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    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


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    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

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    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
                      4.2.1.1  Water
                      4.2.1.2  Soil
                      4.2.1.3  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
                      7.2.2.1  Rat
                      7.2.2.2  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
                      7.5.1.1  Rat feeding studies
                      7.5.1.2  Rat gavage studies
                      7.5.1.3  Dog inhalation studies
               7.5.2. Teratogenicity and embryotoxicity
                      7.5.2.1  Mouse gavage studies
                      7.5.2.2  Rat gavage studies

                      7.5.2.3  Rat feeding studies
                      7.5.2.4  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
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    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

    1.1.6.1  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
    1.1.6.5).

    1.1.6.2  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.

    1.1.6.3  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.

    1.1.6.4  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.

    1.1.6.5  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.

    1.1.6.6  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.

    1.1.6.7  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.

    1.1.6.8  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:

    CHEMICAL STRUCTURE 1

    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.

    FIGURE 1

         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

    4.2.1.1  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).

    4.2.1.2  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.

    4.2.1.3  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).

    FIGURE 2

         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).

    FIGURE 3

    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

    7.2.2.1  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.

    7.2.2.2  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).

    7.5.1.1  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.

    7.5.1.2  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.

    7.5.1.3  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

    7.5.2.1  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).

    7.5.2.2  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).

    7.5.2.3  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).

    7.5.2.4  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).

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    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
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    Wainwright M & Pugh GJF (1974) The effect of fungicides on certain
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    Ward RS & Scott RC (1992) Benomyl:  in vitro absorption of a 500 g
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    Warheit DB, Kelly DP, Carakostas MC, & Singer AW (1989) A 90-day
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    Wheeler J (1985) Hydrolysis of [phenyl-14C(U)]benomyl. Wilmington,
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    WHO (1993) Environmental Health Criteria 149: Carbendazim. Geneva,
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    Wiechman BE (1982) Long term feeding study with methyl
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    Yoshida K & Nishiuchi Y (1972) Toxicity of pesticides to some water
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    Zbozinek JV (1984) Environmental transformations of DPA, SOPP,
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    Zelesco PA, Barbieri I, & Graves JAM (1990) Use of a cell hybrid
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    Zoran MJ, Heppner TJ, & Drewes CD (1986) Teratogenic effects of the
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    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:
       Toxicological Abbreviations
       Benomyl (HSG 81, 1993)
       Benomyl (ICSC)
       Benomyl (PDS)
       Benomyl (WHO Pesticide Residues Series 3)
       Benomyl (WHO Pesticide Residues Series 5)
       Benomyl (Pesticide residues in food: 1983 evaluations)
       Benomyl (JMPR Evaluation 1995 Part II Toxicological and environmental)