<b>Bijsluiter</b>. De hyperlink naar het originele document werkt niet meer. Daarom laat Woogle de tekst zien die in dat document stond. Deze tekst kan vreemde foutieve woorden of zinnen bevatten en de opmaak kan verdwenen of veranderd zijn. Dit komt door het zwartlakken van vertrouwelijke informatie of doordat de tekst niet digitaal beschikbaar was en dus ingescand en vervolgens via OCR weer ingelezen is. Voor het originele document, neem contact op met de Woo-contactpersoon van het bestuursorgaan.<br><br>====================================================================== Pagina 1 ======================================================================

<pre>Benomyl
(CAS No: 17804-35-2)
Health-based Reassessment of Administrative Occupational Exposure Limits
Committee on Updating of Occupational Exposure Limits,
a committee of the Health Council of the Netherlands
No. 2000/15OSH/094 The Hague, March 30, 2004
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<pre>Preferred citation:
Health Council of the Netherlands: Committee on Updating of Occupational
Exposure Limits. Benomyl; Health-based Reassessment of Administrative
Occupational Exposure Limits. The Hague: Health Council of the Netherlands,
2004; 2000/15OSH/094.
all rights reserved
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<pre>1     Introduction
      The present document contains the assessment of the health hazard of benomyl
      by the Committee on Updating of Occupational Exposure Limits, a committee of
      the Health Council of the Netherlands. The first draft of this document was
      prepared by N Smits M.Sc. (Wageningen University and Research Centre,
      Wageningen, the Netherlands)*.
          The evaluation of the toxicity of benomyl has been based on reviews
      published by the American Conference of Governmental Industrial Hygienists
      (ACG99) and in the ‘Handbook of Pesticide Toxicology’ (Edw91). Where
      relevant, the original publications were reviewed and evaluated as will be
      indicated in the text. In addition, in December 1999, literature was searched in
      the on-line databases Medline, Toxline, and Chemical Abstracts, covering the
      period 1964-1966 until December 1999, and using the following key words:
      benomyl and 17804-35-2. Data of unpublished studies were generally not taken
      into account. Exceptions were made for studies that were summarised and
      evaluated by international bodies such as the World Health Organization and the
      Food and Agricultural Organization/World Health Organization (FAO/WHO:
      Joint Meeting of the FAO Working Party of Experts and the WHO Expert
      Committee on Pesticide Residues (JMPR) (FAO96), the International
      Programme on Chemical Safety/World Health Organization (IPCS/WHO)
      (WHO93a, WHO93b), and the Health Effects Division (HED) of the US
      Environmental Protection Agency (EPA) as part of its hazard identification
      assessment review (Sme01). The final literature search was carried out in
      Medline and Toxline in September 2003.
          In October 2003, the President of the Health Council released a draft of the
      document for public review. No comments were received.
*     Current address: Institute of Risk Assessment Sciences, University of Utrecht, Utrecht, the Netherlands.
094-3 Benomyl
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<pre>2     Identity
      name                :   benomyl
      synonyms            :   methyl 1-[1-(butylamino)carbonyl]-1H-benzimidazol-2-ylcarbamate;
                              carbamic acid, [1-(butylamino)carbonyl]-1H-benzimidazol-2-yl]-, methyl ester;
                              methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate;
                              Benlate, Tersan, Fungicide 1991, Fundazol
      molecular formula   :   C14H18N4O3
      structural formula  :
      CAS number          :   17804-35-2
3     Physical and chemical properties
      molecular weight    :  290.36
      boiling point       :  decomposes just after melting
      melting point       :  140oC
      flash point         :  not available
      vapour pressure     :  at 25oC: <5.0 x 10-6 Pa
      solubility in water :  at 25oC and pH 5: 3.6 mg/L
      log Poctanol/water  :  2.12 (experimental); 2.24 (estimated)
      conversion factors  :  not applicable
      Data from: NLM02, WHO93a, http://esc.syrres.com.
      Benomyl is a white crystalline solid, with a faint acrid odour (NLM02).
4     Use
      Benomyl is a systemic fungicide belonging to the class of benzimidazoles. It has
      been registered in more than 50 countries for the use on more than 70 crops,
      including cereals, cotton, grapes, bananas and other fruits, ornamentals,
094-4 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>      plantation crops, sugar beet, soybeans, tobacco, turf, vegetables, mushrooms, and
      many other crops. It can be used under almost all climatic conditions. Benomyl is
      formulated as a wettable powder (WP) and dry flowable or dispersible granules.
      In some countries, the latter formulation is no longer available (WHO93a).
          According to the database of the Dutch Pesticide Authorisation Board
      (CTB)*, benomyl is at present not permitted in the Netherlands for use as an
      active ingredient in pesticides. In the USA, given that production ceased in 2001
      and sale and distribution of benomyl products ended on December 31, 2002, US
      EPA** expected that any use of benomyl-containing products would end in
      2003.
5     Biotransformation and kinetics
      In vivo
      Blood levels of benomyl and its metabolites carbendazim (MBC) and methyl
      (5-hydroxy-1H-benzimidazol-2-yl)-carbamate (5-HBC), ranging from 0.25 to
      2.3 mg/L, were measured in male rats following inhalation exposure to 320 or
      3300 mg/m3 for 0.5 to 6 hours. The main urinary metabolite was identified as
      5-HBC (Tur79). A dermal absorption study was conducted in male ChR-CD rats
      (n=4/group/exposure time), using benomyl in the form of Benlate (50% WP).
      The test material was applied at dose levels of 0.2, 2, 20, or 200 mg of Benlate,
      equivalent to 0.1, 1, 10, or 100 mg of [2-14C]-benomyl. The exposure durations
      were 0.5, 1, 2, 4, and 10 hours. Blood concentrations of benomyl and its
      metabolites carbendazim (MBC) and 5-HBC peaked at 2-4 hours. The amount of
      benomyl absorbed ranged from 0.031 to 3.5 % from the highest to the lowest
      dose, respectively, following the maximum exposure period of 10 hours (Bel79).
          In a metabolism and tissue distribution study, one rat was fed benomyl for 12
      days at a dose level equivalent to 125 mg/kg bw/day, and then received a single
      oral dose of 7.7 mg [2-14C]-benomyl. After 72 hours, 99% of the radioactivity
      had been recovered, mainly in the urine (85.8% of the administered dose), with
      13.1%, 0.2%, and 0.02% in the faeces, the liver and the gastrointestinal tract, and
      the carcass, respectively. The main urinary metabolite was 5-HBC (Gar74). In a
      more recent study, rats (n=5/sex/group) were given oral (gavage) doses of 50 or
      1000 mg/kg bw [phenyl (U)-14C]-carbendazim, the primary metabolite of
      benomyl. A third group of rats (n=5/sex) was fed 50 mg/kg bw of carbendazim
*     At: http://www.ctb-wageningen.nl.
**    At: http://www.epa.gov/pesticides/reregistration/status.htm.
094-5 Benomyl
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<pre>      for 14 days, and then received a single oral dose of 50 mg/kg bw of radiolabelled
      carbendazim. In all groups, more than 98% of the radioactivity was recovered
      within 72 hours after dosing. Urinary excretion accounted for 54-66% or 41% of
      the administered dose in animals given the low or high dose, respectively.
      Elimination in the faeces accounted for all of the remaining radiolabel. The
      amount of radiolabel retained in tissues was less than 1% of the applied dose.
      The major urinary metabolites were the sulphate conjugate of 5-HBC (21-43% of
      dose) in the male rats and [2-[(methoxycarbonyl)amino]-6-oxo-6H-
      benzimidazol-5-yl] β-D-glucopyranosiduronic acid-N-oxide (5,6-HOBC-N-
      oxide-G; 10-19% of dose) in the female rats. Unchanged carbendazim
      represented 10-15% of the dose in the faeces of rats in the high-dose group
      (Mon90).
      In vitro
      An in vitro study on the penetration of a 50% WP formulation through the human
      skin showed that absorption at recommended spray strength solution is poor.
      Even less penetration was detected when dry concentrated benomyl was applied.
      No quantitative data were reported (War92).
6     Effects and mechanism of action
      Human data
      Occupational exposure to benomyl may occur by inhalation of dusts and by skin
      contact with dusts, emulsions, sprays, and sprayed products. In a field study,
      maximal dermal and respiratory exposure occurred during loading and mixing
      for aerial application. Essentially all of the exposure was dermal, resulting in 26
      mg per mixing cycle, while the average respiratory exposure was 0.08 mg
      (duration of exposure not given) (Eve82).
          Inhalation of dust may result in irritation of the nose, throat, and respiratory
      tract. Benomyl can be mildly irritating to the skin and the eyes (Bur93). The
      chemical caused occasionally contact dermatitis and dermal sensitisation in
      agricultural workers (Joo83, Küh85, Lis86). A relatively high incidence of
      contact dermatitis has been reported in women who worked in warm, moist
      greenhouses where benomyl had been repeatedly sprayed (ACG99).
          No poisoning cases with benomyl of agricultural or factory workers have
      been documented in the scientific literature (Edw91, Gou83). Factory workers
      engaged in the manufacture of benomyl did not show statistically significant
094-6 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>      differences in white and red blood cell count or haemoglobin and haematocrit
      levels compared to controls. No exposure data were reported (Eve79).
          No chromosomal aberrations were found in peripheral lymphocytes of 14
      spaymen, using Fundazol 50 WP in greenhouses for up to 2 years (Des90).
      Normal birth rates were observed in spouses of male workers (n=298), who were
      engaged in the manufacture of benomyl for up to 8 years. Spermatogenesis in the
      workers was not examined, and no exposure levels were reported (Goo78).
      Levels of inhalation exposure to benomyl and carbendazim experienced by the
      workers during manufacture of benomyl over the period 1986-1989 were less
      than 0.3 mg/m3 (WHO93a). A possible case of teratogenicity of Benlate DF has
      been reported of a woman who delivered a boy without eyes. While pregnant, the
      woman had had contact with benomyl, while walking in a tomato and strawberry
      field, previously sprayed with the formulation (Fra98).
      Animal data
      Irritation and sensitisation
      Technical-grade benomyl was slightly irritating to the eyes of rabbits, when
      tested as a 50% WP and as a suspension in mineral oil. Temporary mild
      conjunctival irritation and minor transitory corneal opacity were shown after 48
      to 96 hours (Fra72). In a later study, similar results were obtained with Benlate
      PNW, another 50% WP (Gar83a). No dermal irritation was observed 4 or 24
      hours after application of 0.5 g of Benlate 50 DF (containing 50% benomyl) to 6
      New Zealand white rabbits. After 48 hours, 2 rabbits had slight to mild irritation,
      which was still evident after 72 hours (Vic87). In another study, application for
      24 hours of aqueous suspensions containing 10, 25, or 50% dilutions of
      technical-grade benomyl on intact and abraded skin of guinea pigs resulted in
      mild irritation (ACG99, FAO96). Benlate PNW (50% benomyl) was a skin
      sensitiser in albino guinea pigs, when challenged with 8 or 80% Benlate PNW
      after 4-weekly intradermal injections of a 0.1% solution in saline (Gar84). In
      another study, technical-grade benomyl produced sensitisation in all 10 guinea
      pigs tested in a maximisation test (Mat81).
      Acute toxicity
      Results of acute lethal toxicity of benomyl (as 50% WP) are summarised in
      Table 1.
094-7 Benomyl
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<pre>      Table 1 Summary of acute toxicity studies for benomyl (as 50% WP) in mammals.
      exposure route             species            LC50/LD50               reference
      (duration)
      inhalation      (4 h)      rat                >820 mg/m3              Hor69
                      (4 h)      rat                >4010 mg/m3             Bus68a
                      (4 h)      dog                >1650 mg/m3             Lit69
      dermal                     rabbit             >10000 mg/kg bw         Bus68b
                                 rabbit             >2000 mg/kg bw          Bro87, Gar83b
      oral                       rat                >10,000 mg/kg bwa       She69a
                                 rat                >1000 mg/kg bw          She69a
                                 rat                >1200 mg/kg bw          Hos77
                                 rat                >5000 mg/kg bw          Sar87
                                 rabbit             >3400 mg/kg bw          Fri69
                                 dog                >1000 mg/kg bwa         She69b
      a
           Administered as technical-grade benomyl (purity: >95%).
      The clinical signs of toxicity after treatment were generally non-specific. Gross
      and microscopic examination performed in some of these studies revealed
      testicular degeneration, necrosis of germinal epithelium, and aspermatogenesis
      in rats and dogs (no doses presented) (FAO96).
           Male dogs (n=10/group) were exposed by inhalation to 0, 650, or 1650
      mg/m3 for 4 hours, and 5 animals/group were killed at post-exposure days 14 and
      28, respectively. Apart from an effect on spermatogenic activity on day 14 (see
      Section ‘Reproduction toxicity’), there was only a significantly decreased liver
      weight in dogs of the high-concentration group sacrificed at day 28 (Lit69).
           In an acute neurotoxicity study, Sprague-Dawley rats (n=10/sex/group)
      received single oral (gavage) doses of 0, 500, 1000, or 2000 mg/kg bw benomyl
      (purity: 97.4%). Functional and motor activity were assessed during the week
      before the day of treatment and at 2 hours, one day, and 7 and 14 days after
      dosing, after which rats were killed and grossly examined. Histological
      examination was limited to central and peripheral nervous system tissues from 6
      control and all high-dose rats. No treatment-related mortality was observed.
      Effects noted were a transient reduction in body weight gain and in food intake in
      all treated groups. Motor activity was reduced, only on the day of treatment, in
      high-dose females. No abnormalities were recorded in the battery of functional
      neurotoxicity tests, including reactivity, excitability, gait, sensorimotor
      coordination, forelimb and hind limb grip strength, abnormal clinical signs, and
      body temperature, or in the post-mortem examinations. It was concluded that
      benomyl did not induce acute neurotoxicity at doses up to 2000 mg/kg bw
      (Fos93).
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<pre>      Subacute and subchronic toxicity
      In an inhalation study, Sprague-Dawley rats (n=20/sex/group) were exposed
      nose-only to benomyl aerosols at concentrations of 0, 10, 50, or 200 mg/m3, 6
      hours/day, 5 days/week, for approximately 14 weeks. The mass median
      aerodynamic diameters (MMAD) were in the range of 1.7 to 2.0 µm at 10 and
      200 mg/m3, with greater than 93% of all particles being less than 10 µm in
      aerodynamic diameter. Therefore, an adequate concentration of benomyl reached
      the lungs. No compound-related mortality was reported. In the high-
      concentration group, effects observed were decreased mean body weights and
      reduced food consumption in male rats and reduced body weight gains in male
      (days 2-37 only) and female rats (days 64-92 only). No significant differences
      were observed in haematology, clinical chemistry, and urinalysis results between
      exposed and control animals. Ophthalmoscopic examination did not reveal
      lesions related to benomyl exposure. After 45 days, 10 animals/sex/group were
      sacrificed for macroscopic and microscopic examination. A compound-related
      minimal to mild degeneration of the olfactory epithelium was observed in the
      nasal cavity of all males and of 8 out of 10 female rats exposed 200 mg/m3. In 2
      males exposed to 50 mg/m3, less severe olfactory degeneration was observed. At
      the end of the approximate 90 days of exposure, all rats exposed to 200 mg/m3
      and 3 males exposed to 50 mg/m3 showed minimal to mild degeneration of the
      olfactory epithelium. Testicular lesions were observed in 2 rats in the high-
      concentration group. However, only the quantity of spermatids was affected, and
      no abnormalities were observed in spermatogenesis and in maturation to
      spermatozoa. Furthermore, the effect observed was unilateral and compatible
      with a prior unilateral fluid alteration in efferent ducts. Therefore, the lesions
      were not considered to be a toxic, compound-related effect on spermatogenesis.
      No other compound-related organ or tissue lesions, including lungs, testes,
      epididymides, prostate, and seminal vesicle, were observed in any rat at any
      exposure level. Based on olfactory degeneration in the nasal cavity, the NOAEL
      was 10 mg/m3 for male rats and 50 mg/m3 for female rats (War89).
          In a dermal study in New Zealand white rabbits (n=5/sex/group), a 50%
      benomyl formulation was applied to abraded dorsal skin at doses equivalent to 0,
      50, 250, 500, 1000, and 5000 mg/kg bw/day active ingredient, 6 hours/day, 5
      days/week, for 3 weeks. Moderate skin irritation was reported for all dose
      groups. At the 2 highest dose levels, body weight gains were reduced and
      diarrhoea, oliguria, and haematuria were observed, indicating functional
      disturbances of the alimentary tract and the kidneys. At 500 and 1000 mg/kg bw,
      decreased absolute and relative testes weights were observed. This was not seen
094-9 Benomyl
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<pre>       at 5000 mg/kg bw, probably because of the small number of animals examined
       (n=2). The decrease at 500 mg/kg bw was not considered of biological
       significance. No histological changes were observed apart from focal testicular
       degeneration in one rabbit in the 5000-mg/kg bw group. The NOAEL was 500
       mg/kg bw/day, based on biologically significantly decreased absolute and
       relative testes weights (Hoo69).
           Sprague-Dawley rats (n=16/sex/group) were fed benomyl (purity: 72%) via
       the diet, at dose levels equivalent to 0, 5, 25, and 125 mg/kg bw/day, for 96-103
       days. No signs of intoxication or treatment-related effects on body weight gain,
       haematological, liver function (plasma alkaline phosphatase and ALAT), or
       urine parameters were observed. Females in the high-dose group showed an
       increase in relative liver weight. Gross and microscopic examination of tissues
       and organs did not reveal treatment-related abnormalities (She67).
           In a neurotoxicity study, functional and motor activity were assessed in
       Sprague-Dawley rats (n=11/sex/group), fed benomyl (purity: 97.4%) at dietary
       doses equivalent to 0, 5, 125, or 375 mg/kg bw/day, for 90 days. There was no
       treatment-related mortality during the study. At the high-dose, body weight gain
       and food intake were reduced and motor activity was increased. No changes were
       noted in functional activity, including reactivity, excitability, gait, and
       sensorimotor coordination. Microscopic examination of tissues from the central
       and peripheral nervous system did not reveal treatment-related abnormalities.
       The NOAEL was 25 mg/kg bw (Fos94).
           Beagle dogs (n=4/sex/group) were fed benomyl (50% WP) at dietary dose
       levels equivalent to 0, 2.5, 12.5, and 62.5 mg/kg bw/day (active ingredient), for 3
       months. No mortality, signs of intoxication, or effects on growth, food
       consumption, or urine parameters were observed. There were no dose-related
       effects on haematological parameters, but in the high-dose group, plasma
       alkaline phosphatase and ALAT activities were increased and plasma
       albumin:globulin ratio was significantly decreased in both sexes. In addition,
       increases in thyroid weights and decreases in thymus weights were also noted in
       both sexes. One female in the high-dose group had an enlarged spleen, which
       was consistent with the decreased erythrocyte count, haemoglobin concentration,
       and haematocrit values. Microscopic examination revealed myeloid hyperplasia
       of the spleen and bone marrow and erythroid hyperplasia in the same animal.
       Three males in the high-dose group had reduced prostate weights in comparison
       with controls. Microscopic examination of tissues and organs showed no
       consistent effect. The NOAEL was 12.5 mg/kg bw/day (She68).
           In another study, conducted by the same author, beagle dogs (n=4/sex/group)
       were fed benomyl (50% WP) at dietary dose levels equivalent to 0, 2.5, 12.5, and
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<pre>           62.5 mg/kg bw/day (active ingredient), for 2 years. There were no treatment-
           related effects on mortality and no signs of intoxication were observed.
           Haematological and urine parameters in treated groups were similar to those of
           the controls. In the male and female animals of the high-dose group, there were
           decreased body weight gain, food consumption, and plasma albumin:globulin
           ratio and increased plasma alkaline phosphatase and ALAT activities - generally
           more marked in males than in females - while an increase in plasma cholesterol
           and a decrease in total protein levels were seen in males only. The clinical
           chemistry observations indicated adverse effects on the liver, characterised as
           cirrhosis. Four out of 6 dogs in the high-dose group had slight-to-marked bile
           duct proliferation. No hepatocyte vacuolation was observed at 2.5 or 12.5
           mg/kg bw/day, indicating that benomyl is not hepatotoxic at these doses. Focal
           testicular degeneration was seen in all groups, including controls, with marked
           testicular degeneration (reduced testicular weight, absence of spermatozoa, and
           spermatic giant cells) in one out of 3 dogs at 65 mg/kg. The NOAEL was 12.5
           mg/kg bw/day, based on effects on the liver, clinical chemistry parameters, and
           body weight gain at 62.5 mg/kg bw/day (She70).
                 The effect of benomyl on hepatic enzymes was studied in male and female
           CrL-CD mice and CD-1 rats. Animals were fed benomyl at dietary doses
           equivalent to 0, 10, 30, 100, 300, 1000, or 3000 mg/kg bw/day, for 28 days.
           Significantly elevated liver weight was found in females in the high-dose group,
           but no liver toxicity or effect on body weight was observed. Enzyme induction of
           epoxide hydrolase was observed at the 2 high-dose levels and of glutathione
           S-transferase at 3000 mg/kg bw/day in both sexes of mice and rats (Gue81).
           The results of short-term toxicity studies in rats, rabbits, and dogs are
           summarised in Table 2.
Table 2 Summary of subacute and subchronic studies for benomyl.
exposure    species                 concentrations/     exposure critical effect       NOAEL        reference
route       (strain; sex;           dose levels         duration
            number)
inhalation  rat                     0, 10, 50, 200      14 weeks degeneration of       10 mg/m3     War89
            (Sprague-Dawley;        mg/m3                        olfactory epithelium
            n=20/sex/group)
dermal      rabbit                  0, 50, 250,         3 weeks  changes in testes and 500 mg/kg bw Hoo69
            (New Zealand; n=5/sex/ 500,1000, 5000                bone marrow
            group)                  mg/kg bw
oral        rat                     0, 5, 25, 125 mg/ 14 weeks   increase in liver     25 mg/kg bw  She67
            (Sprague-Dawley;        kg bw                        weight
            (n=16/sex/group)
094-11     Benomyl
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<pre>        rat                     0, 5, 125, 375     13 weeks motor activity   125 mg/kg bw  Fos94
        (Sprague-Dawley;        mg/kg bw
        (n=11/sex/group)
        rat                     0, 10, 30, 100,    4 weeks  liver enzyme     300 mg/kg bw  Gue81
        (CD-1)                  300, 1000, 3000             induction
                                mg/kg bw
        mice                    0, 10, 30, 100,    4 weeks  liver enzyme     300 mg/kg bw  Gue81
        (CrL-CD)                300, 1000, 3000             induction
                                mg/kg bw
        dog                     0, 2.5, 12.5, 62.5 3 months abnormal liver   12.5 mg/kg bw She68
        (beagle; n=4/sex/group) mg/kg bw                    function
        dog                     0, 2.5, 12.5, 62.5 2 years  effects on liver 12.5 mg/kg bw She70
        (beagle; n=4/sex/group) mg/kg bw
       Chronic toxicity and carcinogenicity
       Groups of Charles River albino weanling rats (n=36/sex/group) were given
       benomyl (purity: 50-70%) via the diet at doses equivalent to 5, 25, or 125
       mg/kg bw/day, for 104 weeks. No treatment-related mortality, signs of toxicity,
       or changes in food consumption, body weights, or absolute or relative organ
       weights, or in haematological, liver function, or urine parameters were observed.
       Upon gross and microscopic examination, no treatment-related neoplastic or
       non-neoplastic lesions were found in any of the treated groups. As a high
       incidence of testicular degeneration was observed in control males, no
       conclusion could be drawn about compound-related effects on male gonads.
            The NOAEL was 125 mg/kg bw/day, the highest dose tested (Lee77).
       Groups of CD-1 mice (n=80/sex/group) were given benomyl (purity: 99%) via
       the diet at doses equivalent to 0, 75, 225, or 750 mg/kg bw/day, for 2 years. Mice
       in the high-dose group initially (first 37 weeks) received 1125 mg/kg bw/day. No
       treatment-related mortality or signs of toxicity were observed. At the mid- and
       high-dose levels, there were decreases in body weights in both sexes and in
       haematological parameters and absolute and relative liver weights in males.
       Except for slight decreases in erythrocyte counts in females of the high-dose
       group and in haemoglobin, haematocrit, and erythrocyte values in males of the
       mid-dose group, no remarkable changes were seen in the results of
       haematological examinations. The most significant changes in organ weights
       included decreased absolute and relative liver weights in high-dose females and
       mid- and high-dose males as well as reduced absolute testicular weights in high-
       dose males. The incidence of hepatocellular carcinomas and benign hepatic
       neoplasms was significantly increased in a dose-related fashion in treated
       females and in the low- and mid-dose male groups, but not in the high-dose
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<pre>       males. The latency for liver tumour induction was not different between treated
       and control groups. The incidences of lung tumours were significantly increased
       - being within the range of historical controls - in males, but not in females, at the
       mid and high dose. Non-neoplastic changes observed were confined to the liver
       (degeneration, pigment, cytomegaly), the thymus (atrophy), and the testes,
       epididymides, and prostate (degeneration of seminiferous tubules, atrophy,
       aspermatogenesis, distended ascini) in high-dose males, to the spleen
       (haemosiderosis) in high-dose females, and to the trachea (submucosal
       lymphocytic infiltration) in mid-dose females. A NOAEL could not be identified
       in this study (Wie82).
           Several carcinogenicity studies in different strains of mice were conducted
       with carbendazim, the primary metabolite of benomyl. Like benomyl,
       carbendazim induced liver tumours (hepatocellular carcinomas and adenomas) in
       both CD-1 and SPF Swiss mice. In contrast, there was no evidence of a
       carcinogenic effect from carbendazim when administered in the diet of NMRKf
       (SPF71) mice (WHO93b).
       Mutagenicity and genotoxicity
       Mutagenicity assays comprised tests for the detection of gene mutations in
       bacteria (in vitro), in mammalian cells (in vitro), and in Drosophila, cytogenicity
       in mammalian cells (in vitro and in vivo), e.g., sister chromatid exchanges (SCE),
       structural chromosomal effects (chromosome aberrations and micronucleus
       formation), numerical chromosomal effects (e.g., aneuploidy), and dominant
       lethal mutations (in vivo), and other genotoxicity assays, e.g., tests for DNA
       damage (in vitro).
       •   In vitro tests:
           • Gene mutation assays. Benomyl did not induce reverse mutations in
               several strains of S. typhimurium (TA98, TA100, TA1530, TA1535,
               TA1537, TA1538, TA1950), when tested at concentrations up to 5000
               µg/plate, with and without metabolic activation by a rat liver microsomal
               S9 preparation (Car78, Don81, Fis78, Ric83, Rus78, Shi78). In one study,
               positive results were found in S. typhimurium TA1535, in E. coli WP2,
               and E. coli WP2 uvrA, but not in S. typhimurium TA1538, when tested at
               benomyl concentrations up to 1 µg/mL (Kap76). In A. nidulans, benomyl
               was mutagenic when tested at concentrations up to 0.4 µg/mL (Kap74). In
               a host-mediated assay in rats or mice given benomyl as a single oral dose
               of 4000 mg/kg bw, no gene mutations were induced in S. typhimurium
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<pre>             TA1950 (Fis78). Negative results were also obtained in S. typhimurium
             G46 his-, in host-mediated assays in mice given benomyl as a single
             intraperitoneal injection of 2000 mg/kg bw (Shi78) or as 3 consecutive
             subcutaneous injections of 500 mg/kg bw each (Fis78).
             Benomyl (purity: 99.9%) did not induce gene mutations in the HPRT
             forward mutation assay in cultured Chinese hamster ovary cells (CHO)
             when tested at concentrations of 17 or 172 µmol/L in the absence or 3 or
             120 µmol/L in the presence of S9 activation (Fit80). The TK+/- mutation
             assay in cultured L5178Y mouse lymphoma cells was negative at
             benomyl concentrations up to 20 µmol/L without or 25 µmol/L with
             metabolic activation (Ama79). Negative results in the mouse lymphoma
             L5178Y assay were also obtained in an unpublished study, when benomyl
             was tested at concentrations up to 25 µmol/L in the absence of metabolic
             activation. However, in the presence of S9, an increased gene mutation
             rate was found (McC83), while in a third study, results were positive, both
             with and without metabolic activation, at concentrations in the range of 5
             to 100 mg/L (with), and 2 to 40 mg/L benomyl (without) (Jon84).
             No sex-linked recessive lethal mutations were found in D. melanogaster
             when fed a suspension of 1 mg benomyl/mL (Lam80).
          • Cytogenicity assays. Weakly positive SCE results were seen in cultured
             CHO cells when tested at benomyl concentrations above 150 mg/L,
             without metabolic activation (Eva80). In another study, positive results of
             SCE in CHO cells were observed, with and without metabolic activation,
             at benomyl concentrations in the range of 3 to 36 mg/L (with S9), or 0.6 to
             2.5 mg/L (without S9) (Jon84). The frequency of SCEs in cultured human
             lymphocytes was slightly increased at concentrations ranging from 1 to
             100 mg/L in the absence of S9 (Dol92). When tested in cultured human
             lymphocytes, the frequency of chromosome aberrations remained
             unaffected when tested at benomyl concentrations up to 2000 mg/L with
             and without metabolic activation (Gup75). In another study, an increased
             chromosome aberration rate was found at 10 mg/L without S9, but not in
             the presence of S9 (Pil83). Using the same test system, a not statistically
             significant, not dose-related increase in the frequency of micronuclei was
             found at concentrations ranging from 1 to 100 mg/L (Bia97). An increase
             in chromosome aberration rate was observed in benomyl-treated cultured
             Chinese hamster lung cells, when tested at concentrations up to 90
             mg/mL, with and without metabolic activation (Sas88). Using cultured rat
             hepatocytes, benomyl induced a significant dose-related increase in the
             frequency of micronuclei (concentration range tested: 0.5-25 µg/mL)
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<pre>             (Pia94).
             In several studies, a dose-related increase in numerical chromosomal
             aberrations was observed in benomyl-treated cell lines. Aneuploidy and/or
             polyploidy was observed in human/mouse monochromosomal hybrid
             cells at concentrations in the range of 1.5 to 15 mg/L (Ath85), in V79/AP4
             Chinese hamster cells (Rai89), in CHO cells (Eas89), in human
             lymphocytes, at concentrations above 0.1 (Geo90) or 1 mg/L (Ben00), and
             in Chinese hamster/human hybrid cells, at concentrations above 2.0 mg/L
             (Zel90).
             When fed at a suspension of 1 mg/mL, benomyl did not significantly
             increase non-disjunction, chromosome breaks, or chromosome loss in
             D. melanogaster. However, there was a relatively high incidence of
             sterility, which could be expected from a spindle inhibitor (Lam80).
          • Other assays. The rec-assay for DNA repair was negative in B. subtilis at
             2000 µg benomyl/plate (Shi78). Benomyl did not increase unscheduled
             DNA synthesis in rat and mouse hepatocytes at concentrations in the
             range of 0.5 to 500 mg/L (Ton81). Benomyl did not induce non-
             disjunction in A. nidulans when tested at concentrations up to 2.8 mmol/L.
             However, spindle inhibition was observed (DeB80).
       •  In vivo tests:
          • No dominant lethal mutations were induced in ChR-CD rats, given
             benomyl via the feed at doses equivalent to 0, 25, 125, or 250
             mg/kg bw/day, for 7 days (She75). Negative results were also found in
             other studies, when Wistar rats were given benomyl via the feed at doses
             equivalent to 0, 0.05, 0.31, or 10 mg/kg bw/day for 70 days (Bar83) or by
             gavage at daily doses of 0, 10, or 50 mg/kg bw for 70 days (Geo90).
             Benomyl, orally (gavage) administered at single doses of 0, 500, 1000,
             1500, 1750, or 2000 mg/kg bw, induced an increased frequency of
             hyperploid cells in oocytes collected from superovulated female ICR mice
             (n=20-59/group) 17 hours after administration, at all doses. Treatment did
             not cause an increase in structural chromosome aberrations (Mai92).
             When male (probably Wistar) rats (n=4-5/group) were orally given doses
             of Fundazol (containing 80% benomyl as active ingredient) 0, 10, 50, or
             200 mg/kg bw, for 70 days, a small (1.7 fold over control values),
             statistically significant increase in the frequency of micronuclei was found
             in the bone marrow of animals sacrificed 24 hours after last administration
             of 50 mg/kg bw. No effects were seen at 10 or 200 mg/kg bw (Geo90).
             In ICR mice given 2 oral (gavage) doses of 0, 500, or 1000
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<pre>             mg benomyl/kg bw each, 24 hours apart, a statistically significant
             increased incidence of micronuclei in polychromatic erythrocytes was
             observed at the top dose only (Sei76). In another study, an increase in the
             frequency of micronuclei was observed in bone marrow cells obtained
             from B6D2F2/Cr-1Br mice (n=5/sex/group) harvested 48 hours after
             administration of single oral doses of 2500 or 5000 mg/kg bw. Analysis
             revealed that on average 82% of the total micronucleated polychromatic
             erythrocytes showed positive kinetochore staining, indicating benomyl-
             induced aneuploidy. No effects were seen at 100 mg/kg bw (Ben92,
             Sar94). A single oral dose of 1000 mg/kg bw caused a small but
             significant induction of micronuclei in the bone marrow cells of male
             Swiss mice, with a maximum (3.6-fold over control values) at 38 hours
             after administration. Animals were sacrificed at 5 time points between 6
             and 48 hours after administration, and bone marrow samples of at least 4
             animals were analysed per time point. Polyploid cells were significantly
             increased over control levels (at t=0) only at 30 hours whereas some
             hyperploid cells (not statistically significant) were observed at 38 hours
             (Bar93). An increased frequency of micronuclei was also found in
             polychromatic erythrocytes of mice, given oral (gavage) doses of 2500 or
             5000 mg/kg bw (Sas90) or intraperitoneally injected with single doses
             ranging from 250 to1000 mg benomyl/kg bw (Jon84).
             No increased incidences of chromosome aberrations were found in bone
             marrow cells of rats, given benomyl at daily oral (gavage) doses up to 500
             mg/kg bw for 8 days (Ruz76). No increase in structural chromosomal
             aberrations was observed in bone marrow cells of B6D2F2/Cr-1Br mice,
             given single oral (gavage) doses of benomyl of 625, 1250, 2500, and 5000
             mg/kg bw (Sta90). A single dose of 1000 mg/kg bw did not increase the
             percentage of aberrant cells without gaps analysed in bone marrow cells
             of male Swiss mice at time points ranging from 6 to 48 hours (n= at least 4
             animals/time point) (Bar93). Single oral (gavage) doses of 500 and 1000
             mg/kg bw or 5 daily doses of 500 mg/kg bw did not induce an increased
             frequency of chromosome aberrations in bone marrow cells of male mice
             (time of sacrifice 20 hours after final administration) (Pil80). Following
             intraperitoneal injections, twice at intervals of 24 hours, of doses of 250,
             500, or 1000 mg/kg bw, a dose-dependent increase in the total breakage
             frequency and in the frequency of aberrant cells, both being statistically
             significantly different from controls at the high dose only, was observed in
             bone marrow cells of male Wistar rats (n=5/group) (Adh88).
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<pre>       •   Other tests:
           Using the Syrian hamster embryo cell transformation assay, a test thought to
           detect carcinogens with known or suspected aneuploidy-inducing activity,
           benomyl did not induce a significant increase in the frequency of
           morphologically transformed colonies, when exposed to concentrations
           ranging from 0.25 to 2.1 mg/L for 7 days. However, exposure for 24 hours
           caused a significant increase in the transformation frequency at 2 mg/L
           benomyl (Gib95).
       In summary, in vitro tests showed that benomyl induced SCEs and numerical
       chromosomal effects. Results of the induction of gene mutations or structural
       chromosomal aberrations in benomyl-treated mammalian cells were conflicting.
           In vivo, benomyl-treated mice had an increased incidence of micronuclei or
       aneuploidy. Numerical chromosome changes might be a result of the interference
       of benomyl, and its primary metabolite carbendazim, with cell division (Alb93,
       Dav77).
       Reproduction toxicity
       Several studies have been conducted on the effects of benomyl on male
       reproductive organs.
           When male dogs (n=10/group) were exposed by inhalation to 0, 650, or 1650
       mg/m3 for 4 hours, there was only a slight reduction in spermatogenic activity in
       the high-concentration group at post-exposure day 14 when half of the animals
       were killed, but not at day 28 when the remainder of the animals were sacrificed
       (Lit69). In a follow-up study, gross and microscopic changes in testes and
       epididymides, including spermatocyte degeneration, were seen in male Sprague-
       Dawley rats exposed to 1900 mg/m3 for 4 hours (no more data presented)
       (War89). In Sprague-Dawley rats (n=20/group) exposed to 10, 50, or 200 mg/m3,
       6 hours/day, 5 days/week, for 14 weeks, no compound-related effects on male
       reproductive organs or tissue were seen in any of the groups (see also Section
       ‘Subacute and subchronic toxicity’) (War89).
           The effects on the testes following single oral administration were studied in
       adult Sprague-Dawley rats (n=20/group), by giving doses of 0, 25, 50, 100, 200,
       400, or 800 mg/kg bw benomyl (95% active ingredient) in corn oil by gavage.
       Animals were sacrificed at 2 and 70 days after treatment for microscopic
       examination. At day 2 after treatment, testicular swelling, occlusions of the
       efferent ductules, and premature release of germ cells (sloughing) in the tubules
       were observed at doses of 100 mg/kg bw and above. Testis weight was
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<pre>       significantly increased at 200 mg/kg bw and above. At day 70 after treatment,
       dose-dependent increases in seminiferous tubular atrophy and in the number of
       reproductive tracts containing occluded efferent ductules were seen, starting at
       50 mg/kg bw. The NOAEL was 25 mg/kg bw, based on biologically significant
       sloughing and occlusions of the efferent ductules of the testes (Hes91).
           In another study, 65-day-old male Sprague-Dawley rats received 10 daily
       doses of benomyl at 0, 200, or 400 mg/kg bw/day by gavage. There were no
       compound-related effects on body weights and on the absolute weights of liver,
       kidney, adrenal, testis, or seminal vesicles. However, treatment-related
       reductions were observed in caudate epididymal weights, in epididymal sperm
       count, and in vas deferens sperm concentration. Microscopic examination
       revealed that at the high-dose, hypospermatocytogenesis with generalised
       disruption of all stages of spermatogenesis occurred. No NOAEL was
       established in this study (Car82).
           Adult male Wistar rats (n=27/group) were fed benomyl at dietary levels
       equivalent to 0, 0.1, 0.6, or 20 mg/kg bw/day, for 70 days. At the high-dose,
       sperm counts in ejaculate were statistically significantly reduced. There was a
       dose-related decrease in relative testis weights and in fertility index, starting at
       0.1 mg/kg bw/day. No change in copular behaviour was noted, and the
       compound did not induce dominant lethal mutations. Plasma testosterone and
       gonadotropin levels were not influenced by the treatment. The treatment-related
       effects were completely reversible, 70 days after the end of dosing. The LOAEL
       was 0.1 mg/kg bw/day (Bar83). However, the breeding technique used in this
       study may have compromised the results. Male and female animals did not
       receive food (to avoid female exposure) over an 8-hour period during the normal
       dark cycle, apparently for up to 7 or more consecutive days, which may have
       influenced breeding patterns. Furthermore, these fasting periods may have
       exacerbated the testicular effects. Fasted rats appeared to be markedly more
       susceptible to testicular effects induced by a single dose of the benomyl
       metabolite MBC than non-fasted animals (Lin88).
           In another study, 102-day-old Wistar male rats (n=12/group) were given
       benomyl at 0, 1, 5, 15, or 45 mg/kg bw/day by gavage, for 62 days. They were
       then bred to untreated females, and sacrificed at days 14-17 after the end of
       treatment. No differences between treated and control groups were seen in
       reproductive behaviour parameters (number of copulatory plugs, sperm-positive
       smears, total litters, pups per litter, and implantation sites per litter, percentage of
       resorptions per litter, and male and female pup weights), seminal vesicle weight,
       prostate weight, sperm motility, and in the concentrations of serum testosterone,
       luteinising hormone (LH), follicle-stimulating hormone (FSH), prolactin, or
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<pre>       androgen binding protein. At the high-dose, decreased testicular and epididymal
       weights, decreased sperm production, and increased numbers of decapitated
       spermatozoa and of seminiferous tubules containing multinucleated giant cells
       were observed. The NOAEL was 15 mg/kg bw/day (Lin88).
       In a study performed according to (and meant to evaluate) OECD Guideline 421
       (Reproductive Toxicity Screening Test), male and female Wistar-derived (WU)
       rats (n=10/sex/group) were given oral (gavage) doses of benomyl (in corn oil) of
       0, 10, 30, or 90 mg/kg bw/day, for a 14-day pre-mating period and a 14-day (1:1)
       mating period, after which the males were killed and the females were treated for
       another 28 days until terminal sacrifice at post-partum day 6. Apart from
       increases in food consumption and body weight gain in high-dose females during
       the first 2 treatment weeks, no differences between groups were seen regarding
       food consumption and body weight. No other treatment-related changes were
       reported in parental animals during the exposure period. Upon post-mortem
       examination, sex organs were not affected in females. In males, there were no
       changes in testes and epididymides weights, but a dose-dependent increase of
       incidence and severity of testicular epithelial degeneration was seen, becoming
       significant in the high-dose group. Mating resulted in 8, 8, and 4 pregnancies in
       the low-, mid-, and high-dose group, respectively, vs. 5 in the control group.
       There were no differences in time to conception and pregnancy duration between
       groups, and all pregnancies resulted in normal birth. Pup weights at birth and on
       post-natal day 6 were significantly lower in the high-dose group. Pre-
       implantation and post-implantation losses seemed to be increased in treated
       animals, but there were no differences between groups in numbers of corpora
       lutea, pup sex ratio, and post-natal mortality or in incidences of fetal anomalies
       or malformations. Compared with a concomitant developmental study (see
       below), no eye malformations or high incidences of prenatal deaths were
       observed at the high dose of 90
       mg/kg bw. Piersma et al. stated that modulation of maternal metabolic enzyme
       activity during the pre-mating exposure period might have played a role in this
       effect (Pie95).
           In a 2-generation reproduction study, Sprague-Dawley rats (numbers not
       given) were given benomyl in the diet at levels equivalent to 0, 6, 30, 190, or 350
       mg/kg bw/day for 71 days prior to mating, and F1 offspring were fed treated
       diets at levels of 0, 8, 20, 250, or 1000 mg/kg bw/day for at least 105 days after
       weaning, before being bred for production of F2a and F2b litters. No treatment-
       related parental mortality was observed. At the high-dose, F0 and F1 parental
       body weights, body weight gain, and food consumption were depressed, and
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<pre>       male rats had decreased testicular weights. Sperm count was reduced at 190 and
       350 mg/kg bw for F0 parents, and at 250 and 1000 mg/kg bw for F1 parents.
       Microscopic examination revealed atrophy and degeneration of the seminiferous
       tubules in the testes and oligospermia in F0 and F1 parents at the 2 high-dose
       levels. There were no treatment-related changes in mating, fertility, or gestation
       indices. No abnormalities were noted in the percent of pups born alive. However,
       at the high-dose, all male and female offspring (F1 and F2) had decreased birth
       weights and F2a and F2b offspring had significantly decreased viability indices.
       F2 offspring also had significantly depressed body weights on days 14 and 21 of
       lactation at 250 mg/kg bw/day. The parental and reproductive NOAEL were 30
       and 20 mg/kg bw/day, respectively (Meb90).
       Benomyl was evaluated for developmental toxicity in rats, mice and rabbits. In a
       study with pregnant Wistar rats, the animals (n=27-28/group) were given
       benomyl (purity: 99%) via the diet, at doses equivalent to 0, 169, 298, or 505
       mg/kg bw/day, on days 7-16 of gestation. On day 20 of gestation, all pregnant
       animals were sacrificed and fetuses delivered by Caesarean section. Food
       consumption and body weight gain were reduced in dams at the mid- and high-
       dose levels. At these doses, the weight gain of fetuses was also reduced and the
       percentage of fetuses with enlarged renal pelvis was increased, compared with
       animals in the control group. At the high dose, a significant decrease was
       observed in the ossification of the supraoccipital bone. The developmental and
       maternal NOAEL was 169 mg/kg bw/day (Kav82).
            In another study by the same authors, pregnant rats (n=12-30/group) received
       benomyl (purity: 99%) at oral (gavage) doses 0, 15.6, 31.2, 62.5, or 125
       mg/kg bw/day, on days 7-16 of gestation. Pups were delivered by Caesarean
       section on day 21 of gestation. A significant reduction in maternal body weight
       was observed at the highest dose level. The frequency of fetal resorptions and the
       fetal mortality was increased at 125 mg/kg bw/day. Fetal weight was
       significantly reduced at 62.5 and 125 mg/kg bw/day. At these levels, there were
       also increased incidences of skeletal, visceral, and brain malformations, e.g.,
       decreased numbers of sternal and caudal vertebrae, increased percentages of
       enlarged renal pelvis, increased percentages of enlarged lateral ventricles,
       hydrocephaly, and microphthalmia. The maternal NOAEL was 62.5
       mg/kg bw/day and the developmental NOAEL 31.2 mg/kg bw/day (Kav82).
            In a separate study, the authors also evaluated the effect of low levels of
       benomyl as the pups aged. Groups of pregnant Wistar rats were given benomyl
       by gavage at doses of 0, 15.6 or 31.2 mg/kg bw/day from day 7 of gestation until
       delivery, and from delivery to day 22 of lactation. The litters were each reduced
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<pre>       to 4 male and 4 female pups on day 3 of lactation, and weighed on days 8, 15, 22,
       29, and 100 after parturition. The locomotor activity was evaluated periodically
       throughout the study. At 100 days of age, several organs were weighed. No
       treatment-related effects were noted on litter size at birth or weaning or on body
       weights of fetuses. Growth, survival, locomotor activity, and organ weights
       (adrenals, liver, kidneys, ovaries) of offspring were comparable to those of
       controls. However, weights of testes, ventral prostate, and seminal vesicles were
       significantly decreased at the high dose. The NOAEL for developmental toxicity
       was 15.6 mg/kg bw/day (Kav82).
           In another developmental toxicity study, benomyl (purity: 99.2%) was given
       to pregnant Sprague-Dawley rats (n=27/group) at oral (gavage) doses of 0, 3, 10,
       30, 62.5, or 125 mg/kg bw/day, on days 7-16 of gestation. No treatment-related
       maternal effects were observed. However, embryofetal mortality was increased
       at 125 mg/kg bw and fetal body weight decreased at 62.5 and 125 mg/kg bw/day.
       Major skeletal malformations were observed at 62.5 and 125 mg/kg bw/day and
       included misaligned and unossified sternebrae, fused ribs, and thoracic arches.
       Malformations, including microphthalmia, anophthalmia, and hydrocephaly
       (distended lateral ventricles) were observed at doses of 10 mg/kg bw/day and
       above, being dose related at the higher dose levels. Microscopic changes of the
       eyes were also found at these doses, but not at 30 mg/kg bw/day. The authors
       considered microphthalmia at 10 mg/kg bw/day to be related to treatment in view
       of the severity of microscopic changes. The maternal NOAEL was >125
       mg/kg bw/day, and the developmental NOAEL 3 mg/kg bw/day (Sta80).
           In a follow-up study by the same authors, conducted to determine a no-effect
       level for external hydrocephaly and microphthalmia, benomyl (purity: 99.1%)
       was orally (gavage) administered to pregnant Sprague-Dawley rats (n=50/
       group), on days 7-16 of gestation, at dose levels of 0, 3, 6.25, 10, 20, 30, or 62.5
       mg/kg bw/day. Mean fetal body weight was decreased and incidental
       observations of microphthalmia and hydrocephaly occurred at 62.5
       mg/kg bw/day. No other maternal or developmental effects were noted. The
       maternal NOAEL was >62.5 mg/kg bw, and the NOAEL for microphthalmia 30
       mg/kg bw/day (Sta82).
           In another study, groups of pregnant Sprague-Dawley rats were treated with
       0 or 62.5 mg/kg bw/day by gavage, on days 7-16 (8 rats) or 7-20 of gestation (13
       rats). No maternal effects were noted. Hydrocephaly was noted in 35% of fetuses
       examined on day 16 and in 76% of fetuses examined on day 20 (Ell87, Ell88).
           In a comparable study, ocular anomalies (microphthalmia, anophthalmia,
       retinal dysplasia, cataracts) occurred in 43% of the fetuses of dams (Sprague-
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<pre>       Dawley) given (by gavage) 62.4 mg/kg bw/day of benomyl on days 7-21 of
       gestation. The NOAEL was 31.2 mg/kg bw/day (Hoo91).
           In female Wistar-derived (WU) rats (n=10/group) given oral (gavage) doses
       of 0, 90, or 270 mg/kg bw on days 6 to 15 of gestation, body weight gain was
       significantly reduced in both treated groups. In both groups, post-implantation
       loss and the number of total litter resorptions and of fetuses with ophthalmic
       abnormalities were increased and the body weights of surviving near-term
       fetuses decreased. Numbers of corpora lutea and pre-implantation loss did not
       differ among groups (Pie95).
           Groups of pregnant CD-1 mice were given benomyl by gavage at 0, 50, 100,
       or 200 mg/kg bw/day, on days 7-17 of gestation. No treatment-related maternal
       effects were observed. A dose-related increase in fetal mortality and a decrease
       in fetal weight were observed at all levels. The numbers of caudal and sternal
       ossifications were decreased and the incidences of enlarged lateral ventricles
       were increased at the high dose. A dose-related increase in the incidence of
       enlarged renal pelvises was observed at 100 and 200 mg/kg bw/day and an
       increase in the occurrence of supernumerary ribs at all levels. The numbers of
       abnormal litters and fetuses were significantly increased at 100 and 200
       mg/kg bw/day. Major anomalies included brain defects, e.g., hydrocephaly,
       exencephaly, and skeletal abnormalities, e.g., fused ribs, fused vertebrae. No
       NOAEL was established for fetotoxicity and teratogenicity (Kav82).
           Groups of pregnant New Zealand white rabbits (n=20/group) were given oral
       doses of 0, 15, 30, 90, or 180 mg/kg bw/day of benomyl, on days 7-28 of
       gestation. Maternal mortality, occurring at 0, 30, 90, and 180 mg/kg bw, was not
       related to benomyl toxicity. At 180 mg/kg bw, food consumption was
       significantly reduced and 2 animals aborted during the study. Fetal mortality and
       fetal weights were not affected by the treatment and there were no treatment-
       related external malformations. However, a treatment-related decrease in renal
       papillae was observed in 2 viable fetuses in 2 litters. The NOAEL for both
       maternal and developmental toxicity was 90 mg/kg bw/day (Mun95).
       A summary of oral reproductive and developmental toxicity studies with
       benomyl is shown in Table 3.
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<pre>Table 3 Summary of oral reproductive and developmental toxicity studies with benomyl.
species               dose level           exposure duration       critical effect                  NOAEL       reference
(strain; number; sex) (mg/kg bw)
rat                   0, 25, 50, 100, 200, 1 day                   testicular effects;              25          Hes91
(Sprague-Dawley;      400, 800 (gavage)                            occluded efferent ductules
n=20/males/group)
rat                   0, 200, 400          10 days                 reduced epididymus weight,       LOAEL: 200  Car82
(Sprague-Dawley;      (gavage)                                     sperm count, disrupted
males)                                                             spermatogenesis
rat                   0, 0.1, 0.6, 20      70 days                 reduced testicular weight and    LOAEL: 0.1  Bar83
(Wistar; n=27 males/ (diet)                                        fertility index
group)
rat                   0, 1, 5, 15, 45      62 days                 reduced testicular weight,       15          Lin88
(Wistar; n=12 males/ (gavage)                                      epididymal weight, sperm
group)                                                             production, and effect on
                                                                   spermatogenesis
rat                   0, 10, 30, 90        28 d (males)            paternal: testicular epithelial  30          Pie95
(WU; n=10/sex/group) (gavage)              54 d (females)          degeneration
                                                                   maternal: none                   90
                                                                   fetal: decreased pup weight      30
rat                   F0: 0, 6, 30, 190, 71 days                   maternal: reduced sperm          30          Meb90
(Sprague-Dawley;      330 (diet)                                   counts
males, females)       F1: 0, 8, 20, 250, 105 days                  F1/F2: reduced body weight       20
                      1000 (diet)                                  during lactation
rat                   0, 169, 298, 505     gestational day 7-16    maternal: body weight            169         Kav82
(Wistar; n=27-28      (diet)                                       fetal: enlarged renal pelvis     169
females/group)
rat                   0, 15.6, 31.2, 62.5, gestational day 7-16    maternal: body weight            62.5        Kav82
(Wistar; n=12-30      125 (gavage)                                 fetal: skeletal, visceral, brain 31.2
females/group)                                                     malformations
rat                   0, 15.6, 31.2        gestational day 7-15    maternal: none                   >31.2       Kav82
(Wistar; females)     (gavage)                                     fetal: testicular weight          15.6
                                                                           prostate weight
rat                   0, 3, 10, 30, 62.5, gestational day 7-16     maternal: none                   >125        Sta80
(Sprague-Dawley;      125 (gavage)                                 fetal: external, visceral         3
n=27 females/group)                                                anomalies
rat                   0, 3, 6.25, 10, 20, gestational day 7-16     maternal: none                   >62.5
(Sprague-Dawley;      30, 62.5 (gavage)                            fetal: microphthalmia             30         Sta82
n=50 females/group)
rat                   0, 62.5 (gavage)     gestational day 7-16    maternal: none                   >62.5       Ell87,
(Sprague-Dawley;                           or 7-20                 fetal: hydrocephaly              LOAEL: 62.5 Ell88
n=8-13 females/group)
rat                   0, 31.2, 62.4        gestational day 7-21    maternal: none                   >62.4       Hoo91
(Sprague-Dawley;      (gavage)                                     fetal: microphthalmia            31.2
females)
094-23       Benomyl
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<pre>rat                   0, 90, 270 (gavage) gestational day 6-15 maternal: decreased body        LOAEL: 90 Pie95
(WU; n=10 females/                                             weight                          LOAEL: 90
group)                                                         fetal: increases in post-
                                                               implantation loss, total litter
                                                               resorptions, fetuses with
                                                               ophthalmic abnormalities;
                                                               decreased body weight
mouse                 0, 50, 100, 200     gestational day 7-17 maternal: none                  >200      Kav82
(CD-1; n=20-25                                                 fetal: skeletal, visceral and   LOAEL: 50
females/group)                                                 brain anomalies
rabbit                0, 15, 30, 90, 180  gestational day 7-28 maternal: reduced food          90        Mun95
(New Zealand; n=20                                             consumption; abortions;
females/group)                                                 clinical signs
                                                               fetal: renal papillae           90
                                                               anomalies
            In summary, benomyl induced effects on male reproductive organs in rats.
            Following inhalation, testicular lesions were observed in rats and dogs at single
            4-hour exposures to 1900 (only level tested) and 1650 mg/m3, respectively, while
            no such effects were seen in dogs exposed to 650 mg/m3 for 4 hours or in rats
            exposed to 200 mg/m3 (highest concentration tested), 6 hours/day, 5 days/week,
            for 13 weeks. Following single and repeated oral administration, the NOAELs
            for testicular effects were 25 and 15 mg/kg bw, respectively, and the
            corresponding LOAELs 50 and 45 mg/kg bw, respectively.
                In a 2-generation reproduction toxicity study, the NOAELs for parental and
            reproductive effects were 30 and 20 mg/kg bw, respectively, based on decreased
            sperm counts and reduced body weights of F2 pups during lactation.
                Developmental toxicity studies in rats showed effects at levels substantially
            below those causing maternal toxicity, the NOAELs for maternal and
            developmental toxicity being 62.5 and 15 mg/kg bw, respectively. In mice, fetal
            anomalies were seen at 50 mg/kg bw, the lowest level tested, while no maternal
            toxicity was observed at 200 mg/kg bw, the highest level tested. In rabbits, the
            NOAEL for both maternal and developmental effects was 90 mg/kg bw.
7           Existing guidelines
            The current administrative occupational exposure limit (MAC) for benomyl in
            the Netherlands is 10 mg/m3, 8-hour TWA.
                Existing occupational exposure limits for benomyl in some European
            countries and in the USA are summarised in the annex.
094-24      Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>8      Assessment of health hazard
       The health hazard assessment of benomyl is based to a large extent on toxicology
       reviews issued by the World Health organization/International Programme on
       Chemical Safety (WHO93a), the FAO/WHO Joint Meeting on Pesticide
       Residues for recommendation of an acceptable daily intake (ADI) (FAO96), and
       the Health Effect Division of the United States EPA for reregistration eligibility
       (Sme01). The toxicology profile in these reviews is obtained mainly from
       unpublished reports of toxicology studies, conducted for registration purposes.
           Workers can be exposed to benomyl through inhalation of dust or aerosols or
       by direct skin contact with a formulation of the compound. Benomyl was poorly
       absorbed through human skin in vitro (not quantitated) and through rat skin in
       vivo. In rats, the dermal absorption of benomyl (50% wettable powder) ranged
       from 0.031 to 3.5% and was inversely related to the amount of benomyl applied
       on the skin. No data is available of the percentage uptake of the compound
       through the lungs. The extent of absorption following oral intake is 85-90% in
       rats. Following absorption, the compound was rapidly metabolised into
       carbendazim, which was further metabolised into a range of breakdown products
       (e.g., 5-HBC). These metabolites were excreted in the urine and the faeces within
       72 hours after application. There is no evidence of accumulation of the
       compound in any of the tissues.
           Case studies in humans showed that benomyl caused contact dermatitis and
       dermal sensitisation in agricultural workers. No inadvertent systemic poisoning
       of agricultural or factory workers has been documented. In factory workers, no
       abnormalities in haematological parameters, fertility, or cytogenicity parameters
       have been reported, at benomyl concentrations in air that were generally below
       0.03 mg/m3.
           In experimental animals, the compound is slightly irritating to the eyes and
       the skin and a potent skin sensitiser.
           Based on the results of acute lethal toxicity studies in test animals, the
       committee considers the compound as unlikely to present an acute health hazard.
       Four-hour exposure to benomyl concentrations of 1650 or 1900 mg/m3 caused
       slight testicular effects in rats and dogs, respectively, while single oral doses of
       50 mg/kg bw and higher induced effects such as occlusions of the efferent
       ductules and premature release of germ cells (sloughing) in the tubules. Benomyl
       did not induce acute neurotoxicity in rats at single oral doses up to 2000 mg/kg
       bw, apart from a decrease at the treatment day in motor activity in females given
       2000 mg/kg bw.
094-25 Benomyl
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<pre>           Effects of benomyl exposure in subchronic or chronic studies in rats, mice,
       dogs, and rabbits included: degeneration of olfactory epithelium in a 13-week
       inhalation study in rats (NOAEL: 10 mg/m3); testicular changes in a 3-week
       dermal study in rabbits (NOAEL: 500 mg/kg bw/day); abnormalities in the liver
       in a 14-week oral study in rats (NOAEL: 25 mg/kg bw/day) and in a 2-year study
       in dogs (NOAEL: 12.5 mg/kg bw/day); hepatocellular carcinomas in a 2-year
       study in mice (LOAEL: 75 mg/kg bw/day).
           Benomyl was not mutagenic in bacteria, but results of gene mutation and
       cytogenicity assays in cultured mammalian cells were conflicting. In vitro,
       benomyl induced aneuploidy/polyploidy in mammalian cells. In vivo, benomyl
       did not induce gene mutations in the host-mediated assay, or dominant lethal
       mutations, but the incidence of micronuclei and aneuploidy in blood cells of
       mice was increased. Aneuploidy might have been caused by interference of
       benomyl, or its primary metabolite carbendazim, with cell division. According to
       the committee, there is inadequate evidence for mutagenicity of benomyl. The
       committee comments, however, that the in vivo host-mediated gene mutation
       assay and the dominant lethal mutation assay are insensitive tests, which are not
       used anymore in nowadays mutagenicity testing.
           Carcinogenicity study in rats did not show treatment-related increased
       incidences of neoplastic lesions. In CD-1 mice, however, the incidence of
       hepatocellular carcinomas was significantly increased in a dose-related fashion
       in both sexes. The committee considers the relevance of this increase for man as
       controversial, because of the high incidence of spontaneous liver tumours in this
       strain of mice.
           Subchronic oral studies to examine the effects of benomyl exposure on the
       reproductive organs in male rats showed the following effects: decreased
       testicular and epididymal weights and a decreased sperm production with
       generalised disruption of all stages of spermatogenesis in a 62-day oral study
       (NOAEL: 15 mg/kg bw/day). In a subchronic inhalation study, no such effects
       were seen in rats exposed to 200 mg/m3, the highest concentration tested. In a
       2-generation reproductive toxicity study in rats, the parental and reproductive
       NOAELs were 30 and 20 mg/kg bw/day, on the basis of decreased sperm counts
       in parental males, and reduced body weights of F2 pups, respectively. In
       developmental toxicity studies, teratogenicity was demonstrated in rats at dose
       levels below those causing maternal toxicity. The major abnormalities included
       skeletal, visceral, and brain abnormalities (hydrocephaly and microphthalmia).
       In one study, offspring had decreased testicular and prostate weights. The
       committee concluded that the NOAELs for developmental and maternal toxicity
       were 62.5 and 15 mg/kg bw/day, respectively; in mice, they were ≤50 and ≥200
094-26 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>       mg/kg bw/day, respectively. In rabbits, 90 mg/kg bw/day was the NOAEL for
       both maternal and developmental toxicity.
       Based on the above data, the committee takes the well-performed 14-week
       inhalation study in rats (War89), with a NOAEL of 10 mg/m3, as a starting point
       in deriving a health-based recommended occupational exposure limit
       (HBROEL). In this study, the critical effect was degeneration of olfactory
       epithelium in male animals at concentrations of 50 mg/m3 and above, while no
       treatment-related effects were seen on male reproduction at 200 mg/m3, the
       highest concentration tested. For extrapolation to a HBROEL, an overall
       assessment factor of 8 is established. This factor covers intra- and interspecies
       variation, and the type of critical effect. Thus, applying this factor and the
       preferred value approach, a health-based occupational exposure limit of 1 mg/m3
       is recommended for benomyl.
            The committee considers this HBROEL to be sufficient to protect workers
       from reproduction toxicity effects. Assuming a 60-70-kg worker inhales 10 m3
       during an 8-hour working day and a retention of 100%, this HBROEL would
       lead to a daily intake of approximately 0.15 mg/kg bw/day, which is a factor of
       100 lower than the NOAELs of 15 mg/kg bw/day for developmental and male
       reproduction toxicity found in rat studies
       The committee recommends a health-based occupational exposure limit for
       benomyl of 1 mg/m3, as inhalable dust, as an 8-hour time-weighted average
       (TWA).
            In view of the low penetration ability of benomyl (when tested as a 50%
       wettable powder) through human and rat skin, the committee deems a skin
       notation not necessary.
       References
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ACG03b American Conference of Governmental Industrial Hygienists (ACGIH). 2003 TLVs® and BEIs®
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094-27 Benomyl
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094-29 Benomyl
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<pre>FAO96  Food and Agricultural Organization/World Health Organization (FAO/WHO): Joint Meeting of the
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Fos94    Foss JA. Subchronic neurotoxicity study of DPX-T1991-529 (Benomyl) administered orally via the
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094-31 Benomyl
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094-35 Benomyl
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<pre>              Annex
Occupational exposure limits for benomyl in various countries.
country                               occupational             time-weighted     type of             notea       referenceb
- organisation                        exposure limit           average           exposure limit
                                      ppm       mg/m3
the Netherlands
- Ministry of Social Affairs and      -         10             8h                administrative                   SZW03
Employment
Germany
- AGS                                 -         -                                                                 TRG00
- DFG MAK-Kommission                  -         -                                                                 DFG03
Great Britain
- HSE                                 -         10             8h                OES                              HSE02
                                      -         15             15 min            STEL
Sweden                                -         -                                                                 Swe00
Denmark                               -         5              8h                                                 Arb02
USA
- ACGIH                               -         10             8h                TLV                  A4e         ACG03b
- OSHA                                -         15c            8h                PEL                              ACG03a
                                      -         5d             8h                STEL
- NIOSH                               -         -                                                                 ACG03a
European Union
- SCOEL                               -         -                                                                 EC04
a
     S = skin notation, which means that skin absorption may contribute considerably to body burden; sens = substance can
     cause sensitisation.
b
     Reference to the most recent official publication of occupational exposure limits.
c
     Total dust.
d
     Respirable fraction.
e
     Classified in carcinogen category A4, i.e., not classifiable as a human carcinogen: agents which cause concern that they
     could be carcinogenic for humans but which cannot be assessed conclusively because of lack of data. In vitro or animal
     studies do not provide indications of carcinogenicity which are sufficient to classify the agent into one of the other
     categories.
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