperm counts were reportedly depressed in the 203-mg/kg group during the feeding phase. There was also a decrease in relative testicular weights and a slightly lowered male fertility index in all treated groups. Benomyl did not alter copulatory behaviour and did not induce dominant lethal mutation. Plasma testosterone and gonadotropin levels remained unchanged throughout the study. Identical studies conducted during the recovery phase showed all of the treatment-related effects to be completely reversible. 18.104.22.168 Rat gavage studies The effects of exposure to benomyl on male reproductive development was evaluated in prepubertal Sprague-Dawley male rats (33 days old), which were gavaged daily for 10 days at doses of 0 or 200 mg/kg per day. Eight animals per group were killed at 3, 17, 31, 45 and 59 days after the last treatment. Selected tissues, including liver, kidneys, testes, seminal vesicles and epididymides, were removed, weighed and examined histo logically. Samples of seminal fluid from the vas deferens were also examined. Observation intervals were pre-selected to coincide with stages of spermatogenesis. Data were presented on tissue weights, total epididymal sperm counts, vas deferens sperm concentrations and testicular histology. There were no effects related to treatment (Carter, 1982). In a similar study, adult male Sprague-Dawley rats (65 days old) received 10 daily treatments of 0, 200 or 400 mg benomyl/kg per day by gavage, and, 14 days after the last treatment, body weight, tissue weights, total epididymal sperm counts and sperm concentration in the vas deferens were measured and histological investigations of the testes were carried out. Production of testosterone by the Leydig cells was stimulated by subcutaneous injections of human chorionic gonadotrophin (HCG) 2 h prior to sacrifice. There were no compound-related effects on body weight or on liver, kidney, adrenal, testis or seminal vesicle weights. Caudate epididymal weights were, however, depressed by treatment with benomyl. There were also treatment-related reductions in epididymal sperm count (caput and caudate) as well as in the vas deferens sperm concentration. The study was designed to evaluate alterations in spermatozoa undergoing spermatogenesis in the seminiferous tubules of the testes during exposure to benomyl. Animals exposed to 400 mg/kg per day presented histological evidence of hypospermatocytogenesis with generalized disruption of all stages of spermatogenesis, when compared with controls (Carter & Laskey, 1982). Linder et al. (1988) evaluated the effect of administering 1, 5, 15 or 45 mg benomyl/kg per day by gavage daily to 102-day old Wistar male rats (12/group). The males were bred to untreated females after 62 days of dosing and killed after 76-79 days. Reproductive behaviour, seminal vesicle weight, prostate weight, sperm motility, and serum gonadotropin hormones and serum gonadal hormone levels were no different from those in controls at any dose. At necropsy, males exposed to 45 mg/kg had decreased testis and epididymal weights, reduced cauda sperm reserves, decreased sperm production, increased numbers of decapitated spermatozoa and increased numbers of seminiferous tubules containing multinucleated giant cells. In a study by Hess et al. (1991), adult male Sprague-Dawley rats (100 days of age, 20 rats/dose) were given a single dose of benomyl in corn oil (0, 25, 50, 100, 200, 400 or 800 mg/kg body weight). Eight animals/group were sacrificed at 2 days and 12 animals/group at 70 days (except for the 800 mg/kg group) after treatment. The testis and excurrent ducts were examined each time to determine benomyl effects on spermatogenesis and on the epididymis. The primary effects seen at day 2 were testicular swelling and occlusions of the efferent ductules. Premature release of germ cells (sloughing) was the most sensitive short-term response to benomyl. Sloughing was detected in all treatment groups but was statistically significant (p < 0.05) at doses of 100 mg/kg to 800 mg/kg. Occlusions of the efferent ductules of the testis were dose dependent and correlated with the increase in testis weight on day 2. The greatest increase in testes weight was observed in the 400 mg/kg group. Long-term effects (70 days) were seen in the 100, 200 and 400 mg/kg groups, e.g., decreased testis weight (400 mg/kg), dose-dependent increases in seminiferous tubular atrophy, and increases in the number of reproductive tracts containing occluded efferent ductules. No long-term effects were seen in the 0, 25 or 50 mg/kg groups. 22.214.171.124 Dog inhalation studies Three groups (10 dogs per group) of sexually mature male dogs were exposed to benomyl at air concentrations (aerosol/cloud) of 0, 0.65 and 1.65 mg/litre for a 4-h period. One half of the dogs in each group were killed on day 14 and the remainder on day 28 following the exposure. Histopathological examination revealed a reduction in spermatogonic activity on day 14, but not on day 28, in the high-dose group (Littlefield & Busey, 1969). 7.5.2 Teratogenicity and embryotoxicity 126.96.36.199 Mouse gavage studies Groups of pregnant CD-1 mice (20-25 mice/group) were administered benomyl via gavage at dose levels of 0, 50, 100 and 200 mg/kg per day on days 7 to 17 of gestation. Animals were killed on day 18, pups were delivered by Caesarean section, the number of live, dead and resorbed fetuses was determined, and fetuses were examined for gross abnormalities. Half of the fetuses were examined for visceral abnormalities and the other half for skeletal abnormalities. No maternal toxicity was observed. Fetal development was adversely affected by treatment at all dose levels. The high dose caused an increased supraoccipital score, decreased numbers of caudal and sternal ossifications and increased incidences of enlarged lateral ventricles and enlarged renal pelvis. The latter, while not significant at the lower doses, did demonstrate dose-related increases at all other doses. The occurrence of supernumerary ribs and subnormal vertebral centrums was significantly increased in a dose-related manner at all dose levels. There was an increase in the number of abnormal litters and fetuses, which was significantly different from the controls, at levels of 100 and 200 mg/kg per day. Fetal weights were also decreased at these dose levels. Major abnormalities included exencephaly, hydrocephaly, cleft palate, hydronephrosis, polydactyly, oligodactyly, umbilical hernia, fused ribs, fused vertebrae and short/kinky tail (Kavlock et al., 1982). 188.8.131.52 Rat gavage studies In a study by Staples (1980), benomyl (99.2% a.i.) was adminis tered by gavage to groups of pregnant rats (ChR-CD) at dose levels of 0, 3, 10, 30, 62.5 and 125 mg/kg per day from days 7 to 16 of gestation. There were 60 dams in the control group and 27 in each of the other test groups; they were observed daily for signs of toxicity and changes in behaviour. No clinical signs of toxicity or mortality were observed among dams in any dose group. Body weight gain was comparable to controls, as were the incidences of pregnancy, corpora lutea and implantation sites, and the sex ratio. However, fetal body weight was significantly decreased at the two highest dose levels. There was also an increased incidence of embryo/fetal mortality at 125 mg/kg per day. The malformations observed included microphthalmia, anophthalmia and hydrocephaly (distended lateral ventricles). These appeared to be compound related at the higher dose levels. Microphthalmia was seen in two fetuses from different litters at 10 mg/kg per day and in one fetus from the control group. Microphthalmia/anopthalmia was observed in only 4-6 out of 4935 fetuses examined in the historical data base at this laboratory. Histological examination of eyes revealed pathological changes consisting of irregular lenses, retro-bulbar glandular adnexa, distorted or compressed retinal layers and thickened nerve fibres in the 10, 62.5 and 125 mg/kg per day treatment groups. Major skeletal malformations observed in the 125 mg/kg dose group included fused ribs, fused sternebrae and fused thoracic arches. Additional skeletal variations were also increased at 62.5 and 125 mg/kg per day; these included misaligned and unossified sternebrae and bipartite vertebral centra (Staples, 1980). In order to determine a no-effect level for microphthalmia and hydrocephaly, groups of 50 pregnant rats of the same strain and from the same supplier were administered benomyl (99.1% a.i.) via gavage at dosage levels of 0, 3, 6.25, 10, 20, 30 and 62.5 mg/kg per day from days 7 to 16 of gestation (Staples, 1982). Each group contained 50 animals except the high-dose group, which contained 20 female rats. Reproductive status was determined on a per-litter basis following gross pathological evaluation. The number of implantation sites, resorptions, dead, live and stunted fetuses, and the mean weight of live fetuses per litter were determined. Only fetal heads were fixed and examined microscopically. Microphthalmia was determined on the basis of the smallest eye in the control group (< 1.8 mm). Mean fetal body weight was significantly lower in the high-dose group. There were only two animals with malformations, both in the 62.5 mg/kg per day group. One fetus exhibited internal hydrocephaly and another, in a separate litter, unilateral microphthalmia. There was no teratogenic response at doses up to 30 mg/kg. The teratogenic potential of benomyl was examined in groups of Wistar rats (12 to 30 pregnant rats/group) orally gavaged at dose levels of 0, 15.6, 31.2, 62.5 and 125 mg/kg per day on days 7 to 16 of gestation. Major anomalies were observed primarily at levels of 62.5 mg/kg per day or more and included encephaloceles, hydrocephaly, microphthalmia, fused vertebrae and fused ribs. A dose level of 31.2 mg/kg per day appeared to be without adverse effects on the developing rat fetus in this evaluation. A significant reduction in maternal body weight was observed at the highest dose level (Kavlock et al., 1982). Kavlock et al. (1982) also evaluated the effect of low levels of benomyl as the pups aged. Benomyl was administered via gavage to groups of Wistar rats at dose levels of 0, 15.6 and 31.2 mg/kg per day from day 7 of gestation to day 15 of lactation (day 22 of gestation was considered day 0 of lactation). The litters were weighed on days 8, 15, 22, 29 and 100 after parturition, and locomotor activity was evaluated periodically throughout the study. At 100 days of age, several organs were weighed, including the adrenals, liver, kidney, ovaries, testes and the ventral prostate plus seminal vesicles. There were no compound-related effects either on litter size at birth or weaning, or on body weights of fetuses. Growth, survival and locomotor activity values were comparable with those of the controls throughout the study. Organ weights were comparable with those of controls, except that testes and ventral prostate/seminal vesicle weights were significantly reduced at 31.2 mg/kg per day (but not at 15.6 mg/kg) (Kavlock et al., 1982). In other studies (Zeman et al., 1986; Hoogenboom et al., 1991), benomyl produced ocular and craniocerebral malformations in Sprague-Dawley rats when administered by gavage at doses of 31.2 and 62.4 mg/kg per day on day 7-21 of gestation. Ocular anomalies (retinal dysplasia, cataracts, microphthalmia and anophthalmia) occurred in 43.3% of fetuses when the dams were administered 62.4 mg/kg per day. The occurrence increased to 62.5% when the dams were given a semipurified protein-deficient diet and the same dose level of benomyl (Hoogenboom et al., 1991). Craniocerebral malformations (consisting primarily of hydrocephaly) occurred in fetuses from dams administered 31.2 mg/kg per day in combination with the semipurified diet (Zeman et al., 1986). 184.108.40.206 Rat feeding studies In a study by Sherman et al. (1972), groups of rats (26-28 pregnant ChR-CD rats/group) were administered a benomyl formulation (53.5% a.i.) in the diet at dosages of 0, 100, 500, 2500 and 5000 mg/kg from day 6 to day 15 of gestation. Average doses were equivalent to 0, 8.6, 43.5, 209.5 and 372.9 mg/kg body weight per day. On day 20 of gestation, all pregnant animals were sacrificed and fetuses were delivered by Caesarean section. There was no mortality attributable to benomyl, no clinical signs of toxicity and no adverse effects on the body weight of dams. Dams in the highest-dose group had a reduced food intake during the period benomyl was administered in the diet, but the intake returned to a level similar to the controls for the remainder of the study. Except for three litters at the highest dose, where there were incidences of hydronephrosis and retarded ossification (interparietal and occipital bones), there were no effects on fetal development related to benomyl administration. In a similar study, groups of pregnant Wistar rats (27-28 rats/group) were fed benomyl at dose levels of 0, 1690, 3380 and 6760 mg/kg diet (time-weighted doses of 0, 169, 298 and 505 mg/kg body weight per day, respectively) from days 7 to 16 of gestation. No dose-related anomalies or major malformations were associated with exposure to benomyl at any of the dose levels used. Reduced fetal weight was observed at the two highest dose levels (Kavlock et al., 1982). 220.127.116.11 Rabbit feeding studies Groups of rabbits (15 artificially inseminated New Zealand albino rabbits/group) were administered a benomyl formulation (50% a.i.) in the diet at dose levels of 0, 100 and 500 mg/kg diet from day 8 to day 16 of gestation. Mortality, clinical observations and food consumption were determined daily and body weights were measured weekly. There were 12 pregnant does in the control group, 13 in the low-dose group and 9 in the high-dose group. Of these, 6, 7 and 5, respectively, were sacrificed on day 29 or 30 and fetuses were delivered by Caesarean section; the remaining does gave birth normally. Maternal toxicity was not observed at any dose level. Except for a marginal increase in rudimentary ribs at 500 mg/kg, developmental toxicity was not observed. The numbers of litters and of fetuses examined were less than adequate to assess the fetotoxic or teratogenic potential of benomyl to pregnant rabbits (Busey, 1968c; FAO/WHO, 1985a). 7.6 Mutagenicity and related end-points Numerous studies have been conducted to assess the mutagenic potential of benomyl, its metabolite carbendazim, and several benomyl formulations. Many of the results are conflicting and many of the study reports do not provide sufficient detail to evaluate the reasons for the conflicting data. This summary will cover only those studies where sufficient experimental detail and data were reported (Table 9). Studies on somatic and germ cells have shown that benomyl does not cause gene mutations or structural chromosomal damage (aberrations) and that it does not interact directly with DNA. This has been demonstrated in both mammalian and non-mammalian systems. However, in the mammalian in vitro studies for gene mutations and structural chromosome aberrations, some positive results were obtained with benomyl. These positive results appear to have been a consequence of the inherent sensitivity of some in vitro mammalian test systems to cytotoxic agents. Results in mammalian in vivo studies for gene mutations and structural chromosome aberrations were negative. Benomyl does cause numerical chromosomal aberrations (aneuploidy and/or polyploidy) in experimental systems both in vitro and in vivo (Table 9). 7.7 Carcinogenicity A number of carcinogenicity studies have been conducted on carbendazim, the main metabolite of benomyl. A description of these studies can be found in Environmental Health Criteria 149: Carbendazim (WHO, 1993). 7.7.1 Rat In a two-year study on benomyl, groups of Charles River albino weanling rats (36 male and 36 female rats) were fed benomyl (50-70% a.i.) in the diet at levels of 0, 100, 500 and 2500 mg/kg diet. At the end of the study all surviving animals were sacrificed and gross and microscopic examinations of tissues and organs were carried out. The most frequently observed tumours involved the pituitary, but these were equally distributed among control and treated groups. Mammary, adrenal and other tumours were also observed; these were scattered among all groups. There were no adverse effects or significant histopathological changes at any dose levels in this study that could be attributable to benomyl (Sherman, 1969c; Lee, 1977). Table 9. Studies on mutagenicity of benomyl End points/Tests Species, strains Concentrationb Activation Result Reference 1. DNA damage and repair Mitotic gene conversion Saccharomyces cerevisiae, NR with and negative Siebert et al. (1970); D4 & D7 without de Bertoldi et al. (1980) Mitotic gene conversion Aspergillus nidulans, 0.35-2.8 mM without negative de Bertoldi & Griselli D7 (1980) Mitotic crossing-over test A. nidulans, P NR without negative Bignami et al. (1977) A. nidulans, D7 NR without negative de Bertoldi & Griselli (1980) Non-disjunction A. nidulans, D7 0.35-2.8 mM without positive de Bertoldi & Griselli (1980) Sister chromatid exchanges human lymphocyte 0.05-2.0 µg/ml NR slight increase in Georgieva et al. (1990) (SCE) cultures SCE, no dose-response relationship Unscheduled DNA synthesis B6C3F1 male mice & 0.5-500 µg/ml NR negative Tong (1981) Fisher-344 male rat hepatocytes 2. Gene mutation a) Bacterial & fungal Salmonella typhimurium 0.125-1.0 µg/ml NR positive(TA1535) Kappas et al. (1976) gene mutation TA1535, TA1538 0.125-1.0 µg/ml NR negative(TA1538) Escherichia coli, 0.125-1.0 µg/ml NR positive Kappas et al. (1976) WP2 uvra E. coli, WP2 uvra 2.5-10.0 µg/ml NR positive Kappas et al. (1976) E. coli, WP2 0.125-1.0 µg/ml NR negative Kappas et al. (1976) Table 9 (contd). End points/Tests Species, strains Concentrationb Activation Result Reference Point mutation A. nidulans 0.25-0.4 µg/ml NR positive Kappas & Bridges (1981) Spot test S. typhimurium, his, 50-5000 µg per negative Fiscor et al. (1978) G 46 & TA1535, TA1530 spot TA1950 S. typhimurium, TA100 50-2000 µg per with negative Fiscor et al. (1978) plate Host-mediated assay S. typhimurium, his, 3 consecutive negative Fiscor et al. (1978) G 46 subcutaneous injections of 500 mg/kg in mice TA1950 an oral dose of 4000 negative Fiscor et al. (1978) mg/kg in rats or mice Spot test S. typhimurium, TA1535, 20 µg/spot with and negative Carere et al. (1978) TA1536, TA1537, TA1538 without Forward spot mutation Streptomyces coelicolor 500 µg/spot without negative Carere et al. (1978) assay Ames plate incorporation S. typhimurium, TA1537 500 µg/plate without marginal Russell (1978a) test positive S. typhimurium, TA1535, 10-500 µg/ with negative Donovan & Krahn (1981); TA1537, TA98, TA100 plate Rickard (1983a,b) 10-200 µg/plate without negative TA1535, TA1537, TA98, 10-500 µg/plate with and negative Russell (1978b) TA100 without Table 9 (contd). End points/Tests Species, strains Concentrationb Activation Result Reference TA1513, TA1537, up to 1000 µg/ with and negative Shirasu et al. (1978) TA1538, TA98, TA100 plate without E. coli, WP2 hcr up to 500 µg/ with and negative Shirasu et al. (1978) plate without recA spot test B. subtilis, M45 & H-17 2000 µg/plate NR negative Shirasu et al. (1978) Host-mediated assay S. typhimurium, his, 2000 mg/kg negative Shirasu et al. (1978) (ICR mice) G46 b) In vitro mammalian gene mutaion HGPRTa gene Chinese hamster ovary 17 & 172 µM with negative Fitzpatrick (1980) cells 3 & 120 µM without negative Mouse lymphoma Mutant colonies in 0.5-20 µM without negative Amacher et al. (1979) L5178Y TK+/- gene RPMl1640/horse serum mutation assays (MLY) 2.5-25 µM with negative MLY > 2.5 µM negative McCooey et al. (1983a) MLY carbendazim, without negative McCooey et al. (1983b) 25-200 µM carbendazim, with negative McCooey et al. (1983b) 12.5-200 µM MLY N-butylisocyanate without negative McCooey et al. (1983b) 2.5-25 µM Table 9 (contd). End points/Tests Species, strains Concentrationb Activation Result Reference c) Insect germ cell gene mutation Sex-linked recessive adult male fed benomyl suspension negative Lamb & Lilly (1980) lethals D. melanogaster (1.5 mg/ml) 3. Chromosomal effects a) Yeast and fungi A. nidulans 0.25 µg/ml without increased frequency of Hastie (1970) (diploid) 0.5 µg/ml segregants A. nidulans 0.25 µg/ml without no increased frequency Hastie (1970) (haploid) 0.5 µg/ml of segregants A. nidulans 0.75 to 1.75 µM without increased frequency of Kappas (1974) (diploid) segregants A. nidulans 0.75 to 1.75 µM without increased frequency of Kappas (1974) (haploid) segregants A. nidulans 0.35 to 2.8 mM without increased De Bertoldi & Griselli (diploid) nondisjunction (1980) Saccharomyces cerevisiae, 30 µg/ml without induced chromosome Albertini (1991) D61.M malsegregation b) In vitro mammalian human lymphocytes 1.0-100.0 µg/ml with negative Pilinskaya (1983) assays human lymphocytes 1.0-100.0 µg/ml without weak positive at Pilinskaya (1983) 10.0 µg/ml only Table 9 (contd). End points/Tests Species, strains Concentrationb Activation Result Reference human/mouse mono- < 15 µg/ml without induced aneuploidy Athwal & Sandhu (1985); chromosomal hybrid and polyploidy Sandhu et al. (1988) cells (R3-5) human/mouse mono- 15 µg/ml without slight increase in Athwal & Sandhu (1985); chromosomal hybrid structural Sandhu et al. (1988) cells (R3-5) chromosomes; 1.5 µg/ml threshold (polyploidy) V79/AP4 Chinese NR without dose-related increase Rainaldi et al. (1989) hamster cells in numerical chromosomal aberrations Chinese hamster NR without dose-related increase Eastmond & Tucker ovary cells in numerical (1989) chromosomal aberrations human lymphocytes NR without dose-related increase Georgieva et al. (1990) in numerical chromosomal aberrations; 0.1 µg/ml threshold (aneuploidy) Chinese hamster- NR without dose-related increase Zelesco et al. (1990) human hybrid cells in numerical chromosomal (EUBI) aberrations; 2.0 µg/ml threshold (aneuploidy) Table 9 (contd). End points/Tests Species, strains Concentrationb Activation Result Reference c) In vivo mammalian rats up to 500 mg/kg no chromosomal Ruziscka et al. (1976) assays for 8 days by gavage aberrations in bone marrow, increase in chromosomal aberrations in embryonic cells at 200 and 500 mg/kg doses Bone marrow micro- ICR mice 2 gavage doses of increase in Seiler (1976) nucleus test 9, 500 or 1000 micronucleated mg/kg 24 h apart polychromatic erythrocytes at 1000 mg/kg only Bone marrow micro- BDF1 mice single gavage dose increase in Sasaki (1990) nucleus test of 0, 1250, 2500 or micronucleated 5000 mg/kg polychromatic erythrocytes at 2500 and 5000 mg/kg Bone marrow chromosomal B6D2F2/Cr-1Br mice single gavage dose no increase in Stahl (1990) aberrations of 0, 625, 1250, 2500, structural chromosomal or 5000 mg/kg aberrations d) In vivo germ cell chromosomal aberration Dominant lethal test ChR-CD rat 0, 500, 2500 or negative Culik & Gibson (1974) 5000 mg/kg feeding for 7 days Table 9 (contd). End points/Tests Species, strains Concentrationb Activation Result Reference Dominant lethal test Wistar rats 0, 1, 6.3 or 203 negative Barnes et al. (1983) mg/kg feeding for 70 days Dominant lethal test Wistar rats 70 daily gavage doses negative Georgieva et al. (1990) of 0, 10 or 50 mg/kg a HGPRT = hypoxanthine-guanine phosphoribosyl transferase b NR = not reported 7.7.2 Mouse Male and female CD-1 mice (80 males and 80 females per group) were fed benomyl (99% a.i.) at dose levels of 0, 500, 1500 and 5000 mg/kg diet (the highest level was reduced from 7500 mg/kg after 37 weeks) for two years. The incidence of hepatocellular adenomas and carcinomas (see Table 10) in female mice was increased in a dose-dependent manner. In male mice, numbers of hepatocellular adenomas and carcinomas were significantly increased at 500 and 1500 mg/kg but not at 5000 mg/kg dose. The increased number of lung alveogenic carcinomas in male mice was still within the range of historical controls (Weichman et al., 1982; Frame & van Pelt, 1990; Hardisty, 1990). 7.8 Special studies 7.8.1 Neurotoxicity Studies performed using White Leghorn hens (10/group) gave no indication of neurotoxic potential with single oral doses of benomyl at levels up to 5000 mg/kg (Goldenthal, 1978; Jessup & Dean, 1979; Jessup, 1979). Studies performed using adult male CFY rats (10 rats/group) gave no indication of altered EEG potentials or behaviour (learning ability assessed with a 4-choice T-maze) after treatment with 250 or 500 mg benomyl/kg per day for 3 months when compared with controls (Desi, 1983). 7.8.2 Effects in tissue culture In a study by Desi et al. (1977), primary monkey kidney cells were incubated with benomyl (Fundazol 50 WP) at levels of 1, 10, 50, 100, 250 and 400 mg/kg. Growth inhibition and syncytium-like appearance of the cell monolayer was observed at 250 mg/kg, and at 400 mg/kg all the cells were killed. 7.9 Factors modifying toxicity; toxicity of metabolites Benomyl degrades to butyl isocyanate (BIC) and carbendazim quite rapidly in water and in many organic solvents used in toxicological testing. The biological activity of benomyl is essentially that of carbendazim both in a toxicological sense and in its use as a fungicide. A detailed discussion of carbendazim toxicology is given in Environmental Health Criteria 149: Carbendazim (WHO, 1993). Table 10. Incidence and latency of hepatic tumours in benomyl-treated CD-1 micea Male mice Female miceb Benomyl concentration (mg/kg diet): 0 500 1500 5000 0 500 1500 5000 Hepatocellular adenoma Number of animals with tumours 9 8 10 10 2 2 5 7 Latent time to first tumour (days) 530 556 541 627 744 641 650 644 Average latent period (days) 687 707 695 726 744 688 717 722 Hepatocellular carcinoma Number of animals with tumours 14 26c 41d 17 2 7 7 14e Latent time to first tumour (days) 545 470 590 508 744 640 736 426 Average latent period (days) 693 705 721 711 744 708 736 695 a From: Wiechman (1982) b P < 0.05 Dose/response increase of adenomas and carcinomas c P < 0.05 d P < 0.001 e P < 0.01 BIC is toxic by inhalation. In 4-month subchronic inhalation studies, the no-observed-effect levels (NOEL) for BIC in rats and mice were determined to be 0.32 and 1.5 ppm, respectively (Gurova et al., 1976). An industrial hygiene survey found that workers experienced severe eye irritation and lacrimation at BIC exposure levels of 5-10 ppb (Kelly, 1989). 7.10 Mechanisms of toxicity - Mode of action Biochemical studies on the mechanism of action of benzimidazole compounds have shown that their biological effects are caused by interactions with cell microtubules (Davidse & Flach, 1977). These cellular structures are present in all eukariotic cells and are involved in several vital functions, such as intracellular transports and cell division. Benzimidazol compounds have been used as anticancer drugs and as antihelminthic drugs in animals and humans because they act as spindle poisons by interfering with the formation and/or functioning of microtubules. However, eukaryotes are known to be unequally sensitive to each benzimidazol compound, which explains the use of these compounds in helminthiases. Selective toxicity of benomyl and carbendazim for fungi has been explained by comparing their binding to fungal and mammalian tubulin. The different sensitivity of several fungi has also been explained by the different affinity of benomyl and carbendazim for fungal tubulin. Benomyl has been found to bind to fungal tubulin but not to porcine brain tubulin, indicating that mammalian tubulin has no, or at least low, affinity for benomyl (Davidse & Flach, 1977). This is in agreement with the observation that benomyl at concentrations that are lethal for sensitive fungi does not interact with in vitro microtubule assembly in these brain extracts. In vitro ID50 values for several mycelial extracts of various fungal species sensitive to benomyl were all below 5 µmol/litre (Davidse & Flach, 1977). In vitro rat brain tubulin polymerization was inhibited to about 20% at benomyl or carbendazim concentrations of 25 µmol/litre (De Brabander et al., 1976b). For comparison, a standard antitubulin drug in humans such as vincristine inhibited 50% tubulin assembly at 0.1 µmol/litre in the same experiment. The assembly of sheep and calf brain microtubule was also found to be unaffected by carbendazim concentrations higher than 100 µmol/litre (Ireland et al., 1979). Mitotic arrest by benzimidazole and six analogues at metaphase was evaluated in human lymphocyte cultures. Structure-activity relationships indicate that antimitotic activity is related to C6 substitution of the benzimidazole moiety (Holden et al., 1980). In this study, however, benomyl and carbendazim were not tested. The question of whether all C6 unsubstituted benzimidazoles, such as benomyl and carbendazim, have no effect on mitosis of human lymphocytes in cell cultures is therefore unresolved. A link between the effects of benomyl and carbendazim on brain tubulin and their teratogenic effects has been postulated (Ellis et al., 1987, 1988). 8. EFFECTS ON HUMANS 8.1 General population exposure No references to benomyl poisoning in the general population have been documented in the scientific literature. Recent data used to estimate dietary exposure based on food consumption patterns within the USA indicate exposures well below the NOELs in animal toxicity tests. 8.2 Occupational exposure 8.2.1 Acute toxicity Benomyl has a very low acute toxicity. No inadvertent poisoning of agricultural or factory workers has been documented (Goulding, 1983). 8.2.2 Effects of short- and long-term exposure Benomyl causes contact dermatitis and dermal sensitization in some farm workers (van Joost et al., 1983; Kuehne et al., 1985). In controlled patch tests of agricultural, ex-agricultural and non-agricultural workers (total of 200 subjects), only one agricultural worker showed any contact dermatitis to 0.1% benomyl (Lisi et al., 1986). A survey of cross-sensitization between benomyl and other pesticides was conducted in Japan on a group of 126 farmers who applied benomyl to their crops. Thirty-nine of the farmers gave positive test results with benomyl, the highest incidence being among female farmers. There were cross-reactions between benomyl and other pesticides, such as diazinon, saturn, daconil and Z-bordeaux (Matsushita & Aoyama, 1981). Selected blood profiles from 50 factory workers involved in the manufacture of benomyl were compared to those of a control group of 48 workers who were not exposed to carbendazim. White blood cell count, red blood cell count and haemoglobin and haematocrit values were comparable among the two groups. There were no quantitative estimates of exposure given for the factory workers (Everhart, 1979; FAO/WHO, 1985a). A study was performed to determine whether exposure to benomyl and carbendazim had an adverse effect on the fertility of 298 male manufacturing workers exposed to benomyl between 1970 and 1977. The workers ranged from 19 to 64 years of age (79% were between 20 and 39, and 78% of the spouses were similarly aged between 20 and 39 years). Exposure duration ranged from less than one month to 95 months, and more than 51% of the workers were potentially exposed from 1 to 5 months. The birth rates of exposed workers' spouses were compared with those of four comparison populations from the same county, state, region and country (USA). There was no reduction in fertility as shown by the birth rates for the study population, which were generally higher than those of the comparison populations. Spermatogenesis among workers was not examined (Gooch, 1978; FAO/WHO, 1985a). In studies on agricultural spraymen using benomyl (Fundazol 50 WP), 14 spraymen working in greenhouses were followed, some of them for two years. General medical check-up and routine blood and urine tests were performed. Electrocardiograms were recorded and blood cholinesterase activity was monitored. Lymphocytes from peripheral blood were examined for chromosomal aberrations both before and during the study. There was no difference in structural chromosomal aberration between the spraymen and controls. After benomyl exposure, numerical chromosomal aberrations were higher in the spraymen than in controls. However, the spraymen had a higher level of numerical chromosome aberration than the controls even before benomyl exposure (Desi et al., 1990; Nehez et al., 1992). 9. EFFECTS ON ORGANISMS IN THE LABORATORY AND FIELD 9.1 Microorganisms Soil respiration has been found to be little influenced by benomyl at concentrations below 10 mg/kg, which is the maximum soil concentration expected after use at recommended application rates (Hofer et al., 1971; van Fassen, 1974; Peeples, 1974; Weeks & Hedrick, 1975). A study on the influence of benomyl on soil nitrogen mineralization showed that the release of ammonia was not decreased by benomyl, whereas the influence of the fungicide on nitrification varied from a stimulation (van Fassen, 1974), through no effect (Mazur & Hughes, 1975), to a decreased nitrification (Hofer et al., 1971; Wainwright & Pugh, 1974). The differences may be related to the soil composition since Hofer et al. (1971) found a greater effect in sandy than in organic soil. Benomyl, in combination with eleven other pesticides that were used in an orchard spray programme, had only a minimal and short-term effect on respiration, ammonification and nitrification at concentrations expected after recommended use of benomyl over a spraying season. Ten times the recommended application rates had a pronounced effect on both respiration and nitrification, which lasted for more than 4 weeks (Helweg, 1985). The influence of benomyl and carbendazim on soil microbial activity was studied in Sweden following repeated annual applications during autumn to winter cereals for a period of 3 to 5 years. The effects of the fungicides on straw decomposition, balance of straw fungal flora and nitrogen mineralization in the soil were investigated in field and laboratory experiments. The decomposition of straw in the field was not affected in clay soils by annual applications of up to 2 kg/ha. In sandy soils, rates of up to 0.5 kg/ha had no effect, but in one case at 2 kg/ha the initial stages of straw decomposition were slightly inhibited. All doses tested in both clay and sandy soils caused changes in the composition of the straw fungal flora (Torstensson & Wessen, 1984). Benomyl had no effect on soil bacterial populations in laboratory studies, but fungi and actinomycetes populations were reduced (Siegel, 1975). Under greenhouse conditions (Kaastra-Howeler & Gams, 1973) or field conditions (Peeples, 1974) at application rates of up to 89.6 kg a.i./ha, little effect on microbial populations was observed following benomyl treatment. 9.2 Aquatic organisms The effect of benomyl was monitored using the green alga Selenastrum capricornutum in an OECD guideline test (201). The EC50 (based on total growth) at 72 h was 2.0 mg/litre and at 120 h was 3.1 mg/litre. The no-observed-effect concentration (NOEC) was 0.5 mg/litre. To study whether benomyl was algistatic or algicidal, organisms were recultured at the end of the initial 120 h incubation. Regrowth occurred in the control but not in the test cultures (8.0 mg/litre) after a period of 7 days. Benomyl was, therefore, considered to be algicidal (Douglas & Handley, 1988). In another study using Chlorella pyrenoidosa, the 48-h EC50 for growth inhibition was calculated as 1.4 mg/litre (Canton, 1976). The acute toxicity of benomyl to a variety of aquatic organisms is summarized in Table 11. For 96-h tests, LC50 values ranged from 0.006 mg/litre for channel catfish ( Ictalurus punctatus; yolk-sac fry) to > 100 mg/litre for crayfish ( Procambarus sp.) (Mayer & Ellersieck, 1986). 9.3 Terrestrial organisms Several field studies have investigated the toxicity of benomyl to earthworms (Table 12). In one study, the 48-h LC50 for E. foetida was 9.1 µg/cm3 soil (Roberts & Dorough, 1984). Van Gestel et al. (1992) exposed red earthworms ( Eisenia andrei) to benomyl added to artificial soil and used final concentrations of 0, 0.1, 0.32, 1.0, 3.2 and 10 mg/kg dry soil. The worms had been acclimatized for 1 week in the artificial soil, which contained 4 g/kg cow dung as food for the worms. At the highest concentration of 10 mg/kg, high mortality occurred; LC50 values of 6.0 and 5.7 mg/kg dry soil were calculated after 3 and 6 weeks of incubation, respectively. Growth was significantly reduced at 3.2 mg/kg soil. EC50 values for the effect of benomyl on cocoon production did not differ significantly for the two test periods, and the EC50 was 1.6 (1.2-2.3) mg/kg for the entire 6-week test period. Zoran et al. (1986) and Drewes et al. (1987) have shown that the conduction velocity for medial and lateral giant nerve fibres is affected by exposure of earthworms to sublethal concentrations of benomyl. Zoran et al. (1986) have also shown that segmental replication is affected in amputated earthworms exposed to sublethal concentrations of benomyl. The benomyl metabolite carbendazim (99.3% purity) was evaluated for acute contact toxicity after thoracic application to honey-bees (Apis mellifera). Each treatment level consisted of four replicates of ten bees each. Forty bees served as positive control (using carbaryl) and forty as negative control. No deaths occurred after application of carbendazim at 50 µg/bee, the highest rate tested. Carbendazim is, therefore, classified as "relatively non-toxic" to the honey-bee (Meade, 1984). Table 11. Toxicity of benomyl to aquatic organisms Organism Size/ Stat/ Temperature Hardnessb pH Duration LC50 Reference age flow (°C) (mg/litre) (h) (mg/litre) Freshwater Water flea adult stat 25 3 14 Yoshida & Nishiuchi (1972) (Daphnia magna) < 24 h stat 20 87 8.5 48 0.11c Hutton (1989) < 24 h stat 20 48 0.64 Canton (1976) < 24 h stat 17 40 7.4 48 2.8 Mayer & Ellersieck (1986) Scud (Gammarus pseudolimnaeus) adult stat 17 40 7.4 96 0.75 Mayer & Ellersieck (1986) Crayfish (Orconectes nais) instar stat 22 40 7.4 96 >10 Mayer & Ellersieck (1986) Crayfish (Procambarus sp.) immature stat 22 40 7.4 96 >100 Mayer & Ellersieck (1986) Midge (Chironomus plumosus) instar stat 22 40 7.4 48 7.0 Mayer & Ellersieck (1986) Rainbow trout 3 months stat 15 48 0.48 Canton (1976) (Oncorhynchus mykiss) 0.8 g stat 7 44 7.4 96 0.17 Mayer & Ellersieck (1986) 0.8 g stat 12 44 7.4 96 0.20 Mayer & Ellersieck (1986) 0.8 g stat 17 44 7.4 96 0.28 Mayer & Ellersieck (1986) 1.2 g stat 12 44 6.5 96 0.16 Mayer & Ellersieck (1986) 1.2 g stat 12 44 7.5 96 0.19 Mayer & Ellersieck (1986) 1.2 g stat 12 44 8.5 96 0.88 Mayer & Ellersieck (1986) 0.6 g stat 12 44 7.4 96 0.23 Mayer & Ellersieck (1986) 0.6 g stat 12 320 7.4 96 0.60 Mayer & Ellersieck (1986) fingerling stat 12 44 7.4 96 0.12 Mayer & Ellersieck (1986) swimup fry stat 12 44 7.4 96 0.16 Mayer & Ellersieck (1986) yolk-sac fry stat 12 44 7.4 96 0.28 Mayer & Ellersieck (1986) 1 g stat 12 44 7.4 96 0.31c Mayer & Ellersieck (1986) Table 11 (contd). Organism Size/ Stat/ Temperature Hardnessb pH Duration LC50 Reference age flow (°C) (mg/litre) (h) (mg/litre) Fathead minnow 0.9 g stat 22 44 7.4 96 2.2 Mayer & Ellersieck (1986) (Pimephales promelas) 0.5 g stat 22 45 7.1 96 1.3 Mayer & Ellersieck (1986) 0.5 g stat 22 44 7.4 96 1.9c Mayer & Ellersieck (1986) Channel catfish 1.2 g stat 22 44 7.4 96 0.029 Mayer & Ellersieck (1986) (Ictalurus punctatus) 0.05 g stat 22 44 7.4 96 0.013 Mayer & Ellersieck (1986) 0.15 g stat 22 44 7.4 96 0.024 Mayer & Ellersieck (1986) swimup fry stat 22 44 7.4 96 0.012 Mayer & Ellersieck (1986) yolk-sac fry stat 22 44 7.4 96 0.006 Mayer & Ellersieck (1986) 1.2 g stat 22 44 7.4 96 0.028c Mayer & Ellersieck (1986) Bluegill (Lepomis macrochirus) 0.9 g stat 12 44 7.4 96 0.75 Mayer & Ellersieck (1986) 0.9 g stat 17 44 7.4 96 1.3 Mayer & Ellersieck (1986) 0.9 g stat 22 44 7.4 96 1.3 Mayer & Ellersieck (1986) 0.6 g stat 22 44 6.5 96 1.3 Mayer & Ellersieck (1986) 0.6 g stat 22 44 7.5 96 1.2 Mayer & Ellersieck (1986) 0.6 g stat 22 44 8.5 96 6.4 Mayer & Ellersieck (1986) 0.6 g stat 22 44 7.4 96 1.3 Mayer & Ellersieck (1986) 0.6 g stat 22 320 7.4 96 2.3 Mayer & Ellersieck (1986) 0.6 g stat 22 44 7.4 96 1.2c Mayer & Ellersieck (1986) Carp (Cyprinus carpio) 5 cm stat 25 48 7.5 Yoshida & Nishiuchi (1972) Killifish (Fundulus sp.) 2.5 cm stat 25 48 11 Yoshida & Nishiuchi (1972) Loach 10 cm stat 25 48 14 Yoshida & Nishiuchi (1972) Table 11 (contd). Organism Size/ Stat/ Temperature Hardnessb pH Duration LC50 Reference age flow (°C) (mg/litre) (h) (mg/litre) Guppy (Poecilia reticulata) 3 weeks stat 24 48 3.4 Canton (1976) Tadpole (Bufo sp.) < 1 month stat 25 48 4.3 Yoshida & Nishiuchi (1972) Estuarine and Marine Eastern oyster (Crassostrea virginica) 25-50 mm flow 17-19 96 1.37d Boeri (1988a) Grass shrimp (Palaemonetes pugio) 18 mm stat 18 96 45.8c Bionomics Inc. (1972) Mysid shrimp (Mysidopsis bahia) stat 23 96 0.175 Boeri (1988c) Dungeness crab (Cancer magister) larvae 96 7.6 Armstrong et al. (1976) Sheepshead minnow (Cyprinodon variegatus) 0.14 g stat 22 96 3.88 Boeri (1988b) a stat = static conditons (water unchanged for duration of test); flow = flow-through conditions (benomyl concentration in water continuously maintained) b hardness given as mg CaCO3/litre c 50% wettable powder d EC50 based on rate of shell deposition Table 12. Summary of earthworm toxicity data on benomyl in field studiesa Crop/soil Dosage Estimated soil Time Effect Reference type (kg/ha) concentration (mg/kg) (days) Grass 0.125 0.9 63 8% reduction in number Ammon (1985) 1.25 3.6 43% reduction in number Grass 7.8 22.2 21 95% reduction in number Tomlin & Gore (1974) 91% reduction in biomass Grass 0.56 1.6 49 79% reduction in cast Keogh & Whitehead (1975) production Grass 0.56 1.6 13 50% reduction in number Tomlin et al. (1980, (0.18)b 28 40% reduction in number 1981) 180 70% reduction in number 328 67% reduction in number 1.12 (0.90)b 28 35% reduction in number (0.20)b 180 46% reduction in number 328 50% reduction in number 2.24 6.4 13 75% reduction in number 28 50% reduction in number (0.30)b 180 50% reduction in number 328 64% reduction in number Grass/loam 2.0 5.7 30 70% reduction in number Edwards & Brown (1982) 180 22% reduction in number 365 1% reduction in number Grass/sandy loam 5.0 14.3 30 89% reduction in number Edwards & Brown (1982) 180 59% reduction in number 365 32% reduction in number Table 12 (contd). Crop/soil Dosage Estimated soil Time Effect Reference type (kg/ha) concentration (mg/kg) (days) Grass/loamy sand 10.0 28.6 30 80% reduction in number 180 89% reduction in number 365 89% reduction in number Grass/sandy loam 5.0 14.3 30 91% reduction in number; Edwards & Brown (1982) 99% reduction in L. terrestris; 29% reduction in L. festivus 180 90% reduction in L. terrestris; 200% increase in L. festivus 365 99% reduction in L. terrestris; 1457% increase in L. festivus a From: Van Gestel (1992) b Results of analysis of the top 15-cm layer carried out by the author, recalculated as the concentration in the top 2.5 cm layer The acute toxicity for several birds is listed in Table 13. Benomyl is of low toxicity to birds. Table 13. Acute toxicity of benomyl to birds Species LD50a 5-day LC50b Reference (mg/kg) (mg/kg) Bobwhite quail > 10 000 Busey (1968e) Mallard duck > 10 000 Busey (1968e) Japanese quail > 5000 c Hill & Camardese (1986) Starling > 100 Schafer (1972) Redwinged blackbird 100 Schafer (1972) a LD50 = single oral dose expressed as mg/kg body weight b LC50 = 5-day dietary exposure followed by 3 days on a "clean" diet expressed as mg/kg diet c 50% active ingredient; no overt signs of toxicity were observed 9.4 Population and ecosystem effects Under certain conditions benomyl may have an effect on populations of earthworms. In apple orchards where foliage has been treated repeatedly at a rate of 0.28 kg/ha and has fallen to the ground, earthworms may be eliminated after two years of benomyl use (up to 13 sprayings). The earthworms Lumbricus terrestris and Allolobophora chlorotica were most affected. Populations of other species recovered within two years of the termination of spraying. Orchard yields were unaffected, as were earthworm populations adjacent to the orchards, because of the immobility of benomyl in the soil (Stringer & Wright, 1973; Stringer & Lyons, 1974). Van Gestel (1992) has summarized reports of the toxicity of benomyl on earthworms from field studies with different soil types, application rates and crops (Table 12). Estimated soil concentrations in Table 12, for the various uses of the fungicide, are based on application rates; they assume no mobility of the compound beyond the top 2.5 cm of soil and homogeneous distribution of benomyl in this layer. For orchard application, it was further assumed that 50% of the applied active ingredient reached the soil. Reported effects include reduced numbers and reduced activity of worms. Application rates are within the recommended rates for benomyl as a fungicide on these crops. 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT Benomyl and carbendazim are two different fungicides in their own right. However, carbendazim is also the main metabolite of benomyl in mammals and the degradation product of benomyl in the environment. Butyl isocyanate is the chemical moiety removed from benomyl when carbendazim is formed. Given the similar toxicities caused by benomyl and carbendazim and the different toxicological profile of butyl isocyanate, the two fungicides are evaluated together in this monograph. 10.1 Evaluation of human health risks The two primary routes of exposure to humans are through diet and through manufacturing or use of the product. There is limited information on actual dietary exposure to benomyl and carbendazim. Dietary exposure has been estimated in the Netherlands and the USA. In the USA, exposure based on dietary habits, measured residue levels, and the percentage of crop treated has been calculated for various subgroups of population. These calculations indicate that the estimated benomyl exposure is 0.144-1.479 µg/kg per day (section 5.2.1). In the Netherlands, the mean dietary intake was estimated to be 0.83 µg/kg per day (0.05 mg/day per person). These levels of exposure are below the recommended ADI of 0.01 (carbendazim) and 0.02 (benomyl) mg/kg body weight. The average air levels of benomyl and carbendazim have been determined in a manufacturing facility (section 5.3) and found to be less than 0.2 and 0.3 mg/m3, respectively. Both values are below the Threshold Limit Value of 5-10 mg benomyl/m3 established by a number of governmental agencies. In one study, potential respiratory and dermal exposure to benomyl wettable powder formulation was determined under several agricultural use situations (section 5.3). The highest rates of exposure occurred in situations of mixing and loading in preparation for aerial application; dermal and respiratory exposures were, respectively, 26 and 0.08 mg/person per cycle. Home users and agricultural workers re-entering treated fields were estimated to be exposed to about 1 mg/person per cycle and 5.9 mg/person per hour, principally through the dermal route. Because of the low mammalian toxicity, acute benomyl or carbendazim poisonings are unlikely to occur under conditions of normal use. Several studies of agricultural workers (section 8.2) have shown some cases of contact dermatitis after exposure to benomyl. These effects can be significantly reduced or eliminated by wearing long-sleeved shirts, long trousers and gloves. There is little information on health effects in humans as a consequence of exposure to either benomyl or carbendazim. Two studies have been conducted on factory workers involved in the manufacture of benomyl (section 8.2). In one study, haematological profiles from 50 factory workers involved in the manufacture of benomyl were comparable to those from a control group of 48 workers. A second study found no decrease in the birth rate of the wives of 298 factory workers exposed to benomyl. Extensive studies of various species of laboratory animals show reproductive, developmental, mutagenic and carcinogenic effects associated with both benomyl and carbendazim. The effects observed on rat fetuses were microphthalmia, hydrocephaly and encephaloceles. The no-observed-effect levels (NOEL) for developmental toxicity are equal to or greater than 10 mg/kg body weight per day, depending upon the species and route of administration. Similarly, the NOEL of benomyl for reproductive effects in the male rat appears to be 15 mg/kg body weight per day after gavage dosing. In feeding studies with both benomyl and carbendazim, the NOEL appears to be 500 mg/kg diet (equivalent to 25 mg/kg body weight per day). However, one benomyl feeding study reported a NOEL of less than 1 mg/kg diet (0.05 mg/kg body weight per day) for male reproductive effects. The reason for the discrepancy between the NOEL in this latter study and other investigations is unknown. The only consistent genotoxic effect noted in animal studies is the induction of numerical chromosomal aberrations. These effects are consistent with the interaction of benomyl and carbendazim with microtubule formation. Rat carcinogenicity studies did not show any carcinogenic effect for either compound. Benomyl and carbendazim induce hepatocellular tumours in CD-1 and SPF Swiss mice but not in NMRKf mice. This finding in mice is not considered to be a result of a direct genotoxic action. Rather, it appears to be associated with liver toxicity in strains of mice that are highly susceptible to tumour formation at this site. Benomyl and carbendazim are spindle poisons. Effects on target cells are consequences of binding to microtubules, giving toxicities similar to those of other spindle poisons such as colchicine and vincristine. Benzimidazol compounds in general and benomyl and carbendazim in particular have selective effects on the microtubules of different eukaryotes. Reasons for this selectivity include the binding capability to different tubulins and pharmacokinetic differences across species. In vitro concentrations of benomyl used to kill sensitive fungi were found to be ineffec tive in disturbing mammalian microtubular functions. These studies on the mechanism of action of benomyl and carbendazim indicate a selective effect of these compounds for target species. In summary, the LD50, as determined in a number of test species, for benomyl ranges from > 2000 to > 12 000 mg/kg and for carbendazim from > 2000 to > 15 000 mg/kg. There are no known reports of human poisoning for either compound. This, coupled with the low estimated environmental levels of both compounds, would suggest that the possibility of acute poisoning by benomyl or carbendazim is very remote. Similarly, the data available on test species make it unlikely that either benomyl or carbendazim is carcinogenic for humans. The NOELs for both reproductive and teratogenic effects of benomyl and carbendazim (i.e. 10-15 mg/kg) do raise a possibility that an accidental ingestion of either fungicide could adversely alter reproductive outcome in humans, but the likelihood that such poisoning would occur is remote. The selectivity of these two benzamidazol compounds for the tubulin of the target species (fungi) and their relative ineffectiveness to disturb mammalian microtubule function further reduce the possibility of their having toxic effects in humans. 10.2 Evaluation of effects on the environment Benomyl is rapidly converted to carbendazim in various environmental compartments, the half-lives being 2 and 19 h in water and soil, respectively. Therefore, data from studies on both benomyl and carbendazim are relevant for the evaluation of environmental effects. Carbendazim persists on leaf surfaces and in leaf litter. In soil the half-life is between 3 and 12 months, and the compound may be detected for up to 3 years. However, in many cases, major residues will be lost within a single season. Residues of carbendazim and its metabolites are strongly bound or incorporated into soil organic matter. The strong adsorption (Koc = approximately 2000) of carbendazim to soil and sediment particles reduces its bioavailability to terrestrial and aquatic organisms. Similarly, the mobility of carbendazim in soil is limited, and it is not expected to leach to ground water. Benomyl and carbendazim are highly toxic to some aquatic organisms in laboratory tests, the most sensitive species being the channel catfish with a 96-h LC50 for yolk-sac fry of 0.006 mg benomyl/litre. However, this toxicity is unlikely to be manifest in the environment for most aquatic organisms because of the low bioavailability in surface waters. The exposure of sediment-living organisms could be greater, but no test results are available for these organisms. Benomyl and carbendazim affect groups of fungi in soil but do not seem to modify the overall microbial activity of the soil when used at normal field rates. In both the laboratory and field, benomyl and carbendazim applied at recommended rates cause deaths and sublethal reproductive effects on earthworms of many different species. Surface-feeding species eating leaf litter are most at risk. Populations may take more than 2 years to recover. There are no studies available on other litter and soil invertebrates. Benomyl and carbendazim have low toxicity for birds and carbendazim is classified as "relatively non-toxic" to honey-bees. 10.3 Conclusions Benomyl causes dermal sensitization in humans. Both benomyl and carbendazim represent a very low risk for acute poisoning in humans. Given the current exposures and the low rate of dermal absorption of benomyl and carbendazim, it is unlikely that they would cause systemic toxicity effects either in the general population or in occupationally exposed subjects. These conclusions are drawn from animal data and from the limited human data available, but these extrapolations are supported by the understanding of the mode of action of carbendazim and benomyl in both target and non-target species. Further elucidation of the mechanism of toxicity of carbendazim and benomyl in mammals will perhaps permit a better determination of no-observed-effect levels. Binding studies on tubulins of target cells (testis and embryonic tissues) will facilitate comparisons across species. Carbendazim is strongly adsorbed to soil organic matter and remains in the soil for up to 3 years. It persists on leaf surfaces and, therefore, in leaf litter. Earthworms have been shown to be adversely affected (population and reproductive effects) at recommended application rates. There is no information on other soil or litter arthropods that would be similarly exposed. The high toxicity to aquatic organisms in laboratory tests is unlikely to be seen in the field because of the low bioavailability of sediment-bound residues of carbendazim. However, no information is available on sediment-living species which would receive the highest exposure. 11. FURTHER RESEARCH 1. Comparative binding studies of carbendazim to tubulins of target tissues from various species should be undertaken. 2. Further clarification of the fate of 1,2-diaminobenzene and bound residues in the environment is needed. 3. The effects of benomyl and carbendazim on sediment-dwelling organisms needs to be investigated. 12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES Benomyl was evaluated by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) in 1973, 1975, 1978, 1983 and 1988. The 1978 meeting agreed that the MRLs for benomyl, carbendazim and thiophanate-methyl should be combined and expressed as carbendazim. Benomyl residues were last evaluated by the 1988 meeting (FAO/WHO, 1988a,b) and the MRLs were updated at that time. These MRLs (expressed as carbendazim) are listed in Table 4. The 1983 meeting (FAO/WHO, 1985a) evaluated benomyl toxicology and set the following benomyl NOEL levels and ADI: Rat: 2500 mg/kg in the diet, equivalent to 125 mg/kg body weight Dog: 100 mg/kg (carbendazim) in the diet, equivalent to 2.5 mg/kg body weight Rat: teratology - 30 mg/kg body weight per day The estimated ADI for benomyl was established at 0-0.02 mg/kg body weight. Benomyl has not been evaluated by the International Agency for Research on Cancer (IARC). REFERENCES Albertini S (1991) Reevaluation of the 9 compounds reported conclusive positive in yeast Saccharomyces cerevisiae aneuploidy test systems by the Gene-Tox Program using strain D61.m of Saccharomyces cerevisiae. Mutat Res, 260: 165-180. Amacher DE, Paillet S, & Ray VA (1979) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells: Application to genetic toxicological testing. Mutat Res, 64: 391-406. Ammon HU (1985) Worm toxicity tests using Tubifex tubifex. In: Comportement et effets secondaires des pesticides dans le sol. Les Colloques de l'INRA 31, Versailles, 4-8 juin 1984. Paris, Institut national de la Recherche agronomique, pp 303-317. Armstrong DA, Buchanan DU, & Caldwell RS (1976) A mycosis caused by Lagenidium sp. in laboratory-reared larvae of the Dungeness crab, Cancer magister, and possible chemical treatments. J Invertebr Pathol, 28: 329-336. Arthur MF, Schweitzer KL, Fadel LC, Marsh BH, & Marsh SS (1989a) Aerobic aquatic metabolism of [phenyl(U)-14C]benomyl in Greenville, Mississippi, water and sediment (Battelle Study No. N-0966-7301). Columbus, Ohio, Battelle, Environmental Sciences Department (Unpublished report No. AMR-1452-89, prepared for E.I. Du Pont de Nemours and Co., Inc.). Arthur MF, Marsh BH, Fadel LC, & Zwick TC (1989b) Anaerobic aquatic metabolism of [phenyl(U)-14C]benomyl in West Jefferson, Ohio, pond water and sediment (Battelle Study No. NO799-8800). Columbus, Ohio, Battelle, Environmental Sciences Department (Unpublished report No. AMR 770-87, prepared for E.I. Du Pont de Nemours and Co., Inc.). Athwal RS & Sandhu SS (1985) Use of human X mouse hybrid cell line to detect aneuploidy induced by environmental chemicals. Mutat Res, 149: 73-81. Barbo EC & Carroll KS (1972) Oral ALD test (S-triazine, 3-butylbenzimidazolo[1,2-a],-2,4[1H,3H]-dione. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 227-72). Barefoot AC (1988) Vapor pressure of benomyl. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Research & Development Division, Experimental Station (Unpublished report No. AMR 1078-88). Barnes TB, Verlangieri AJ, & Wilson MC (1983) Reproductive toxicity of methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate (benomyl) in male Wistar rats. Toxicology, 28: 103-115. Baude JF, Gardiner JA, & Han JCY (1973) Characterization of residues on plants following foliar spray applications of benomyl. J Agric Food Chem, 21: 1084-1090. Baude FJ, Pease HL, & Holt RF (1974) Fate of benomyl on field soil and turf. J Agric Food Chem, 22: 413. Belasco IJ (1979a) Study showing the absence of acetylcholinesterase inhibition with a wettable powder formulation (50% Benomyl). Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. B/TOX-4). Belasco IJ (1979b) 2-14C-Benomyl (50 WP) adsorption through rat skin. Part II: Effect of time and dose applied with supplement. Wilmington, Delaware, Du Pont de Nemours and Co., Inc. (Unpublished report No. B/ME-47 and supplement No. HLR 117-79). Belasco IJ, Kirkland JJ, Pease HL, & Sherman H (1969) Studies with 2-14C-labelled methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) in rats. Wilmington, Delaware, Du Pont de Nemours and Co., Inc., Biochemical Department, Research Division, Experimental Station (Unpublished report No. B/ME-36). Bignami M, Aulicino F, Velcich A, Carere A, & Morpurgo G (1977) Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans. Mutat Res, 46: 395-402. Bionomics, Inc. (1972) Acute toxicity of Benlate to grass shrimp ( Palaemonetes vulgaris). Wareham, Massachusetts, Bionomics, Inc. (Unpublished report No. HOL 275-72, prepared for E.I. Du Pont de Nemours and Co., Inc.). Boeri RL (1988a) Flow through acute toxicity of benomyl technical to the eastern oyster, Crassostrea virginica. Marblehead, Massachusetts, Enseco (Unpublished report No. HLO 13-89, prepared for E.I. Du Pont de Nemours and Co., Inc.). Boeri RL (1988b) Static acute toxicity of benomyl technical to the sheepshead minnow, Cyprinodon variegatus. Marblehead, Massachusetts, Enseco (Unpublished report No. HLO 9-89, prepared for Du Pont de Nemours and Co., Inc.). Boeri RL (1988c) Static acute toxicity of benomyl technical to the mysid, Mysidopsis bahia. Marblehead, Massachusetts, Enseco (Unpublished report No. HLO 828-88, prepared for E.I. Du Pont de Nemours and Co., Inc.). Bolton EE, Anderson JJ, & Koeppe MK (1986a) Metabolism of [phenyl(U)-14C] benomyl in field-grown soybeans. Wilmington, Delaware, Du Pont de Nemours and Co., Inc., Agricultural Products Department, Experimental Station (Unpublished report No. AMR-531-86). Bolton EE, Anderson JJ, & Koeppe MK (1986b) Metabolism of [phenyl(U)-14C] benomyl in paddy rice. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Agricultural Products Department, Experimental Station (Unpublished report No. AMR-507-86). Brock WJ (1987) Acute dermal toxicity study with Benlate 50 DF fungicide in rabbits. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 505-87). Busey WM (l968a) Acute dermal LD50 test and dermal irritation test on rabbits using a wettable powder formulation (50% benomyl) with histological addendum. Falls Church, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. MRO 581-239, prepared for E.I. Du Pont de Nemours and Co., Inc.). Busey WM (l968b) Acute inhalation exposure test in rats using a wettable powder formulation (50% benomyl). Falls Church, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. MRO 1126-1, prepared for E.I. Du Pont de Nemours and Co., Inc.). Busey WM (l968c) Teratology study in rabbits using a wettable powder formulation (50% benomyl). Falls Church, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. MRO 1079, prepared for E.I Du Pont de Nemours and Co., Inc.). Busey WM (l968d) Repeated dermal application test on rabbits using a wettable powder formulation (50% benomyl). Falls Church, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. HLO 298-68, prepared for E.I. Du Pont de Nemours and Co., Inc.). Busey WM (1968e) Acute dietary administration - mallard ducklings and bobwhite quail: Fungicide 1991. Falls Church, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. MRO 581-239, prepared for E.I. Du Pont de Nemours and Co., Inc.). Bushway RJ, Hurst HL, Kugabalasooriar J, & Perkins LB (1991) Determination of carbendazim in blueberries by reversed-phase high performance liquid chromatography. J Chromatogr, 555: 321-324. Canton JH (1976) The toxicology of benomyl, thiophanate-methyl, and BCM to four freshwater organisms. Bull Environ Contam Toxicol, 16: 214-218. Carere A, Ortali VA, Cardamone G, Torracca AM, & Raschetti R (1978) Microbiological mutagenicity studies of pesticides in vitro. Mutat Res, 57: 277-826. Carter SD (l982) Effect of benomyl on the reproductive development in the prepubertal male rat. Raleigh, North Carolina, North Carolina State University (Thesis). Carter SD & Laskey JW (l982) Effect of benomyl on reproduction in the male rat. Toxicol Lett, 11: 87-94. Chang WM (1985) Soil column leaching studies with [phenyl- 14C(U)]benomyl. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Agricultural Chemicals Department, Experimental Station (Unpublished report No. AMR 426-85). Chiba M & Veres DF (1981) Fate of benomyl and its degradation compound methyl 2-benzimidazole carbamate on apple foliage. J Agric Food Chem, 29: 588-590. Clermont Y (1972) Kinetics of spermatogenesis in mammals: "seminiferous epithelium cycle and spermatogonial renewal". Physiol Rev, 52: 198-236. Colburn CW (1969) Skin irritation and sensitization tests on guinea pigs using technical benomyl (> 95% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 84-69). County Natwest Woodmac (1992) The fungicide market. London, County Natwest Woodmac, Agrochemical Service, pp 49-51. Culik R (1981a) Determination of benomyl/methyl-2-benzimidazole carbamate (MBC) concentrations in maternal blood and in the concepti of rats exposed to benomyl and Benlate by diet. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 916-80). Culik R (1981b) Determination of benomyl/methyl-2-benzimidazole carbamate (MBC) 4-OH MBC and 5-OH MBC concentrations in maternal blood and in the concepti of rats exposed to benomyl by gavage. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 970-80). Culik R & Gibson JR (1974) "Benlate" dominant lethal study in male rats with supplemental statistical report (22-77). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 72-74). Cummings AM, Harris ST, & Rehnberg GL (1990) Effects of methyl benzimidazole carbamate during early pregnancy in the rat. Fundam Appl Toxicol, 15: 528-535. Dashiell OL (1972) Acute oral test (benzimidazole, 2-(3-butylureido)). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 456-72). Davidse LC & Flach W (1977) Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J Cell Biol, 72: 174-193. De Bertoldi M & Griselli M (1980) Different test systems in Aspergillus nidulans for the evaluation of mitotic gene conversion, crossing-over and nondisjunction. Mutat Res, 74: 303-324. De Bertoldi M, Griselli M, Giovannetti M, & Barale R (1980) Mutagenicity of pesticides evaluated by means of gene-conversion in Saccharomyces cerevisiae and in Aspergillus nidulans. Environ Mutagen, 2: 359-370. De Brabander M, Van de Veire R, Aerts F, Geuens S, & Hoebeke J (1976a) A new culture model facilitating rapid quantitative testing of mitotic spindle inhibition in mammalian cells. J Natl Cancer Inst, 56: 357-363. De Brabander M, Van de Veire R, Aerts F, Brogers M, & Janssen PAJ (1976b) The effect of methyl [5(2-thienylcarbonyl)-1H-benzimidazol- 2-yl carbamate (17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer Res, 36: 905-916. Delp CJ (1980) Coping with resistance to plant disease control agents. Plant Dis, 64: 652-657. Dési I (1979) Hygienic-toxicological evaluations of pesticides. Budapest, National Institute of Hygiene (D.Sc. Thesis). Dési I (1983) Neurotoxicological investigation of pesticides in animal experiments. Neurobehav Toxicol Teratol, 5: 503-515. Dési I, Dura G, Szlobodnyik J, & Csuka I (1977) Testing of pesticide toxicity in tissue culture. J Toxicol Environ Health, 2: 1053-1066. Dési I, Nehéz M, Palotas M, Tempfli A, Hogye A, & Vetro G (1990) Experience of health status surveillance of pesticide workers in Hungary. Med Lav, 81(6): 517-523. Donovan SD & Krahn DF (1981) Mutagenic evaluation in Salmonella typhimurium. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 343-81). Douglass MT & Handley JW (1988) The algistatic activity of benomyl technical. Huntingdon, United Kingdom, Huntingdon Research Centre Ltd (Unpublished report No. DPT 171(n)/88371, prepared for E.I. Du Pont de Nemours and Co., Inc.). Draize JH, Woodard G, & Gawery HO (1944) New methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharmacol Exp Ther, 82: 377-390. Drewes CD, Zoran MJ, & Callahan CA (1987) Sublethal neurotoxic effects of the fungicide benomyl on earthworms (Eisenia fetida). Pestic Sci, 19: 197-208. Du Pont (1972) Residue studies - fish: benomyl, MBC, and 2-AB. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Biochemicals Department (Unpublished report). Du Pont (1987) Determination of octanol water partition coefficient for benomyl. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Biochemicals Department (Unpublished report No. B/PC-55-CA). Eastmond DA & Tucker JD (1989) Kinetochlore localization in micronucleated cytokinesis-blocked Chinese hamster ovary cells: A new and rapid assay for identifying aneuploidy-inducing agents. Mutat Res, 224: 517-525. Edwards PJ & Brown SM (1982) Use of grassland plots to study the effect of pesticides on earthworms. Pedobiologia, 24: 145-150 Eickhoff JC, Petersen BJ, & Chaisson CF (1989) Anticipated residues of benomyl in food crops and potential dietary exposure and risk assessment. Washington, DC, Technical Assessment System, Inc. (Unpublished report No. TAS 000-005, prepared for E.I. Du Pont de Nemours and Co., Inc.). Ellis WG, Semple JL, Hoogenboom ER, Kavlock RJ, & Zeman FJ (1987) Benomyl-induced craniocerebral anomalies in fetuses of adequately nourished and protein -deprived rats. Teratog Carcinog Mutagen, 7: 357-375. Ellis WG, De Roos F, Kavlock RJ, & Zeman FJ (1988) Relationship of periventricular overgrowth to hydrocephalus in brains of fetal rats exposed to benomyl. Teratog Carcinog Mutagen, 8: 377-391. Evans EL & Mitchell AD (1980) An evaluation of the effect of benomyl on sister chromatic exchange frequencies in cultured Chinese hamster ovary cells. Menlo Park, California, SRI International (Unpublished report prepared for E.I. Du Pont de Nemours and Co., Inc.). Everhart LP (1979) Benlate dust exposure survey. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Biochemical Department (Unpublished report No. B/TOX 6). Everhart LP & Holt RF (1982) Potential benlate fungicide exposure during mixer/loader operations, crop harvest and home use. J Agric Food Chem, 30: 222-227. FAO/WHO (1985a) Benomyl. In: Pesticide residues in food - Evaluations 1983. Rome, Food and Agriculture Organization of the United Nations, pp 7-46 (FAO Plant Production and Protection Paper 61). FAO/WHO (1985b) Carbendazim. In: Pesticide residues in food - Evaluations 1983. Rome, Food and Agriculture Organization of the United Nations, pp 89-121 (FAO Plant Production and Protection Paper 61). FAO/WHO (1988a) Benomyl. In: Pesticide residues in food - Evaluations 1988. Part I: Residues. Rome, Food and Agriculture Organization of the United Nations, pp 5-15 (FAO Plant Production and Protection Paper 93/1). FAO/WHO (1988b) Carbendazim. In: Pesticide Residues in Food - Evaluations 1988: Part I: Residues. Rome, Food and Agriculture Organization of the United Nations, pp 41-54 (FAO Plant Production and Protection Paper 93/1). Fielding M, Barcelo D, Helweg A, Galassi S, Torstensson L, van Zoonen P, Wolter R, & Angeletti G (1992) Pesticides in ground and drinking water. Brussels, Commission of the European Communities, Directorate-General for Science, Research and Development, Environmental and Waste Recycling, 135 pp (Water Pollution Research Report 27). Fiscor G, Bordas S, & Stewart SJ (1978) Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanine in Salmonella typhimurium in vitro and in rodent host-mediated assays. Mutat Res, 51: 151-164. Fitzpatrick K (1980) Chinese hamster ovary cell assay for mutagenicity. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 438-80). Frame SR & Van Pelt CS (1990) Oncogenicity studies with benomyl and MBC in mice: Supplemental peer review. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 20-82 and supplement No. HLR 70-82). Frank KM (1969) Skin irritation and sensitization tests on guinea pigs using a wettable powder formulation (50% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 85-69). Frank KM (1972) Eye irritation test in rabbits using a wettable powder formulation (50% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 233-72). Fritz SB (1969) Acute oral ALD test in rabbits using a wettable powder formulation (50% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 109-69). Fritz SB & Sherman H (1969) Acute oral ALD test in rats using technical 2-AB (> 95% 2-AB). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 51-69). Gargus JL & Zoetis T (1983a) Primary irritation study in rabbits (Benlate PNW). Vienna, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. HLO 510-83, prepared for E.I. Du Pont de Nemours and Co., Inc.). Gargus JL & Zoetis T (1983b) Eye irritation test in rabbits (EPA pesticide registration - Benlate PNW). Vienna, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. HLO 511-83, prepared for E.I. Du Pont de Nemours and Co., Inc.). Gargus JL & Zoetis T (1983c) Acute skin absorption LD50 test on rabbits (EPA pesticide registration guidelines - Benlate PNW). Vienna, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. HLO 512-83, prepared for E.I. Du Pont de Nemours and Co., Inc.). Gargus JL & Zoetis T (1984) Primary skin irritation and sensitization test on guinea pigs (Benlate PNW). Vienna, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. HLO 67-84, prepared for E.I. Du Pont de Nemours and Co., Inc.). Georgieva V, Vachkova R, Tzoneva M, & Kappas A (1990) Genotoxic activity of benomyl in different test systems. Environ Mol Mutagen, 16: 32-36. Goldenthal EI (1978) Neurotoxicity study in hens [using technical benomyl (< 95% benomyl). Mattawan, Michigan, International Research and Development Corporation (Unpublished report and addendum No. HLO 28-79, prepared for E.I. Du Pont de Nemours and Co., Inc.). Gooch JJ (1978) Fertility of workers potentially exposed to benomyl. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. B/TOX 7). Goodman NC (1975) Intraperitoneal LD50 test in rats. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 847-74). Goulding R (1983) Poisoning on the farm. J Soc Occup Med, 33: 60-65. Guengerich FP (1981) Enzyme induction with Du Pont compounds H11, 202-02 and H10, 962-02. Nashville, Tennessee, Vanderbilt University, School of Medicine (Unpublished report No. HLO 850-81, prepared for E.I. Du Pont de Nemours and Co., Inc.). Gurova AI, Alekseeva NP, Gorlova OE, & Chernyshova RA (1976) [Data for substantiation of the maximum permissible level of m-(trifluoromethyl) phenyl isocyanate and butyl isocyanate in the air of work areas.] Gig Tr Prof Zabol, 3: 53-55 (in Russian). Gustafson DI (1989) Groundwater ubiquity score: A simple method for assessing pesticide leachability. Environ Toxicol Chem, 80: 339-357. Han JCY (1978) Metabolism of 14C-labeled benomyl in the mouse and hamster. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Biochemicals Department (Unpublished report No. B/ME-65). Han JCY (1979) 2-14C-Benomyl (50% WP) rat study - intravenous injection. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Biochemicals Department (Unpublished report No. B/ME-41). Han JCY (1980) Metabolism of 2-14C-benomyl in the lactating nanny goat. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Biochemicals Department (Unpublished report No. B/ME-39). Hardesty PT (1982) Attempts to characterize liver residues from 14C-benomyl dosed goat. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc., Biochemicals Department (Unpublished report No. AMR 71-82). Hardisty JF (1990) Oncogenicity studies with benomyl and MBC in mice. Peer-review of liver neoplasms. Research Triangle Park, North Carolina, Experimental Pathology Laboratories, Inc. (Unpublished report No. 129-012, prepared for E.I. Du Pont de Nemours and Co., Inc.). Hastie AC (1970) "Benlate"-induced instability of Aspergillus diploids. Nature (Lond), 226: 771. Helweg A (1977) Degradation and absorption of carbendazim and 2-aminobenzimidazole in soil. Pestic Sci, 8: 71-78. Helweg A (1985) Side effects caused by pesticide combinations. In: Jensen V, Kjoller A, & Sorensen LH ed. Proceedings of the FEMS Symposium on Microbial Communities in Soil. Amsterdam, Oxford, New York, Elsevier Science Publishers, pp 385-395. Hess RA, Moore BJ, Forrer J, Linder RE, & Abuel-Atta AA (1991) The fungicide benomyl (methyl) 1-(butylcarbomyl)-2-benzimidazole carbamate causes testicular dysfunction by inducing the sloughing of germ cells and occlusion of efferent ductules. Fundam Appl Toxicol, 17: 733-745. Hill EF & Camardese MB (1986) Lethal toxicities of environmental contaminants and pesticides to Coturnix. Washington, DC, US Department of the Interior, Fish and Wildlife Service (Fish and Wildlife Technical Report No. 2). Hofer I, Beck T, & Wallnöfer P (1971) [The influence of the fungicide benomyl on soil microflora.] Z Pflanzenkr Pflanzenschutz, 78: 399-405 (in German). Holden HE, Crider PA, & Wahrenburg MG (1980) Mitotic arrest by benzimidazole analogs in human lymphocyte cultures. Environ Mutat, 2: 67-73. Hood DB (1969) Fifteen exposure dermal tests on rabbits using a wettable powder formulation (50% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 211-69). Hoogenboom ER, Ransdell JF, Ellis WG, Kavlock RJ, & Zeman FJ (1991) Effects on the fetal rat eye of maternal benomyl exposure and protein malnutrition. Curr Eye Res, 10: 601-612. Hornberger CS (1969) Acute dust inhalation test in rats using a wettable powder formulation (50% benomyl) with report on spermatogenesis effects. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 95-69). Hostetler KH (1977) Oral LD50 test (Benlate OD). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 527-77). Hutton DG (1989) Static acute 48-hour EC50 of Benlate fungicide 50 DF to Daphnia magna. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 541-88). Hutton DG, Kasprzak DJ, & Priester TM (1984) Laboratory studies of [2-14C] carbendazim bioconcentration in Bluegill sunfish. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 428-84). ILO (1991) Occupational exposure limits for airborne toxic substances, 3rd ed. Geneva, International Labour Office (Occupational Safety and Health Series No. 37). Ireland CM, Gull K, Gutteridge WE, & Pogson CI (1979) The interaction of benzimidazole carbamates with mammalian microtubule protein. Biochem Pharmacol, 28: 2680-2682. Jessup CD (1979) Acute delayed neurotoxicity study in chickens using technical benomyl (> 95% benomyl). Mattawan, Michigan, International Research and Development Corporation (Unpublished report No. HLO 674-79, prepared for E.I. Du Pont de Nemours and Co., Inc.). Jessup DC & Dean W (1979) Acute delayed neurotoxicity study in chickens using technical benomyl (less than 95% benomyl). Mattawan, Michigan, International Research and Development Corporation (Unpublished report No. HLO 29-79, prepared for E.I. Du Pont de Nemours and Co., Inc). Johnson JD (1988) Determination of the plateau level of bound [phenyl(U)-14C] carbendazim residues in goat liver. Columbus, Ohio, Batelle, Columbus Division (Unpublished report No. AMR 779-87, prepared for E.I. Du Pont de Nemours and Co., Inc.). Kaastra-Howeler LH & Gams W (1973) Preliminary study on the effect of benomyl on the fungal flora in a greenhouse soil. Neth J Plant Pathol, 79: 516-518. Kappas A & Bridges BA (1981) Induction of point mutations by benomyl in DNA-repair-deficient Aspergillus nidulas. Mutat Res, 91: 115-118. Kappas A, Georgopoulos SG, & Hastie AC (1974) On the genetic activity of benzimidazole and thiophanate fungicides on diploid Aspergillus nidulans. Mutat Res, 26: 17-27. Kappas A, Green MHL, Bridges BA, Rogers AM, & Muriel WJ (1976) Benomyl - a novel type of base analogue mutagen? Mutat Res, 40: 379-382. Kavlock RJ, Chernoff N, Gray LE, Gray JA, & Whitehouse D (1982) Teratogenic effects of benomyl in the Wistar rat and CD-1 mouse, with emphasis on the route of administration. Toxicol Appl Pharmacol, 62: 44-54. Kelly DP (1989) Butyl isocyanate industrial hygiene survey. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. BENO/TOX 18). Keogh RG & Whitehead PH (1975) Observations on some effects of pasture spraying with benomyl and carbendazim on earthworm activity and litter removal from pasture. NZ J Exp Agric, 3: 103-104. Kirkland JJ (1973) Method for high-speed liquid chromatographic analysis of benomyl and/or metabolite residue in cow milk, urine, feces and tissues. J Agric Food Chem, 21: 171-177. Kirkland JJ, Holt RF, & Pease HL (1973) Determination of benomyl residues in soils and plant tissues by high speed cation exchange liquid chromatography. J Agric Food Chem, 21: 368-371. Krechniak J & Klosowska B (1986) The fate of 14C-carbendazim in rat. Xenobiotica, 16(9): 809-815. Krupka RM (1974) On the anti-cholinesterase activity of benomyl. Pestic Sci, 5: 211-216. Kuehne G, Heise H, Plottke B, & Puskeiler T (1985) Dermatitis after benlate contact. Z Gesamte Hyg Grenzgeb, 31: 710-711. Lamb MJ & Lilly LJ (1980) An investigation of some genetic toxicological effects of the fungicide benomyl. Toxicology, 17: 83-95. Lee KP (1977) The two-year feeding study in rats with benomyl with supplemental pathology report. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 66-77). Liesivuori J & Jääskeläinen S (1984) Exposure of greenhouse workers to pesticides. Tampore, Finland, National Board of Labor Protection, pp VIII-IX (Research Report No. 46). Linder RE, Rehnberg GL, Strader LF, & Diggs JP (1988) Evaluation of reproductive parameters in adult male Wistar Rats after subchronic exposure. J Toxicol Environ Health, 25: 285-298. Lisi P, Caraffini S, & Assalve D (1986) A test series for pesticide dermatitis. Contact Dermatitis, 15: 266-269. Littlefield NA & Busey WM (1969) Four-hour acute inhalation exposure test in dogs using a wettable powder formulation (50% benomyl). Falls Church, Virginia, Hazelton Laboratories, Inc. (Unpublished report No. HLR 192-69, prepared for E.I. Du Pont de Nemours and Co., Inc.). McCooey KT, Arce GT, Sarrif AM, & Krahn DF (1983a) L5178Y mouse lymphoma cell assay for mutagenicity (benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 86-83). McCooey KT, Arce GT, Sarrif AM, & Krahn DF (1983b) L5178Y mouse lymphoma cell assay for mutagenicity. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. HLR 253-83). Majut JC (1966) Skin irritation and sensitization tests on guinea pigs using technical benomyl (< 95% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 174-06). Marsh BH & Arthur MF (1989) Aerobic metabolism of [phenyl(U)-14C]benomyl in Keyport Silt Loam. Columbus, Ohio, Battelle Memorial Institute (Unpublished report No. AMR 1112-88, prepared for E.I. Du Pont de Nemours and Co., Inc.). Marvin CH, Birndle ID, Hall CD, & Chiba M (1991) Rapid on-line precolumn high performance liquid chromatographic method for the determination of benomyl, carbendazim and aldicarb species in drinking water. J Chromatogr, 555: 147-154. Matsushita T & Aoyama K (1981) Cross reactions between some pesticides and the fungicide benomyl in contact allergy. Ind Health, 19: 77-83. Mayer FL & Ellersieck MR (1986) Manual of acute toxicity: Interpretation and data base for 410 chemicals and 66 species of freshwater animals. Washington, DC, US Department of the Interior, Fish and Wildlife Service, pp 45-56 (Resource Publication No. 160). Mazur AR & Hughes TD (1975) Nitrogen transformation in soil as affected by the fungicides benomyl, dyrene and maneb. Agron J, 67: 755-758. Meade AB (1984) Acute contact LD50 toxicity study in honey bees ( Apis mellifera) with INE 965-212 (MBC). Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMB 84-5). Mebus CA (1990) Reproductive and fertility effects with DPX-1991-529 (benomyl). Multigeneration reproduction study in rats. Newark, Delaware, Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 765-90). Monson KD (1985) Metabolism of [2-14C]benomyl in the lactating dairy cow. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 247-84). Monson KD (1986a) Metabolism of [2-14C]benomyl and [phenyl(U)- 14C]benomyl in laying hens. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 391-85). Monson KD (1986b) Metabolism of [2-14C]carbendazim laying hens. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 264-84). Monson KD (1990) Metabolism of [phenyl(U)-14C]carbendazim in rats. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 1141-88). Monson KD (1991) Release and characterization of bound benomyl and carbendazim metabolites in animal tissues via Raney nickel desulfurization and acid dehydration. J Agric Food Chem, 39: 1808-1811. Nakai M, Hess RA, Moore BJ, Guttroff RF, Strader LF, & Linder RE (in press) Acute and long-term effects of the fungicide carbendazim (methyl 2-benzimidazole carbamate, MBC) on the male reproductive system in the rat. J Androl. Nehéz M, Palotas M, Zimanyi M, Boros P, Mohos G, Vetro G, & Desi I (1992) [Repeated cytogenetic examinations of workers using pesticides in Csongrad County.] Egeszsegtudomany, 36: 40-47 (in Hungarian). Newsome WH & Collins PG (1987) Enzyme-linked immunosorbent assay of benomyl and thiobendazole in some foods. J Assoc Off Anal Chem, 70: 1025-1027. Newsome WH & Shields JB (1981) A radioimmunoassay for benomyl and methyl 2-benzimidazolecarbamate on food. J Agric Food Chem, 29: 220-222. Palawski DU & Knowles CO (1986) Toxicological studies of benomyl and carbendazim in rainbow trout, channel catfish and bluegills. Environ Toxicol Chem, 5: 1039-1046. Parsons DW & Witt JM (1988) Pesticides in groundwater in the United States of America: A report of a 1988 survey of State lead agencies. Corvallis, Oregon, Oregon State University Extension Service. Peeples JL (1974) Microbial activity in benomyl-treated soil. Phytopathology, 64: 857-860. Pilinskaya MA (1983) Investigation of the cytogenetic action of the pesticides captan and benomyl in a culture of human peripheral blood lymphocytes in the absence and presence of a system of metabolic activation. Cytol Genet, 17: 29-33. Powley CR (1985) Aqueous photolysis of [phenyl-14C(U)]benomyl. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 420-85). Priester TM (1984) Hydrolysis of carbendazim [2-14C]. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 265-84). Priester TM (1985) Batch equilibrium (adsorption/deabsorption) and soil thin-layer chromatography studies with [phenyl- 14C(U)]benomyl. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 425-85). Rainaldi G, Flori L, Colella CM, Mariani T, Piras A, Simi S, & Simili M (1989) Analysis by BrUdR-labelling technique of induced aneuploidy in mammalian cells in culture. Mutat Res, 177: 255-260. Reinke RE (1966) Eye irritation test in rabbits using technical benomyl (> 95% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 81-66). Rhodes BC (1987) Greenhouse crop-rotation study with [2-14C]carbendazim. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 495-86). Rickard LB (1983a) Mutagenicity evaluation in Salmonella typhimurium. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 97-83). Rickard LB (1983b) Mutagenicity evaluation in Salmonella typhimurium. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 98-83). Roberts BL & Dorough HW (1984) Relative toxicities of chemicals to the earthworm Eisenia foetida. Environ Toxicol Chem, 3: 67-78. Russell JF (1978a) Mutagenic activity of 2-benzimidazolecarbamic acid, 1-(butylcarbamoyl)-methyl ester in the Salmonella/microsome assay. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 18-78). Russell JF (1978b) Mutagenic activity of 2-benzimidazolecarbamic acid, 1-(butylcarbamoyl)-methyl ester in the Salmonella/microsome assay. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 31-78). Ruzicska P, Peter S, Laczi J, & Czeizel E (1976) Study of the chromosome mutagenicity of Fundazol 50 WP. Egeszegtudomany, 20: 74-83. Ryan DL (1989) Soil column leaching of [phenyl(U)-14C]benomyl in a rice paddy soil. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 1512-89). Sandhu SS, Gudi RD, & Athwal RS (1988) A monochromosomal hybrid cell assay for evaluating the genotoxicity of environmental chemicals. Cell Biol Toxicol, 4: 495-506. Sarver JW (1987) Acute oral toxicity study with IN-T1991 in male and female rats. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. HLR 334-87). Sasaki YFX (1990) Benomyl: Micronucleus test in mice. Tokyo, Institute of Environmental Toxicology, Kodaira Laboratories (Unpublished report No. IET 89-0046, prepared for E.I. Du Pont de Nemours and Co., Inc.). Schafer EW (1972) The acute oral toxicity of 369 pesticidal pharmaceuticals and other chemicals to wild birds. Toxicol Appl Pharmacol, 21: 315-330. Seiler JP (1976) The mutagenicity of benzimidazole and benzimidazole derivatives. VI. Cytogenetic effects of benzimidazole derivatives in the bone marrow of the mouse and the Chinese hamster. Mutat Res, 40: 339-348. Sherman H (1968) Three-month feeding study in dogs using a wettable powder formulation (50% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 269-68). Sherman H (1969a) Acute oral LD50 test in rats using technical benomyl (> 95% benomyl) and a wettable powder formulation (50% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 17-69). Sherman H (1969b) Acute oral ALD test in a dog using technical benomyl (> 95% benomyl). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 168-69). Sherman H (1969c) Long term feeding study in rats with 1-butylcarbamoyl-2-benzimidazolecarbamic acid, methyl ester (INT-1991). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 232-69). Sherman H (1970) Long-term feeding study in dogs with 1-butycarbamoyl-2-benzimidazolecarbamic acid, methyl ester (INT-1991). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 48-70). Sherman H (1972) Long-term feeding studies in rats and dogs with 2-benzimadazolecarbamic acid, methyl ester (INE-965) (50% and 70% MBC wettable powder formulations): Parts I and II. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 195-72). Sherman H & Krauss WC (1966) Acute oral test [benomyl]. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 100-66). Sherman H, Barnes JR, & Krauss WC (1967) Ninety-day feeding study with 1-butylcarbamoyl-2-benzimidazolecarbamic acid, methyl ester (INT-1991). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell, Laboratory (Unpublished report No. HLR 11-67). Sherman H, Culik R, & Jackson RA (1975) Reproduction, teratogenic and mutagenic studies with benomyl. Toxicol Appl Pharmacol, 32: 305-315. Shirasu Y, Moriya M, & Kato K (1978) Mutagenicity testing on fungicide 1991 in microbial systems. Tokyo, Institute of Environmental Toxicology, Kodaira Laboratories (Unpublished report prepared for E.I. Du Pont de Nemours and Co., Inc.). Shukla Y, Antony M, & Mehrota NK (1989) Studies on gamma-glutamyl transpeptidase in rodents exposed to benomyl. Bull Environ Contam Toxicol, 42: 301-306. Siebert D, Zimmermann FK, & Lemperle E (1970) Genetic effects of fungicides. Mutat Res, 10: 533-543. Siegel MR (1975) Benomyl-soil microbial interactions. Phytopathology, 65: 219-220. Snee DA (1969) Acute oral ALD test in rats and ten-dose subacute oral test in rats using technical 5-HBC (> 95% 5-HBC) (with pathology on the acute oral ALD test described in HLR 43-69). Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. 134-69). Stahl RG Jr (1990) In vivo evaluation of INT-1991-259 for chromosome aberrations in mouse bone marrow. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 401-90). Staples RE (1980) Teratogenicity study in the rat after administration by gavage of technical benomyl (> 95% benomyl): Parts I, II and III. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 649-80). Staples RE (1982) Teratogenicity study in the rat using technical benomyl (>95% benomyl) administered by gavage and supplement with individual animal data. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 587-82). Staub T & Sozzi D (1984) Fungicide resistance: A continuing challenge. Plant Dis, 68: 1026-1031. Stevenson IE (1985) Metabolism of [phenyl(U)-14C]benomyl in peaches. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 443-85). Stringer A & Lyons C (1974) The effect of benomyl and thiophanate-methyl on earthworm populations in apple orchards. Pestic Sci, 5: 189-196. Stringer A & Wright MA (1973) The effect of benomyl and some related compounds on Lumbricus terrestris and other earthworms. Pestic Sci, 4: 165-170. Tolle DA (1988) Metabolism of [phenyl(U)-14C] benomyl in sugar beets. West Jefferson, Ohio, Battelle Columbus DW (Unpublished report No. AMR 620-86, prepared for E.I. Du Pont de Nemours and Co., Inc.). Tomlin AD & Gore FL (1974) Effects of six insecticides and a fungicide on the numbers and biomass of earthworms in pasture. Bull Environ Contam Toxicol, 12: 487-492. Tomlin AD, Tolman JH, & Thorn GD (1980/1981) Suppression of earthworm ( Lumbricus terrestris) populations around an airport by soil application of the fungicide benomyl. Prot Ecol, 2: 319-323. Tong C (1981) Hepatocyte primary culture/DNA repair assay on compound 10, 962-02 (benomyl) using mouse hepatocytes in culture. Valhalla, New York, Naylor Dana Institute (Unpublished report No. HLO 741-81, prepared for E.I. Du Pont de Nemours and Co., Inc.). Torstensson L & Wessen B (1984) Interactions between the fungicide benomyl and soil microorganisms. Soil Biol Biochem, 16: 445-452. Turney RT (1979) Rat inhalation study - Benlate. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 116-79). Van Faassen HG (1974) Effect of the fungicide benomyl on some metabolic processes and on numbers of bacteria and actinomycetes in the soil. Soil Biol Biochem, 6: 131-133. Van Gestel CAM (1992) Validation of earthworm toxicity tests by comparison with field studies: A review of benomyl, carbendazim, carbofuran, and carbaryl. Ecotoxicol Environ Saf, 23: 221-236. Van Gestel CAM, Dirven-van Breeman EM, Baerselman R, Emans HJB, Janssen JAM, Postuma R, & van Vliet PJM (1992) Comparison of sublethal and lethal criteria for nine different chemicals in standardized toxicity tests using the earthworm Eisenia andrei. Ecotoxicol Environ Saf, 23: 206-220. Van Joost TH, Naafs B, & van Ketel WG (1983) Sensitization to benomyl and related pesticides. Contact Dermatitis, 9: 153-154. Vick DA & Brock WJ (1987) Primary dermal irritation study with benlate 50 DF fungicide in rabbits. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 300-87). Wainwright M & Pugh GJF (1974) The effect of fungicides on certain chemical and microbial properties of soils. Soil Biol Biochem, 6: 263-267. Ward RS & Scott RC (1992) Benomyl: in vitro absorption of a 500 g kg-1 WP formulation through human epidermis. Fernhurst, Naslemere, Surrey, Imperial Chemical Industries (ICI) (Unpublished report No. CTL/P/3659). Warheit DB, Kelly DP, Carakostas MC, & Singer AW (1989) A 90-day inhalation toxicity study with benomyl in rats. Fundam Appl Toxicol, 12: 333-345. Weeks RE & Hedrick HG (1975) Influence of a systemic fungicide on oxygen uptake by soil microorganisms. Soil Sci, 119: 280-284. Wheeler J (1985) Hydrolysis of [phenyl-14C(U)]benomyl. Wilmington, Delaware, E.I. Du Pont de Nemours and Co., Inc. (Unpublished report No. AMR 419-85). WHO (1993) Environmental Health Criteria 149: Carbendazim. Geneva, World Health Organization. Wiechman BE (1982) Long term feeding study with methyl l-(butylcarbamoyl)-2-benzimidazolecarbamate in mice (INT-1991; >95% benomyl): Parts I, II and III. Newark, Delaware, E.I. Du Pont de Nemours and Co., Inc., Haskell Laboratory (Unpublished report No. HLR 20-82). Yoshida K & Nishiuchi Y (1972) Toxicity of pesticides to some water organisms. Bull Agric Chem Insp Stn, 12: 122-128. Zbozinek JV (1984) Environmental transformations of DPA, SOPP, benomyl and TBZ. Residue Rev, 92: 113-155. Zelesco PA, Barbieri I, & Graves JAM (1990) Use of a cell hybrid test system to demonstrate that benomyl induces aneuploidy and polyploidy. Mutat Res, 242: 329-335. Zeman FJ, Hoogenboom ER, Kavlock RJ, & Semple JL (1986) Effects on the fetus of maternal benomyl exposure in the protein-deprived rat. J Toxicol Environ Health, 17: 405-417. Zoran MJ, Heppner TJ, & Drewes CD (1986) Teratogenic effects of the fungicide benomyl on posterior segmental regeneration in the earthworm, Eisenia fetida. Pestic Sci, 17: 641-652. Zweig G, Gao R, & Popendorf W (1983) Simultaneous dermal exposure to captan and benomyl by strawberry harvesters. J Agric Food Chem, 31: 1109-1113. RESUME ET CONCLUSIONS 1. Résumé 1.1 Identité, propriétés physiques et chimiques et méthodes d'analyse Le bénomyl, un solide cristallin de couleur ambrée, est un fongicide endothérapique qui appartient à la famille du benzimidazole. Il se décompose juste au-dessus de son point de fusion de 140 °C et sa tension de vapeur est < 5 x 10-6 Pa (< 3,7 x 10-8 mmHg) à 25 °C. Le bénomyl est pratiquement insoluble dans l'eau à pH 5 et à 25 °C, sa solubilité étant de 3,6 mg/litre. Il est stable dans les conditions normales de stockage mais se décompose en carbendazime dans l'eau. L'analyse des résidus de même que celle des prélèvements effectués dans l'environnement comporte une extraction au moyen d'un solvant organique, une purification de l'extrait par partage liquide-liquide et la transformation du résidu obtenu en carbendazime. Le dosage de ces résidus peut s'effectuer par chromatographie en phase liquide à haute performance ou par titrage immunologique. 1.2 Sources d'exposition humaine et environnementale En 1988, on estimait à environ 1700 tonnes la quantité de bénomyl utilisée dans le monde. Il s'agit d'un fongicide très largement utilisé, homologué dans 50 pays pour le traitement de plus de 70 cultures. Le bénomyl est présenté sous forme de poudre mouillable. 1.3 Transport, distribution et transformation dans l'environnement Le bénomyl se transforme rapidement en carbendazime dans l'environnement avec une demi-vie respective de 2 et 19 heures dans l'eau et le sol. On peut donc utiliser aussi bien les résultats des études sur le bénomyl que sur le carbendazime pour l'évaluation des effets sur l'environnement. Dans l'environnement, le carbendazime se décompose avec une demi-vie de 6 à 12 mois sur le sol nu, de 3 à 6 mois sur le gazon et de 2 à 25 mois dans l'eau en aérobiose et en anaérobiose, respectivement. Le carbendazime est principalement décomposé par les microorganismes. Le 2-aminobenzimidazole (2-AB) en est l'un des principaux produits de dégradation et il est à son tour décomposé par les microorganismes. Lors de la décomposition de bénomyl marqué au 14C sur le noyau phényle, on a constaté que 9% seulement du carbone-14 étaient éliminés sous forme de CO2 en une année d'incubation. Le carbone-14 restant était principalement récupéré sous forme de carbendazime et de résidus liés. L'étude de la destinée d'un éventuel produit de dégradation (1,2-diaminobenzène) pourrait peut-être permettre de mieux définir la voie de dégradation des fongicides benzymidazoliques dans l'environnement. Des études effectuées sur le terrain ou sur colonnes ont montré que le carbendazime restait dans les couches superficielles du sol. On n'a pas mesuré l'adsorption du carbendazime dans le sol, mais on pense qu'elle doit être aussi forte que dans le cas du bénomyl, avec des valeurs de Koc allant de 1000 à 3600. Les valeurs de log Kow sont respectivement de 1,36 et de 1,49 pour le bénomyl et le carbendazime. Un modèle de criblage basé sur les données d'adsorption et de persistance n'a pas révélé de risque de lessivage. Cette observation est corroborée par des analyses d'eau de puits effectuées aux Etats-Unis, analyses qui n'ont pas permis de déceler ce composé dans l'un quelconque des 495 puits étudiés ni le carbendazime dans 212 autres (la limite de détection n'a pas été précisée). On estime que le bénomyl et le carbendazime entraînés par ruissellement correspondent uniquement à la fraction adsorbée aux particules de sol; d'ailleurs ces composés sont sans doute fortement adsorbés aux sédiments présents dans l'environnement aquatique. En solution, dans les végétaux et le sol, le bénomyl subit une décomposition en carbendazime (méthyl-1H-benzimidazole-2-carbamate) en 2-AB, STB (3-butyl-1,3,5-triazino[1,2a]-benzimidazole- 2,4(1H,3H)dione) ainsi qu'en BBU (1-(2-benzimidazolyl)-3-n- butylurée). La photolyse du bénomyl est pratiquement inexistante. Chez l'animal, le bénomyl est métabolisé en carbendazime ainsi qu'en d'autres métabolites polaires qui sont rapidement excrétés. On n'a pas observé d'accumulation de bénomyl ni de carbendazime dans aucun système biologique. 1.4 Concentrations dans l'environnement et exposition humaine Il ne semble pas qu'il existe de données résultant d'une surveillance du bénomyl dans l'environnement. Toutefois on peut récapituler ainsi les données tirées d'études portant sur la destinée de ce produit dans l'environnement. Comme le bénomyl et le carbendazime restent stables pendant des semaines sur les végétaux, ils peuvent être ingérés par des organismes qui se nourrissent de feuilles mortes. Des résidus de carbendazime peuvent subsister jusqu'à 3 ans dans le sol et les sédiments. Toutefois, la forte adsorption du carbendazime aux particules de sol et de sédiments réduit l'exposition des organismes terrestres et aquatiques. Ce sont les résidus de bénomyl et de carbendazime présents sur les cultures vivrières qui constituent la principale source d'exposition de la population humaine dans son ensemble. L'analyse de l'exposition par voie alimentaire qui a été effectuée aux Etats-Unis (bénomyl et carbendazime associés) et aux Pays-Bas (carbendazime seul) a montré que la quantité moyenne ingérée était vraisemblablement de l'ordre d'un dixième de la dose journalière acceptable (DJA) qui est, pour le bénomyl de 0,02 mg/kg de poids corporel, et pour le carbendazime de 0,01 mg/kg de poids corporel. L'exposition professionnelle au cours de la production est inférieure à la valeur-seuil. Les ouvriers agricoles qui préparent les mélanges, effectuent le remplissage, ou retournent dans les champs traités par du bénomyl, courent un risque d'exposition cutanée de quelques mg de bénomyl à l'heure. Le port de dispositifs de protection permettrait de réduire encore cette exposition. En outre, étant donné que l'absorption percutanée est vraisemblablement faible, il est très peu probable que le bénomyl puisse avoir des effets toxiques généraux sur les populations humaines en étant absorbé par cette voie. 1.5 Cinétique et métabolisme Après exposition par voie orale ou respiratoire, le bénomyl est rapidement absorbé par l'organisme animal mais cette absorption est bien moindre après exposition par voie cutanée. Une fois absorbé, le bénomyl est rapidement métabolisé, puis excrété dans les urines et les matières fécales. Après avoir administré du bénomyl marqué au carbone-14 à des rats, on a retrouvé dans le sang, et en petites quantités dans les testicules, les reins et le foie, deux de ses métabolites, le carbendazime et le carbamate de méthyl-5-hydroxy-1H- benzimidazole-2-yle (5-HBC). La distribution tissulaire des composés n'était pas révélatrice d'une bioconcentration. Dans l'urine, le principal métabolite était le 5-HBC à côté d'un peu de carbendazime. Dans les 72 heures suivant l'administration, 98% de la quantité administrée avaient été excrétés. Chez des vaches recevant pendant 5 jours des capsules contenant du bénomyl radiomarqué à raison de 50 mg/kg de nourriture, les concentrations équivalentes de bénomyl retrouvées dans les divers organes étaient de 4 mg/kg dans le foie, et de 0,25 mg/kg dans les reins; dans les autres tissus et les graisses, les valeurs n'étaient pas significatives. Pendant la période d'administration, 65% du composé radiomarqué ont été excrétés dans les urines, 21% dans les matières fécales et 0,4% dans le lait. Le principal métabolite présent dans le lait était le 5-HBC. Chez d'autres animaux, on a constaté un métabolisme et des modalités d'élimination similaires. Le bénomyl n'inhibe pas l'acétylcholinestérase in vitro. On a montré qu'il induisait l'époxyhydrolase hépatique, la gamma- glutamyle transpeptidase ainsi que la glutation-S-transférase chez la souris et le rat in vivo. 1.6 Effets sur les mammifères de laboratoire et sur les systèmes d'épreuve in vitro 1.6.1 Exposition unique Le bénomyl présente une faible toxicité aiguë, la DL50 par voie orale chez le rat étant > 10 000 mg/kg, et la CL50 à 4 h par voie respiratoire étant > 4 mg/litre. Des chiens exposés par voie respiratoire pendant 4 heures à la dose de 1,65 mg/litre et examinés 28 jours après l'exposition, présentaient une diminution du poids du foie. Une dose unique administrée à des rats par gavage a entraîné des effets sur la reproduction 70 jours après l'exposition (voir Section 1.6.5). 1.6.2 Exposition de brève durée Administré par gavage pendant de brèves périodes allant jusqu'à 90 jours, en mélange à la nourriture ou en application cutanée, le bénomyl a légèrement augmenté le poids du foie chez le rat (à la dose quotidienne de 125 mg/kg, en mélange à la nourriture) et a produit des effets sur les organes reproducteurs mâles chez le rat: diminution du poids des testicules et de l'épididyme, réduction de la spermatogenèse (dose quotidienne: 45 mg/kg, par gavage, dose sans effet observable = 15 mg/kg), chez le lapin (dose quotidienne: 1000 mg/kg, par voie orale; 500 mg/kg, en appli cation cutanée) et chez le chien beagle (62,5 mg/kg, dose sans effet observable = 18,4 mg/kg par jour, en mélange à la nourriture). Chez les rats exposés par la voie respiratoire à du bénomyl à des concentrations allant jusqu'à 200 mg/m3 pendant 90 jours, on n'a pas observé d'effets sur le foie ni les testicules. 1.6.3 Irritation et sensibilisation au niveau de la peau et des yeux L'application de bénomyl sur l'épiderme de lapins ou de cobayes n'a produit que peu ou pas d'irritation et une sensibilisation modérée de la peau. Chez le rat, l'instillation oculaire a produit une irritation légère et temporaire de la conjonctive. 1.6.4 Exposition de longue durée A l'issue d'une étude prolongée d'alimentation chez des rats, on n'a pas pu mettre en évidence d'effets imputables à ce composé à des doses allant jusqu'à 2500 mg/kg de nourriture (soit 125 mg/kg de poids corporel par jour). Cette étude n'a pas été jugée suffi sante pour permettre une évaluation des effets sur la reproduction. Chez la souris CD-1, le poids du foie était en augmentation à partir de la dose de 1500 mg/kg de nourriture. A la dose de 5000 mg/kg de nourriture, on notait chez les souris mâles une réduction du poids absolu des testicules et une atrophie du thymus. 1.6.5 Reproduction, embryotoxicité et tératogénicité Le bénomyl détermine une diminution du poids des testicules et de l'épidydime, avec réduction des réserves de spermatozoïdes au niveau de la queue de l'épidydime, une diminution de la production des spermatozoïdes et une réduction de la fécondité des mâles. A plus fortes doses, il y a diminution de la spermatogénèse qui est perturbée à tous ses stades. Le bénomyl n'affecte pas le comportement copulatoire, les vésicules séminales, la mobilité des spermatozoïdes ni les hormones sexuelles correspondantes. La concentration de bénomyl la plus faible qui produise un effet statistiquement significatif sur la spermatogénèse du rat mâle a été chiffrée à 45 mg/kg par jour. La dose sans effet observable pour ces effets est de 15 mg/kg par jour. Une seule dose de bénomyl (100 mg/kg ou davantage) administrée par gavage à des rats a produit, 70 jours après l'exposition, des effets consistant notamment en une réduction du poids des testicules et une atrophie des tubes séminifères. Administré par gavage à des rats ChD-CD et Wistar du 7e au 16e jour de la gestation, le bénomyl s'est révélé tératogène pour les 2 souches à 62,5 mg/kg, mais pas à 30 mg/kg pour les ChR-CD, ni à 31,2 mg/kg pour les Wistar. Administré par gavage à des rats Sprague-Dawley du 7e au 21e jour de gestation, le bénomyl s'est révélé tératogène à 31,2 mg/kg. Les effets observés étaient une microphthalmie, une hydrocéphalie et une encéphalocèle. Le développement post-natal des rats était également perturbé aux doses supérieures à 15,6 mg/kg. Chez la souris, l'administration par gavage à des concentrations supérieures ou égales à 50 mg/kg a provoqué l'apparition de côtes surnuméraires et autres anomalies du squelette et des viscères. On n'a pas établi, chez la souris, la valeur de la dose sans effet observable car les doses administrées étaient toutes supérieures à 50 mg/kg. A part une augmentation marginale de la fréquence des côtes surnuméraires chez le lapin, on n'a pas observé, chez cet animal, d'effets tératogènes à des doses atteignant 500 mg/kg de nourriture. 1.6.6 Mutagénicité et autres points d'aboutissement des effets toxiques L'étude des cellules somatiques et germinales montre que le bénomyl n'entraîne pas de mutations géniques ni d'anomalies structurales chromosomiques et qu'il n'y a pas non plus d'interaction directe avec l'ADN (qui provoquerait des lésions de l'ADN et leur réparation). Ces faits ont été mis en évidence à la fois sur des cellules mammaliennes et non mammaliennes. Cependant le bénomyl provoque des aberrations dans le nombre des chromosomes (aneuploïdie et/ou polyploïdie) dans les systèmes d'épreuve (tant in vitro qu' in vivo). 1.6.7 Cancérogénicité Chez les souris CD-1 et Swiss axéniques, qui présentent un taux important de tumeurs spontanées du foie, on a observé ce type de tumeurs après administration de bénomyl ou de carbendazime. En revanche, le carbendazime ne s'est pas révélé cancérogène chez les souris NMRKf, qui n'ont qu'un faible taux de tumeurs hépatiques spontanées. La première étude de cancérogénicité portant sur des souris CD-1 a fait ressortir l'existence d'une augmentation liée à la dose et statistiquement significative des néoplasmes hépatocellulaires chez les femelles et l'on a également observé chez les mâles, aux doses moyennes (1500 mg/kg), une réaction statistiquement significative qui ne s'observait plus aux doses élevées en raison d'un fort taux de mortalité. Une deuxième étude de cancérogénicité portant sur le carbendazime a été effectuée chez une souche génétiquement apparentée de souris Swiss axéniques et exogames à des doses de 0, 150, 300 et 1000 mg/kg (la dernière dose étant portée à 5000 mg/kg au cours de l'étude); elle a révélé un accroissement dans l'incidence de l'ensemble des adénomes et carcinomes hépatocellulaires. Une troisième étude effectuée cette fois sur des souris NMRKf à des doses 0, 50, 150, 300 et 1000 mg/kg (portées ensuite à 5000 mg/kg) n'a pas fait ressortir d'effets cancérogènes. Les études de cancérogénicité portant sur le bénomyl et le carbendazime ont donné des résultats négatifs chez le rat. 1.6.8 Mécanisme de la toxicité - mode d'action On pense que les effets biologiques du bénomyl et du carbendazime résultent de leur interaction avec les microtubules cellulaires. Ces structures interviennent dans des fonctions aussi importantes que la division cellulaire, qui est inhibée par ces deux substances. La toxicité du bénomyl et du carbendazime pour les mammifères est donc liée à une perturbation des fonctions du système microtubulaire. Comme les autres dérivés du benzimidazole, le bénomyl et le carbendazime sont plus ou moins toxiques selon les espèces. Cette sélectivité toxicologique s'explique au moins en partie par le fait que le bénomyl et le carbendazime ne se lient pas de la même manière aux tubulines des espèces visées et des espèces non visées. 1.7 Effets sur l'homme Le bénomyl provoque des dermatites de contact ainsi qu'une sensibilisation cutanée. On n'a pas fait état d'autres effets. 1.8 Effets sur les autres êtres vivant au laboratoire et dans leur milieu naturel Le bénomyl n'a guère d'effet sur l'activité microbienne du sol aux doses d'emploi recommandées. On a cependant signalé l'existence d'effets nocifs vis-à-vis de certains groupes de champignons. On a calculé que la CE50 à 72 heures, fondée la croissance totale, pour les algues bleu-vert du genre Selenastrum capricornutum, était égale à 2,0 mg/litre; la concentration sans effet observable était de 0,5 mg/litre. La toxicité du bénomyl pour les invertébrés aquatiques et les poissons varie dans de larges proportions, les valeurs de la CL50 à 96 heures allant de 0,006 mg/litre pour des poissons-chats du genre Ictalurus (alevins porteurs de leur sac vitellin) à plus de 100 mg/litre pour les écrevisses. Le bénomyl s'est révélé toxique pour les lombrics lors d'expériences de laboratoire qui reproduisaient les conditions réelles d'exposition résultant de l'utilisation recommandée sur le terrain. Il est peu toxique pour les oiseaux et son produit de dégradation, le carbendazime, est "relativement non toxique" pour les abeilles. 2. Conclusions Le bénomyl provoque une sensibilisation cutanée chez l'homme. Le bénomyl et le carbendazime ne font courir à l'homme qu'un très faible risque d'intoxication aiguë. Etant donné les conditions actuelles d'exposition et le faible taux d'absorption percutanée de ces deux composés, il est improbable qu'ils entraînent des effets toxiques généraux dans la population ou chez les personnes exposées de par leur profession. Ces conclusions sont tirées de données relatives à l'animal et, dans une moindre mesure, à l'homme; elles reposent sur la connaissance du mode d'action du carbendazime et du bénomyl, tant chez les espèces visées que chez les espèces non visées. Grâce à une meilleure connaissance du mécanisme de la toxicité du bénomyl et du carbendazime pour les mammifères, on pourra peut-être mieux définir les doses sans effet observable. Des études portant sur la liaison de ces composés aux tubulines des cellules cibles (tissus testiculaires et embryonnaires) faciliteront sans doute les comparaisons interspécifiques. Le carbendazime est fortement adsorbé aux matières organiques du sol et il y persiste pendant des périodes pouvant atteindre 3 ans. Il persiste également à la surface des feuilles et se retrouve par conséquent dans les feuilles mortes. On a montré que les lombrics pouvaient souffrir (dans leur population et dans leur reproduction) de ces composés aux doses d'emploi recommandées. On ne possède aucun renseignement sur les autres arthropodes qui vivent dans le sol ou les débris organiques et qui pourraient être exposés de la même manière. Il est improbable que la forte toxicité vis-à-vis des organismes aquatiques révélée par les épreuves de laboratoire s'observe également dans le milieu naturel, du fait de la faible biodisponi-bilité des résidus de carbendazime liés aux sédiments. Toutefois on ne possède aucune donnée sur les espèces vivant sur les sédiments et qui seraient donc les plus exposées. RESUMEN Y CONCLUSIONES 1. Resumen 1.1 Identidad, propiedades físicas y químicas y métodos analíticos El benomilo es un sólido cristalino de color tostado y acción fungicida sistémica que pertenece a la familia del bencimidazol. Se descompone a una temperatura apenas superior a 140 °C, correspondiente a su punto de fusión, y su presión de vapor a 25 °C es < 5 x 10-6 Pa (< 3,7 x 10-8 mmHg). Prácticamente es insoluble en agua a pH 5 y 25 °C, siendo su solubilidad 3,6 mg/litro. Es un compuesto estable en condiciones de almacenamiento normales, pero en agua se descompone y forma carbendazima. Los análisis de las muestras procedentes de residuos y del medio ambiente se realizan mediante extracción con un disolvente orgánico, purificación del extracto obtenido utilizando un procedimiento de reparto líquido-líquido y conversión del residuo en carbendazima. La valoración de los residuos se puede realizar mediante cromatografía líquida de alto rendimiento o inmunoensayo. 1.2 Fuentes de exposición humana y ambiental En 1988, el uso mundial estimado de benomilo fue unas 1700 toneladas. Es un fungicida muy utilizado, que se encuentra registrado en 50 países en los que se permite su uso en más de 70 cultivos. El benomilo está formulado como polvo humectable. 1.3 Transporte, distribución y transformación en el medio ambiente En el medio ambiente, el benomilo se transforma rápidamente en carbendazima con una semivida de 2 y 19 h en el agua y el suelo respectivamente. Por consiguiente, para la evaluación de los efectos sobre el medio ambiente son importantes los datos obtenidos de los estudios realizados con ambos compuestos. La carbendazima se descompone en el medio ambiente con una semivida de 6 a 12 meses en el suelo desnudo, de 3 a 6 meses en el césped y de 2 y 25 meses en el agua en condiciones aerobias y anaerobias respectivamente. La carbendazima se descompone principalmente por acción de los microorganismos. Un producto importante de su degradación es el 2-aminobencimidazol (2-AB), que luego se descompone de nuevo por la actividad microbiana. En la descomposición del benomilo marcado con un grupo fenilo con 14C, sólo el 9% del 14C formó CO2 durante un año de incubación. El resto del 14C se recuperó sobre todo como carbendazima y en productos unidos a residuos. El destino de un posible producto de degradación (1,2-diaminobenceno) puede aclarar ulteriormente la vía de degradación de los fungicidas bencimidazólicos en el medio ambiente. En estudios de campo y de columna se ha puesto de manifiesto que la carbendazima queda retenida en la capa superficial del suelo. Aunque no se dispone de datos sobre su adsorción en el suelo, se considera que ésta puede ser tan intensa como la del benomilo, con valores de Koc que oscilan entre 1000 y 3600. Los valores del log Kow para el benomilo y la carbendazima son respectivamente 1,36 y 1,49. En la evaluación realizada en un modelo de selección, basado en datos de adsorción y persistencia, se puso de manifiesto que no había riesgo de lixiviación. En los Estados Unidos se han efectuado análisis de agua de pozos que confirman esto, puesto que no se encontraron trazas de benomilo en ninguno de los 495 pozos muestreados ni se detectó carbendazima en ninguno de los 212 (no se dispone del límite de detección). Es de suponer que la escorrentía superficial de ambos compuestos se deba solamente al fungicida adsorbido en las partículas del suelo y que en el medio acuoso estén fuertemente adsorbidos en los sedimentos. En solución, en las plantas y en el suelo, el benomilo se degrada a carbendazima (1H-bencimidazol-2-carbamato de metilo) y a 2-AB, STB (3-butil-1,3,5-triazino[1,2a]-bencimidazol-2,4(1H,3H) diona) y BBU (1-(2-bencimidazolil)-3-n-butilurea). La fotolisis del benomilo es escasa o nula. En los animales, el benomilo se descompone formando carbendazima y otros metabolitos polares, que se excretan rápidamente. No se ha observado que el benomilo o la carbendazima se acumulen en ningún sistema biológico. 1.4 Niveles medioambientales y exposición humana No parece que se disponga de datos de vigilancia ambiental para el benomilo. Sin embargo, los estudios realizados sobre su destino en el medio ambiente pueden resumirse como sigue. Puesto que el benomilo y la carbendazima se mantienen estables en las plantas durante varias semanas, pueden pasar a los organismos que se alimentan de las hojas caídas. El suelo y los sedimentos pueden conservar residuos de carbendazima hasta tres años. Sin embargo, la fuerte adsorción de este compuesto en las partículas del suelo y en los sedimentos reduce la exposición de los organismos terrestres y acuáticos. La principal fuente de exposición para la población humana general se debe a los residuos de benomilo y carbendazima en los cultivos alimentarios. En análisis de la exposición a través de los sedimentos realizados en los Estados Unidos (benomilo y carbendazima combinados) y en los Países Bajos (con carbendazima) se obtuvo una ingesta media prevista de alrededor del 10 por ciento de la ingesta diaria admisible (IDA) recomendada, de 0,02 mg/kg de peso corporal para el benomilo y de 0,01 mg/kg de peso corporal para la carbendazima. La exposición profesional durante el proceso de fabricación es inferior al valor umbral límite. Se considera que los trabajadores agrícolas que se ocupan de mezclar y cargar los plaguicidas o que entran en campos tratados con benomilo sufren exposición cutánea a unos mg de benomilo por hora. Este forma de exposición se podría reducir con algún tipo de protección. Por otra parte, puesto que la absorción cutánea prevista es baja, la probabilidad de que el benomilo tenga efectos tóxicos sistémicos sobre la población humana a través de esta vía es muy escasa. 1.5 Cinética y metabolismo En experimentos con animales se ha puesto de manifiesto que éstos absorben fácilmente el benomilo tras la exposición oral y respiratoria, pero mucho menos después de una exposición cutánea. El benomilo absorbido se metaboliza rápidamente y se excreta en la orina y las heces. En ratas alimentadas con benomilo marcado con 14C, se encontraron en la sangre, y en pequeña cantidad en los testículos, los riñones y el hígado, los metabolitos carbendazima y (5-hidroxi-1H-bencimidazol-2-il)-carbamato de metilo (5-HBC). La distribución en los tejidos demostraba la ausencia de bioconcentración. El metabolito principal en la orina era el 5-HBC, acompañado de una pequeña cantidad de carbendazima. A las 72 h de la administración ya se había excretado el 98% de la cantidad suministrada. En vacas tratadas durante 5 días con cápsulas con benomilo marcado, en dosis equivalentes a una alimentación de 50 mg/kg, se detectó en el hígado una concentración de esta sustancia equivalente a 4 mg/kg, en los riñones 0,25 mg/kg y en el tejido adiposo o en otros niveles no significativos. Cuando se administró con los alimentos, el 65% del compuesto marcado se excretó en la orina, el 21% en las heces y el 0,4% en la leche. El principal metabolito en la leche fue el 5-HBC. El metabolismo y los sistemas de eliminación fueron semejante en otros animales. El benomilo no inhibe la acetilcolinesterasa in vitro. Se ha demostrado en estudios in vivo en ratas y ratones que induce la epoxihidrolasa hepática, la gamma-glutamil transpeptidasa y la glutatión-S-transferasa. 1.6 Efectos en los mamíferos de laboratorio y en sistemas de prueba in vitro 1.6.1 Exposición única El benomilo tiene una toxicidad aguda baja, con una DL50 por vía oral en ratas de > 10 000 mg/kg y una CL50 por inhalación durante 4 h de > 4 mg/litro. La carbendazima, al igual que la sustancia de la que se deriva, tiene una DL50 en ratas de > 10 000 mg/kg. Los perros, expuestos por inhalación a 1,65 mg/litro durante 4 h y examinados 28 días después, mostraron una dismi nución del peso del hígado. La administración por sonda de una sola dosis de benomilo a ratas mostró efectos en la reproducción a los 70 días de la exposición (véase el apartado 1.6.5). 1.6.2 Exposición breve La administración de benomilo mediante sonda, en los alimentos o por exposición cutánea durante un período máximo de 90 días en las ratas aumentó ligeramente el peso del hígado (125 mg/kg al día, con los alimentos) y tuvo efectos sobre los órganos reproductores masculinos (disminución del peso de los testículos y los epidídimos, y reducción de la espermatogénesis) en las ratas (45 mg/kg al día, administrado por sonda; nivel sin efecto observado (NOEL) = 15mg/kg), los conejos (1000 mg/kg al día, por vía oral; 500 mg/kg de peso corporal al día, por vía cutánea) y los perros (62,5 mg/kg; NOEL = 18,4 mg/kg al día, con los alimentos). No se observaron efectos hepáticos ni testiculares en la ratas expuestas por inhalación a concentraciones de benomilo de hasta 200 mg/m3 durante 90 días. 1.6.3 Irritación y sensibilización cutánea y ocular La aplicación cutánea a conejos y cobayos ocasionó una irritación leve o nula y una sensibilización moderada de la piel. Su aplicación ocular en ratas produjo de manera transitoria una irritación conjuntival ligera. 1.6.4 Exposición prolongada En un estudio de alimentación de larga duración en ratas, con dosis de hasta 2500 mg/kg de alimentos (125 mg/kg de peso corporal al día) no se puso de manifiesto ningún efecto relacionado con el compuesto. Este estudio no se consideró adecuado para evaluar los efectos sobre la reproducción. En el ratón CD-1 se observó que, con dosis de 1500 mg/kg de alimento o superiores, se producía un aumento de peso del hígado. En los ratones macho, las dosis de hasta 5000 mg/kg de alimento provocaron una disminución en términos absolutos del peso de los testículos y la atrofia del timo. 1.6.5 Reproducción, embriotoxicidad y teratogenicidad El benomilo produce una disminución del peso de los testículos y el epidídimo, de la producción de esperma y de la tasa de fecundidad de los machos. A dosis más elevadas, provoca hipoespermatogénesis, con interrupción general de todas las fases de la espermatogénesis. No afecta, en cambio, al comportamiento copulatorio, las vesículas seminales, la movilidad del esperma o las hormonas de la reproducción asociadas. La concentración más baja del benomilo capaz de inducir un efecto espermatogénico estadísticamente significativo en ratas macho fue de 45 mg/kg por día. El NOEL para estos efectos fue de 15 mg/kg por día. La administración por sonda de una sola dosis de benomilo (100 mg/kg o más) mostró efectos en ratas a los 70 días de la exposición, que incluyeron descenso del peso de los testículos y atrofia de los túbulos seminíferos. Administrado por sonda a ratas ChD-CD y Wistar durante los días 7 a 16 de la gestación, el benomilo resultó teratogénico a 62,5 mg/kg en ambas estirpes, pero no a 30 mg/kg en ratas ChD-CD y no a 31,2 mg en ratas Wistar. Administrado por sonda a ratas Sprague-Dawley en los días 7 a 21 de la gestación, el benomilo resultó teratogénico en dosis de 31,2 mg/kg. Los efectos que produjo fueron microftal mia, hidrocefalia y encafaloceles. Las dosis superiores a 15,6 mg/kg tuvieron un efecto negativo sobre el desarrollo posnatal. La administración por sonda de 50 mg/kg o concentraciones superiores indujo en ratones la aparición de costillas supernumerarias y de otras anomalías esqueléticas y viscerales. No se ha determinado el NOEL en los ratones porque no se ensayaron dosis inferiores a 50 mg/kg. A excepción de un aumento marginal de las costillas supernumerarias en conejos, no se observaron efectos teratogénicos incluso a dosis de hasta 500 mg/kg de alimento. 1.6.6 Mutagenicidad y otros efectos finales afines En unos estudios realizados en células somáticas y germinales se ha observado que no provoca mutaciones genéticas ni daños en la estructura de los cromosomas (aberraciones) y tampoco tiene un efecto directo sobre el ADN (causante de daños y la reparación del ADN). Esto se ha demostrado tanto en mamíferos como en otros animales. El benomilo, sin embargo, produce aberraciones cromosómicas numéricas (aneuploidía o poliploidía) en sistemas experimentales in vitro e in vivo. 1.6.7 Carcinogenicidad En el primer estudio de carcinogenicidad con ratones CD-1 se puso de manifiesto un aumento estadísticamente significativo de neoplasia hepatocelular relacionado con la dosis en las hembras y también en los machos tratados con una dosis de nivel medio (1500 mg/kg) se observó una respuesta estadísticamente significativa, pero no en los que recibieron dosis elevadas, a causa del elevado índice de mortalidad. En un segundo estudio sobre la carcinogenicidad de la carbendazima en una raza de ratones genéticamente relacionada con la anterior, los ratones SPF (raza aleatoria suiza), con dosis de 0, 150, 300 y 1000 mg/kg (que se aumentó a 5000 mg/kg durante el estudio) se puso de manifiesto un aumento en el número de casos de adenomas y carcinomas hepatocelulares combinados. En un tercer estudio realizado en ratones NMRKf con dosis de 0, 50, 150, 300 y 1000 mg/kg (que se aumentó a 5000 mg/kg durante el estudio) no se produjeron efectos carcinogénicos. El benomilo y la carbendazima causaron tumores hepáticos en dos estirpes de ratones (CD-1 y suizos (SPF)) que presentaban una tasa alta de tumores hepáticos de formación espontánea. Por el contrario, la carbendazima no resultó carcinogénica en ratones NMRKf, que presentan una tasa baja de esos tumores espontáneos. Los estudios de carcinogenidad del benomilo y la carbendazima en ratas fueron negativos. 1.6.8 Mecanismo de toxicidad, modo de acción Se considera que los efectos biológicos de estos compuestos son el resultado de su interacción con los microtúbulos celulares. Estas estructuras participan en funciones esenciales, como la división celular, que inhiben el benomilo y la carbendazima. La toxicidad de estos productos en los mamíferos está vinculada a una disfunción microtubular. El benomilo y la carbendazima, al igual que otros compuestos del bencimidazol, tienen una toxicidad selectiva para distintas especies. Esta se explica, por lo menos en parte, porque el benomilo y la carbendazima se unen de manera distinta a los microtúbulos de las especies específicas en las que actúan y en las que no. 1.7 Efectos en el ser humano El benomilo causa dermatitis por contacto y sensibilización cutánea. No se ha informado de otros efectos. 1.8 Efectos en otros organismos en el laboratorio y en el medio ambiente Con las dosis de aplicación recomendadas, el benomilo tiene pocos efectos sobre la actividad microbiana del suelo. Se han notificado algunos efectos adversos sobre ciertos grupos de hongos. La CE50 a las 72 h, basada en el crecimiento total, para el alga verde Selenastrum capricornutum fue de 2,0 mg/litro; la concen tración sin efecto observado (NOEC) fue de 0,5 mg/litro. La toxicidad del benomilo para los invertebrados acuáticos y los peces varía ampliamente, con una CL50 a las 96 h que oscila entre 0,006 mg/litro para Ictalurus punctatus (alevines con saco vitelino) y > 100 mg/litro para los cangrejos de río. No observaron efectos tóxicos en experimentos de laboratorio en las lombrices de tierra expuestas a concentraciones normales de benomilo y como resultado del uso de la dosis de aplicación recomendada en el campo. Tiene una toxicidad baja para las aves, y la carbendazima, producto de su degradación, es "relativamente no tóxica" para las abejas de miel. 2. Conclusiones El benomilo causa sensibilización cutánea en el ser humano. Tanto el benomilo como la carbendazima representan un riesgo muy pequeño de intoxicación aguda. Dados los niveles de exposición actuales y el bajo índice de absorción cutánea de estos dos compuestos, no es probable que pudieran tener efectos de toxicidad sistémica en la población general o en personas expuestas profesionalmente. Estas son las conclusiones que se pueden sacar de los datos obtenidos en animales y de los limitados datos sobre el ser humano de que se dispone, pero estas extrapolaciones están respaldadas por el conocimiento del modo de acción de la carbendazima y el benomilo en especies en las que actúan y en las que no. Una mayor clarificación del mecanismo de toxicidad de ambos compuestos en los mamíferos permitirá quizás definir mejor los niveles sin efectos observados. El estudio de su unión a los microtúbulos de las células destinatarias (tejidos testicular y embrionario) facilitará la comparación entre distintas especies. La carbendazima se adsorbe fuertemente en la materia orgánica del suelo que la conserva durante un período de hasta 3 años. Persiste en la superficie de las hojas y, por consiguiente, en las hojas caídas. Se ha demostrado que las dosis recomendadas de aplicación afectan negativamente a las lombrices de tierra (con efectos sobre la población y la reproducción). No se dispone de datos acerca de sus efectos sobre otros artrópodos del suelo o de la maleza, que estarían igualmente expuestos. No es probable que se pueda observar en el medio ambiente la elevada toxicidad demostrada en las pruebas de laboratorio para los organismos acuáticos debido a la baja biodisponibilidad de los residuos de carbendazima unidos a los sedimentos. Sin embargo, no se dispone de información acerca de sus efectos en las especies que viven en los sedimentos, que sufrirían la exposición más intensa.
See Also: Benomyl (ICSC) Benomyl (PDS)