609. Benomyl (Pesticide residues in food: 1983 evaluations)

PESTICIDE RESIDUES IN FOOD - 1983

Sponsored jointly by FAO and WHO

EVALUATIONS 1983

Data and recommendations of the joint meeting
of the FAO Panel of Experts on Pesticide Residues
in Food and the Environment and the
WHO Expert Group on Pesticide Residues
Geneva, 5 - 14 December 1983

Food and Agriculture Organization of the United Nations
Rome 1985

BENOMYL

Explanation

Benomyl was reviewed by the Joint Meetings of 1973, 1975 and 1978
(FAO/WHO 1974a,b, 1976a,b, 1979a).¹ An acceptable daily intake (ADI)
was not allocated because of insufficient acute oral, short-term and
long-term studies. Furthermore, a carcinogenic study, as well as
observations in humans, were indicated as desirable information. These
studies have been reviewed and are summarized in the toxicology
section of the following monograph.

In the 1978 report it was recommended that residue guideline
levels for benomyl and carbendazim should be replaced by a single list
of guideline levels for carbendazim residues that occur as metabolic
products of benomyl or thiophanate-methyl or from the direct use of
carbendazim. That procedure is continued in the section on residues of
this evaluation.

Extensive information was available on approved uses of benomyl
in 22 countries, the fate of residues, international maximum residue
limits (MRLs) and the results of a marked-basket survey in the United
States. Residue data from supervised trials were available from the
United States, Africa, the Federal Republic of Germany, the United
Kingdom, Australia and Japan.

TOXICOLOGY

EVALUATION FOR ACCEPTABLE DAILY INTAKE

BIOCHEMICAL ASPECTS

Absorption, Distribution and Excretion

Absorption, distribution and elimination in rats following dermal
application (0.1, 1, 10 and 100 mg) was measured using ¹⁴C-labelled
benomyl. Blood levels, mode of excretion, metabolic products and rate
of penetration were also examined. A separate group of rats, dosed
intravenously with ¹⁴C-benomyl for comparison of blood level
concentrations, revealed that absorption into the bloodstream was
non-linear with respect to dose. Blood values, which were low
(0.004-0.07 ppm), first appeared 30 min. after treatment, peaked at
all treatment levels after 4 h and were eliminated in urine after
30 min. linearly with time. The low blood concentrations resulted from
limited absorption with rapid metabolism and elimination via the
urine. The major urinary metabolite identified was 5-HBC and, to a
lesser extent, MBC. Greater than 95 percent of the radioactivity was
eliminated in 24 h in the urine following intravenous injection. No
radioactivity was found in any body tissues sampled 24 h after
injection (<0.1 percent) (Fisher et al. 1981 ).

In a similar evaluation, blood levels of benomyl and its
metabolites were measured in rats following inhalation exposure to
actual time-weighted averages of 0.32 and 3.3 mg/l. Exposure intervals
of 0.5, 1, 2 and 6 h were utilized. Urinary residues consisted
primarily of 5-HBC, with limited amounts of benomyl/MBC detected. The
methodology did not distinguish between benomyl and MBC. At both
exposure levels blood concentration of benomyl/MBC was greater than
that of 5-HBC after 6 h, with levels ranging from 0.39-2.3 and
0.25-1.2 ppm, respectively. At 18 h post-exposure, 5-HBC was the only
residue detected in the blood (1.1 ppm) and only at the high dose.

Rapid elimination of benomyl was also demonstrated in mice and
hamsters following intragastric intubation. 90 percent of the
radioactivity was eliminated in the urine and faeces in 72 h. Little
to no radioactivity was evident in tissues. Conjugates of 5-HBC and
MBC appeared in the urine and faeces, respectively (Han 1974).

Lactating and non-lactating goats were given five consecutive
daily doses of 2-¹⁴C benomyl by capsule at rates equivalent to 36 and
88 ppm in the total daily diet, respectively. Evaluation of
radioactivity in various tissues, milk (where present), urine and
faeces, as well as characterization of the bound and unbound residues,
revealed a similar metabolism and elimination pattern identified for
rats, dogs, chickens and dairy cows. Most of the radioactivity was
eliminated in the urine and faeces and identified as 5-HBC and 4-HBC.
Milk residues, principally of 5-HBC with minor amounts of 4-HBC and
5-hydroxy-2-aminobenzimidazole (5-HAB), accounted for approximately
2 percent of the total dose. Approximately 25 percent of the milk
radioactivity was reincorporated into the natural milk components
casein and whey protein. There were no detectable residues in muscle
tissue and fat (<0.01 ppm). However, radioactivity detected in liver
and kidney amounted to 3.8 and 0.09 ppm, respectively, with 5-HBC
identified as the principal benomyl metabolite (ca. 6 percent). Much
of the liver residues were reincorporated into glycogen, protein,
fatty acids and cholesterol and accounted for approximately 35 percent
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 percent) ¹⁴C-residue found in goat liver.
No free benomyl, MBC or 5-hydroxy-MBC were determined in the liver
(Han 1980; Hardesty 1982).

¹ See Annex 2 for FAO and WHO documentation.

In a series of metabolic studies, benomyl and/or Benlate were
administered either by gavage or in the diet to pregnant ChR-CD rats
to determine the concentration of benomyl, MBC and two MBC metabolites
(4-OH MBC and 5-OH MBC) in maternal blood and embryonic tissue. The
design of these studies followed typical teratology schedules wherein
dosing was administered on days 7 through 16 of gestation and included
levels of 125 mg/kg/day via gavage or 5 000 - 10 000 ppm ca.
400-800 mg/kg) in the diet. Blood samples from the dams and tissue
samples from their embryos were examined at several time points during
compound administration. Residue analyses revealed that benomyl was
either much more efficiently metabolized or biologically unavailable
following dietary administration. The half-life for benomyl/MBC in
maternal blood, following gavage, was approximately 45 min. and was
less than 45 min. in embryonic tissue. The metabolite 4-OH MBC was not
detectable (< 0.03 ppm), while 5-OH MBC demonstrated a half-life for
maternal blood of 2-3 h and 4-8 h in embryos. Similar analyses
demonstrated that levels of benomyl/MBC in maternal blood varied
from 0.23 to 0.61 ppm, regardless of dose, while 5-OH MBC averaged
0.44-4.4 ppm and 0.33-3.3 ppm in blood and embryo, respectively (Culik
et al 1981).

Effects on Enzymes and Other Biochemical Parameters

A study conducted using acetylcholinesterase from bovine
erythrocytes showed that benomyl did not inhibit the functioning of
this enzyme. The acetylcholinesterase inhibition constant, K₁, for
benomyl was greater than 1 × 10^-3 M (Belasco undated).

TOXICOLOGICAL STUDIES

Acute Toxicity

The acute toxicity of benomyl in several animal species is
summarized in Table 1.

Gross and histopathological changes were evaluated and selected
organs in several species were examined with emphasis on the male
gonads. Testicular degeneration, necrosis of germinal epithelium and
aspermatogenesis were observed in male rats following some acute oral
intubation and acute inhalation exposures. Dogs, exposed via
inhalation for 4 h at 1.65 mg/l, presented evidence of focal
aspermatogenesis and reduced spermiogenesis.

Table 1 Acute Toxicity of Benomyl in Animals

Chemical Species Sex (Number) Route Vehicle mg/kg b.w.¹ Reference

Benomyl Rat M (1/dose) Oral Peanut Oil ALD > 1 000 Zwicker 1965

Rat M (1/dose) Oral Peanut Oil ALD > 9 590 Sherman & Krauss
1966

Rat M/F (10/dose) Oral Peanut Oil LD₅₀ > 10 000 Sherman 1969a

Rabbit M (1/dose) Oral 50% Wettable ALD > 3 400 Fritz 1969
Powder

Dog M (1) Oral Evaporated ALD > 1 000 Sherman 1969b
Milk and
Water (1:1)

Rabbit M/F (4/dose) Dermal 50% Wettable LD₅₀ > 10 000 Busey 1968a
Powder

Rat M (5/dose) Inhal. 50% Wettable LC₅₀ > 0.82 Hornberger 1969
(4 hr) Powder mg/l

Rat M (6/dose) Inhal. 50% Wettable LC₅₀ > 4.01 Busey 1968b
(4 hr) Powder mg/l (analytical)

Dog M (10/dose) Inhal. 50% Wettable LC₅₀ > 1.65 Littlefield 1969
(4 hr) powder mg/l (analytical)

Benlate (53% benomyl) Rat M/F (10 dose) Oral Aqueous suspension LD₅₀ > 10 000 Sherman 1969a

2-Benzimidazole Rat M/F (10/dose) Oral Corn Oil LD₅₀ > 10 000 Goodman 1975
carbamic acid, methyl ester

Table 1 (con't)

Chemical Species Sex (Number) Route Vehicle mg/kg b.w.¹ Reference

5-Hydroxy-2-benzimidazole- Rat M (1 dose) Oral Corn Oil ALD > 7 500 Snee 1969
carbamic acid, methyl ester

2-Aminobenzimidazole Rat M (1/dose) Oral Peanut Oil ALD > 3 400 Sherman & Fritz
1969

¹ Based on active ingredient.

Special Study on Eye and Skin Irritation

The eye irritation properties of benomyl were examined in albino
rabbits in several tests using technical grade benomyl, 50 percent
wettable powder, and a suspension in mineral oil. Mild conjunctival
irritation and minor transitory corneal opacity were reported in all
tests (Reinke 1966; Frank 1986, 1972).

A 50 percent 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 greater than 0.5 g/animal. Albino
guinea pigs similarly exposed to 10, 25 and 40 percent dilutions of
technical grade benomyl in dimethyl phthalate presented only mild
irritation of both intact and abraded skin sites (Busey 1968a; Majkut
1966; Colburn 1969; Frank 1969).

Special Study on Sensitization

Albino guinea pigs exposed to benomyl, either technical material
or a 50 percent sucrose formulation, produced mild to moderate skin
sensitization reactions following both intradermal injections or
repeat applications to abraded skin (Majkut 1966; Colburn 1969; Frank
1969).

Short-term Studies

Rat

Benomyl, intubated into ChR-CD male rats at 200 and 3 400 mg/kg
in peanut oil five times a week for two weeks produced mortality in
4/6 animals at the high dose. Animals dosed with 3 400 mg/kg
demonstrated evidence of degeneration of germinal epithelium,
multinucleated giant cells and reduction or absence of sperm. Very
minor changes were observed in the testes of the animals dosed with
200 mg/kg (Sherman & Krauss 1966). ChR-CD male rats similarly dosed
with 200 mg/kg of the metabolite 5-hydroxy-2-benzimidazole-carbamic
acid (methyl ester) presented no toxic symptoms or evidence of effects
on the testes (Snee 1969).

Groups of rats (16 of each sex, four-week-old ChR-CD rats/group)
were fed benomyl (72 percent a.i.) in the diet at dosage levels of 0,
100, 500 and 2 500 ppm for 90 days. Animals were observed daily for
behavioral changes and body weights and food consumption were 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
urinalyses were performed on the same animals as well as plasma
alkaline phosphatase and glutamic pyruvic transaminase activity. After
96-103 days of continuous feeding 10 male and 10 female rats in each
group were killed and selected organs weighed. Additional organs were
preserved for microscopic examination. The six male and six female

animals remaining in each group after the terminal sacrifice were
subjected to a reproduction study.

There were no gross toxic signs of poisoning and no compound-
related effects on weight gain, food consumption, food efficiency,
haematology, biochemistry or urinalysis determinations. Liver-to-body
weight ratio in females was slightly increased at 2 500 ppm, compared
with control rats. Cross 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 2 500 ppm (Sherman
et al. 1967).

Rabbit

Groups of five male and five female New Zealand albino rabbits,
weighing 2 to 2.4 kg, were exposed to 15 dermal applications of a
50 percent benomyl formulation (equivalent to 1 000 mg/kg b.w.) on
both abraded and intact abdominal skin sites. Animals were exposed for
six hours each day, five days/week for three 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 for both abraded and
intact skin sites. Slight to moderate desquamation was reported
throughout the study. No apparent compound-related body weight or
organ-to-body weight changes were reported. Microscopic examination of
the males demonstrated that administration of 1 000 mg/kg of benomyl
produced degeneration of the spermatogenic elements of the
seminiferous tubules of the testes, including vacuolated and
multinucleated supermatocytes. A slight increase in haematogenic
activity in the bone marrow, as well as acanthosis and hyperkeratosis
of the skin, were reported for treated animals (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, 1 000 and
5 000 mg/kg b.w. applied to non-occluded abraded dorsal skin sites six
hours 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 1 000 mg/kg
and greater. Mild to moderate skin irritation was reported for all
groups but was most notable at the higher doses. Functional
disturbances of the alimentary canal and kidney, including diarrhoea,
oliguria and haematuria, were observed in males and females at 1 000
and 5 000 mg/kg. There was a decreased average haemoglobin
concentration in the 1 000 mg/kg males, although this was not
significant. Decreased average testicular weights and testes-to-body
weight ratios were observed at the 1 000 mg/kg dose only. There were
no histopathological changes reported and the lower testicular weights
were not considered to be directly related to compound administration
(Hood 1969).

Dog

Groups of beagle dogs (four males and four females per group)
were administered benomyl 50 percent wettable powder in the diet at
dosage levels of 0, 100, 500 and 2 500 ppm (based on active
ingredient) for three months. Dogs were 7-9 months old. Food
consumption and body weight data were recorded weekly. 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 all animals were killed,
selected organs weighed and additional organs subjected to gross and
microscopic evaluations. There was no mortality or adverse clinical
observation over the course of the study and growth and food
consumption were normal. Urinalyses were unaffected by treatment.
There were no dose-related effects on the haematological values;
however, alkaline phosphatase and glutamic pyruvic transaminase
activities were increased in high dose males and females. Furthermore,
there were significant decreases in the albumin/globulin ratio in both
males and females fed 2 500 ppm in the diet. Organ-to-body weight
changes were observed in the high dose males and females for the
thymus (decreased) and thyroid (increased). One female fed 2 500 ppm
had an enlarged spleen, which was consistent with the decreased
erythrocyte count, haemoglobin concentration and haematocrit values.
Histopathological examination revealed myeloid hyperplasia of the
spleen and bone marrow with erythroid hyperplasia for this same animal
at the 3-month examination. Group mean values were not significantly
different, however. Three out of four males fed 2 500 ppm had reduced
relative prostate weights when compared with controls. Microscopic
examination of tissues and organs did not indicate a consistent lesion
effect in animals fed benomyl for 90 days. A no effect level (NOEL)
was demonstrated at 500 ppm (Sherman 1968).

Special Studies on Reproduction

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 and
200 mg/kg/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 histologically. 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, or testicular histology. There were no
effects related to treatment (Carter 1982).

In a similar experiment, adult male Sprague-Dawley rats (65 days
old) received 10 daily treatments of 0, 200 or 400 mg benomyl/kg/day
by gavage. At 14 days after the last treatment, body weight, tissue
weights, total epididymal sperm counts, sperm concentration in the
vas deferens and testes histology were performed. Production of
testosterone by the Leydig cells was artificially stimulated by
subcutaneous injections of hypothalamic chorionic gonadotrophin (HCG)
2 h prior to sacrifice. There were no compound-related effects on body
weight, liver, kidney, adrenal, testes 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 spermiogenesis in the seminiferous tubules
of the testes during exposure to benomyl. Animals exposed to
400 mg/kg/day presented histologic evidence of hypospermatocytogenesis
with generalized disruption of all stages of spermatogenesis, when
compared with controls (Carter & Laskey 1982).

Special Studies on Teratogenicity

Mouse

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/day on
days 7 to 17 of gestation. Animals were killed on day 18, pups
delivered by Caesarean section, the number of live, dead and resorbed
foetuses determined, and foetuses examined for gross anomalies. Half
of the foetuses were examined for visceral abnormalities and the other
half for skeletal abnormalities. Maternal indices were unaffected by
treatment. However, foetal mortality increased, foetal weight
decreased and foetal development was adversely affected by treatment.
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 pelves. 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 significant and increased in a dose-
related manner at all dose levels. There was an increase in the number
of abnormal litters and foetuses, which was significantly different
from the control at 100 and 200 mg/kg/day. Major anomalies included:
exencephaly, hydrocephaly, cleft palate, hydro-nephrosis, polydactyly,
oligodactyly, umbilical hernia, fused ribs, fused vertebrae and
short/kinky tail. Although benomyl demonstrated dose-related
foetotoxicity at all levels, it was not teratogenic at 50 mg/kg/day in
mice (Kavlock et al. 1982).

Rat

Groups of rats (ca. 26-28 pregnant ChR-CD rats/group) were
administered benomyl (53.5 percent a.i.) in the diet at dosages of
0, 100, 500, 2 500 and 5 000 ppm from day 6 through day 15 of
gestation. Average doses were equivalent to 0, 8.6, 43.5, 209.5 and
372.9 mg/kg/day. On day 20 of gestation, all pregnant animals were
sacrificed and foetuses delivered by Caesarean section.

Determinations of the number and location of live foetuses, dead
foetuses and resorption sites were made, as well as of body weights,
crown-rump length, sex and external gross examination for visible
abnormalities. Two thirds of the foetuses were prepared for
examination of skeletal abnormalities, and the rest were examined for
visceral and soft tissue anomalies.

There were no mortalities attributable to benomyl, no clinical
signs of toxicity reported and no adverse effects on body weight of
dams. Dams in the 5 000 ppm group had a reduced food intake during the
period benomyl was administered in the diet, but it returned to
comparable control levels for the remainder of the study. The data
related to reproduction (implantation sites, resorption sites and
live/dead foetuses) were not affected by benomyl up to and including
the highest dose level. There were no external or internal
abnormalities reported, except for three litters at the highest dose
with incidences of hydronephrosis and retarded ossification
(interparietal and occipital bones). There were no teratogenic effects
noted with the administration of benomyl in the diet to pregnant rats
during the critical period of organogenesis (Sherman et al. 1970).

Groups of pregnant Wistar rats (27-28 rats/group) were fed
benomyl in the diet at dose levels of 0, 1 690, 3380 and 6 760 ppm
(time-weighted doses of 0, 169, 298 and 505 mg/kg/day, respectively)
from days 7 to 16 of gestation. Foetuses were delivered by Caesarean
section on day 21, weighed, examined grossly and fixed for evaluation
of visceral and skeletal abnormalities. Food consumption decreased at
3 380 and 6 760 ppm with resultant decreases in body weight gain for
dams, significantly so in the high dose group. Weight gain was
significantly reduced in mid- and high-dose group foetuses and a
significant decrease in the ossification of the supraoccipital bone in
the latter. Furthermore, the percentage of enlarged renal pelves was
increased in the two highest dosage groups, compared with the control.
No dose-related anomalies or major malformations were apparent from
exposure to benomyl at any of the dose levels utilized (Kavlock et
al. 1982).

Benomyl (99.2 percent a.i.) was administered by gavage to groups
of pregnant rats (ChR-CD) at dose levels of 0, 3, 10, 30, 62.5 and
125 mg/kg/day from days 7 through 16 of gestation. There were 60 dams
in the control group (corn oil) and 27 in each of the other test
groups. Dams 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 was the incidence of pregnancy, corpora lutea, implantation sites
and sex ratio. However, foetal body weight was significantly decreased
in the 62.5 and 125 mg/kg/day dose groups. There was also an increased
incidence of embryo-foetal mortality at 125 mg/kg/day.

Some foetuses exhibited external and visceral malformations,
which were dose-related and significant at 10 mg/kg/day and greater.
Malformations observed included microphthalmia, anophthalmia and
hydrocephaly (distended lateral ventricles). These occurred
predominantly at the higher doses. Histological examination of eyes
from all groups revealed pathologic 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/day
treatment groups. No alterations occurred in control or 30 mg/kg
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/day, including misaligned and unossified sternebrae and
bipartite vertebral centra.

Benomyl produced teratogenic responses in ChR-CD rate at doses of
10 mg/kg/day and greater. Microphthalmia at 10 mg/kg was reported to
be compound related, on the basis of the severity of the pathologic
changes and the finding that the increased incidence at 62.5 and
125 mg/kg/day was not a direct reflection of reduced foetal body
weight (Staples & Culik 1980).

Groups of pregnant rats were administered benomyl (99.1 percent
a.i.) via gavage at dosage levels of 0, 3, 6.25, 10, 20, 30 and
62.5 mg/kg/day from days 7 through 16 of gestation. Each group
contained 50 animals, except the high dose group, which contained 20
female rate. Reproductive status was determined on a per litter basis
following gross pathological evaluation. The number of implantation
sites, resorptions, dead and live foetuses, stunted foetuses and mean
weight of live foetuses per litter were determined. This study was
performed to determine a no-effect level for microphthalmia and
hydrocephaly; therefore, only foetal heads were fixed and examined.
Microphthalmia was determined on the basis of the smallest eye in the
control group (< 1.8 mm).

Mean foetal body weight was significantly lower in the high dose
group. There were only two incidences of malformations, both in the
62.5 mg/kg/day group. One foetus had internal hydrocephaly and
another, in a separate litter, had unilateral microphthalmia. These
were not statistically different from controls. The only type of
variation reported was subcutaneous haematoma, which was not dose
related. There was no teratogenic response at 30 mg/kg (Staples 1982).

The teratogenic potential of benomyl was examined in groups of
Wistar rats (12 to 30 pregnant rats/group) orally gavaged with dose
levels of 0, 15.6, 31.2, 62.5 and 125 mg/kg/day administered on days
7 to 16 of gestation. Pups were delivered by Caesarean section on day
21 of gestation. Weight gain in high-dose dams was decreased from days
17 to 20 of gestation. Foetal resorption was increased in this group,
with six litters completely resorbed. Foetal weight was also adversely
affected, with significant decreases at 62.5 and 125 mg/kg/day and a
significant increase in foetal mortality at 125 mg/kg/day. There were
several skeletal and visceral variants observed among foetuses in the
62.5 and 125 mg/kg/day group, including: increased supraoccipital
score, decreased number of sternal and caudal vertebrae, increased
percentage of enlarged lateral ventricles and enlarged renal pelves.
Major anomalies observed primarily at 62.5 mg/kg/day and above
included: encephaloceles, hydrocephaly, microphthalmia, fused
vertebrae and fused ribs. A dose level of 31.2 mg/kg/day appeared to
be without adverse effects on the developing rat foetus in this
evaluation (Kavlock et al. 1982).

Benomyl was administered via gavage to groups of Wistar rats at
dose levels of 0, 15.6 and 31.2 mg/kg/day from day 7 of gestation
through day 15 of lactation (day 22 of gestation was considered day
0 of lactation). The litters were reduced to 8 pups/litter, equal in
sex, on day 3 of lactation. The litters were weighed on days 8, 15,
22, 29 and 100, and locomotor activity was evaluated periodically
throughout the study. At 100 days of age, several organs were weighed,
including 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
foetuses (by sex). Growth, survival and locomotor activity were
comparable with controls throughout the study. Organ weights were
comparable with controls except for decreased testes and ventral
prostate/seminal vesicle weights, which were significantly reduced at
31.2 mg/kg/day but not at 15.6 mg/kg (Kavlock et al. 1982).

Rabbit

Groups of rabbits (15 artificially inseminated albino
rabbits/group) were administered benomyl (50 percent a.i.) in the diet
at dosage levels of 0, 100 and 500 ppm from day 8 through day 16 of
gestation. Mortality, clinical observations and food consumption were
determined daily and body weights were measured weekly. On day 29 or

30 of gestation, selected pregnant animals were sacrificed and
foetuses delivered by Caesarean section; the remaining does were
allowed to hutch normally on day 35.

One low level doe and one high level doe aborted on days 3 and 6,
respectively, and were sacrificed and excluded from the study. 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 and foetuses were delivered by Caesarean section; the
remaining does hutched normally.

There was no mortality attributable to benomyl. General
appearance, behaviour, body weight gain and food consumption were
comparable among all groups. The data related to reproduction
(implantation sites, resorption sites and live young) were not
affected by benomyl. There were no external or internal abnormalities
associated with benomyl treatment of pregnant rabbits. Internal
development, including somatic and skeletal development, was normal,
except for a marginal increase in rudimentary ribs at 500 ppm. Numbers
of pregnant rabbits, of litters and of foetuses examined were less
than adequate to assess the foetotoxic or teratogenic potential of
benomyl to pregnant rabbits (Busey 1968c).

Special Studies on Neurotoxicity

Hen

Studies performed using hens gave no indication of neurotoxic
potential at single oral doses up to and including 5 000 mg/kg
(Goldenthal et al. 1978; Jessep & Dean 1979; Jessup 1979).

Long Term Studies

Rat

Groups of weanling rats (36 male and 36 female Charles River
albino rats/group) were administered benomyl (50-70 percent a.i.) in
the diet for 104 weeks at dosage levels of 0, 100, 500 and 2 500 ppm.
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 behavioural changes and mortality. At
periodic intervals over the course of the study, haematologic,
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
2 500 ppm groups were conducted, along with liver, kidney and testes

examinations of animals in the 100 and 500 ppm dose groups. In a
follow-up pathology evaluation, all of the tissues and organs of the
control, low-, intermediate- and high-dose groups were examined
microscopically.

There was no mortality in the study attributable to the presence
of benomyl in the diet. Survival decreased to approximately 50 percent
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 2 500 ppm group was
330 mg/kg b.w./day (initially, M/F), 91-106 mg/kg (at one year) and
70-85 mg/kg (at two years). There were no compound-related clinical
manifestations of toxicity. Haematologic, urinalysis and liver
function examinations were unaffected by treatment. There were no
observed differences in organ weights or organ-to-body weight changes.
Histopathological examinations revealed no differences between
treatment and control groups. The most frequently observed tumors were
mammary, pituitary and adrenal ones, which were equally distributed
among all groups. Hepatic toxicity was similar among all groups,
including controls, with no demonstrated compound-related effects. A
high incidence of testicular degeneration was observed in control
males and, therefore, no conclusion could be made with regard to
compound-related effects on male gonads.

Benomyl was without adverse effects in this study at levels up to
and including 2 500 ppm (Sherman et al. 1969; Lee 1977).

Dog

Groups of beagle dogs (four males and females/group) one to two
years old, were administered benomyl (50 percent a.i.) in the diet at
dosage levels of 0, 100, 500 and 2 500 ppm for two years. Food
consumption and body weight data were obtained weekly and animals were
examined daily for clinical signs of toxicity. Haematological,
biochemical and urinalysis examinations were performed periodically
throughout the study. Interim sacrifice after one year was performed
on one male and one female per group from the control and high-dose
groups. Organ weights, gross necropsy and histopathological
evaluations were performed at the conclusion of the study. Only the
livers and testes were examined histologically in the 100 and 500 ppm
dose groups.

There was no mortality related to treatment. Body weight changes
and food consumption values were similar among all groups, except the
high dose one, which demonstrated both decreased food intake and body
weight gain. The average daily dose was 55-58 mg/kg b.w. (initially,
M&F), 74-79 mg/kg (at one year) and 45-55 mg/kg (at two years). One
dog in the high-dose group lost its appetite and was replaced. No
other clinical signs of toxicity were observed. Haematological
evaluations and urinalyses were similar to the control. Males in the

2 500 ppm group had increased cholesterol, alkaline phosphatase and
GPT values (initially), as well as decreased total protein and
albumin/globulin (A/G) ratio. There were similar, but less marked,
effects in high dose females. Cholesterol and total protein were
similar to controls among the females examined.

The biochemical determinations were indicative of adverse liver
effects, which were demonstrated as liver cirrhosis among animals in
the high dose group. There was also slight to marked bile duct
proliferation in 4/6 dogs at the 2 500 ppm level. Haemosiderosis,
evident in one dog in the 2 500 ppm group at one year, was not seen in
other dogs examined at two years after staining specifically for iron.
Preparation of preserved wet tissue with oil red 0 and sudan black for
hepatocyte vacuolation confirmed that benomyl was not hepatotoxic at
100 and 500 ppm in the diet.

Focal testicular degeneration was present in all treatment
groups, with marked testicular degeneration (reduced testes weight,
absence of spermatozoa and spermatic giant cells) in 1/3 dogs at
2 500 ppm. A complete histological evaluation of the testes and
secondary sex organs of historical control dogs from the testing
facility in conjunction with the findings from the present report
demonstrated that the testicular lesions reported did not appear
to be attributable to benomyl ingestion. However, an outbreak of
inflammatory infection causing orchitis in beagle colonies at that
time may have contributed to the unusually high level in control dogs;
therefore, no clear evidence exists for the absence of testicular
effects. Data from the carbendazim two-year dog study provide
additional confirmatory results for the absence of these effects at
dietary levels of 100 ppm carbendazim. Furthermore, as indicated, only
testes and liver were examined microscopically in all dose groups in
this benomyl two-year dog study. The two-year carbendazim study
provides added assurance for the absence of adverse effects in organs
or tissues in other dose groups. Considering the absence of such data
for benomyl, a NOEL for benomyl of greater than or equal to 100 ppm
can be assigned (Sherman 1970; Lee 1970, 1971a,b,c, 1977).

Special Studies for Carcinogenicity

Mouse

Male and female CD-1 mice (80 males and 80 females per group)
were administered benomyl (99 percent a.i.) in the diet at dose levels
of 0, 500, 1 500 and 5 000 ppm (5 000 ppm was lowered from 7 500 ppm
after 37 weeks) for two years. Mice were 6-7 weeks old at the start of
the study. Animals were examined daily for behaviour and clinical
signs of toxicity, biweekly for palpable masses and regularly weighed
for body weight changes. Food consumption was similarly determined on
a routine basis. Mortality was noted and recorded. Peripheral blood
was collected periodically throughout the study for haematological

examinations. Urine and faecal samples were collected and evaluated
prior to terminal sacrifice. Selected organs were weighed, including
brain, heart, lungs, liver, spleen, kidney, testes and thymus.
Microscopic examination was performed on tissues and organs.

Median survival time was unaffected by treatment. Male and female
mice fed 1 500 and 5 000 ppm benomyl had 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 1 079 mg/kg b.w,/day
(initially), 878 mg/kg (1 yr.) and 679 mg/kg (2 yr.); for females
it was 1 442 mg/kg (initially), 1 192 mg/kg (1 yr.) and 959 mg/kg
(2 yr.). Clinical signs of toxicity were not different between groups.
Alopecia/dermatitis were observed in all groups with equal severity
and occurrence. The aetiology of this symptom was unknown. There were
no apparent differences between treatment and control groups for
palpable masses, number of mice affected or latency period of
discovery. Haematology examinations were unremarkable except for
decreased erythrocyte counts for males at 1 500 and females at
5 000 ppm. Haemoglobin and haematocrit values were also depressed in
males of the intermediate dose group.

Several significant organ weight changes occurred among treated
groups. In male mice, mean absolute thymus weights were depressed at
all levels and relative thymus weights decreased at 500 and 1 500 ppm
(decrease at 5 000 was not significant). Relative thymus weights were
increased among females at 5 000 and 1 500 ppm (not significant).
Relative brain weights were increased in males and females at
5 000 ppm, in males at 500 and in females at 1 500 ppm. The most
significant compound-related organ weight changes involved absolute
and relative liver weights for males at 1 500 and 5 000 ppm and for
females at 5 000 ppm. Male mice also presented decreased absolute
testes weights at the high dose. The liver and testes weight changes
were accompanied by histomorphic changes in these tissues.

The incidence of hepatocellular carcinomas and benign neoplasms
in female mice was increased and dose dependent. Hepatocellular
carcinomas were significant in females at 500 and 5 000 ppm, while
hepatic neoplasms were significant at 1 500 and 5 000 ppm. In male
mice, hepatocellular carcinomas and neoplasms were significantly
increased at 500 and 1 500 ppm, but not at the high dose. Lung tumours
(alveologenic carcinomas) in 500 and 1 500 ppm dose groups were also
significantly increased in males but not in females. Several non-
neoplastic organ changes were increased in males (5 000 ppm), but were
confined to liver (degeneration, pigment, cytomegaly), thymus
(atrophy) and testes, epididymus, prostate (degeneration of
seminiferous tubules, atrophy, aspermatogenesis, distended acini).
Splenic haemosiderosis was significantly increased at 5 000 ppm, as
was submucosal lymphocytic infiltration of the trachea at 1 500 ppm,
in female mice.

The latency period for liver tumour induction (adenomas and
carcinomas) was determined from palpation, gross necropsy and
histopathology performed on all animals throughout the study. These
findings demonstrated that there is no measurable difference in time
elapsed from mean test day to tumour between control animals and
treatment groups with regard to liver neoplasms.

Benomyl, when fed to CD-1 mice at dose levels of 500 and
5 000 ppm in the diet, is oncogenic in male and female animals. This
effect was compound-related in males at the low and intermediate dose
levels. In the liver of female mice, the oncogenic effect was
compound- and dose-related at all levels. A no-effect level was not
observed in male or female mice for hepatocellular carcinomas or
combined hepatocellular neoplasms (Wiechman et al. 1982).

Human Exposure

Potential dermal and respiratory exposure to benomyl under actual
use situations was determined for: mixing procedures for aerial
application, reentry into treated fields and home use (garden,
ornamental and greenhouse). 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 percent). Respiratory exposure averaged 0.08 mg of benomyl.
Reentry data demonstrated dermal and respiratory exposures of 5.9 mg/h
and less than 0.002 mg/h, respectively. Home use situations produced
exposures of 1 mg and 0.003 mg per application cycle for dermal and
respiratory routes, respectively (Everhart & Holt 1982).

Field use conditions involved in spraying fruit orchards were
examined for potential dermal and respiratory exposures of humans
involved in mixing and applying 20 and 100 gallons of Benlate per acre
(1 acre = 0.4 hectares). The application cycle was approximately
70 min. and resulted in total dermal and respiratory exposure of 11 or
15 mg benomyl/cycle. Essentially all exposure was dermal, resulting in
12.2 mg/cycle dermally, with less than 0.05 mg/cycle via the
respiratory route (DuPont 1979a, undated).

Selected blood profiles from 50 factory workers involved in the
manufacture of Benlate were compared to a control group of 48 workers
who were not exposed to Benlate. White blood count, red blood count,
haemoglobin and haematocrit values were comparable among the two
groups. There were no quantitative estimates of exposure given for the
factory workers. There were no female employees included in the
control group (DuPont 1979b).

An epidemiology survey was performed to determine whether
potential exposure to Benlate had an adverse effect on the fertility
of 298 male workers exposed to benomyl between 1970 and 1977. The
workers ranged from 19 to 64 years of age, with 79 percent between 20
and 39. Seventy-eight percent of the spouses were similarly aged
between 20 and 39 years. Exposure duration ranged from less than one
month to 95 months, with more than 51 percent of the workers
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 evidenced by the birth rates
for the study population, which were generally higher than the
comparison populations. Spermatogenesis among workers was not examined
(Gooch 1978).

Special Studies on Mutagenicity

Results of mutagenicity assays of benomyl are summarized in Table 2.

Mouse, micronucleus

Groups of 24 male mice were given two daily doses by gavage of
250, 500 or 1 000 mg benomyl per kg b.w. on consecutive days. A
vehicle control (DMSO) group was also included. Eight animals from
each group were sacrificed 24, 48 or 72 hours after the second dose
was administered. Bone marrow from the femur of each animal was taken
for examination. For each animal 500 polychromatic erythrocytes (PCE)
were examined for micronuclei, and the number of mature erythrocytes
was counted until 200 PCE were found.

The statistical procedure used showed that four groups had
significantly increased numbers of cells with micronuclei. These
groups included the low- and mid-dose groups at 48 h (15/3.500 and
16/4 000, respectively as compared with 5/3 000 cells from vehicle
controls) and in the high-dose group at 24 and 48 h after treatment
(20/3 500 and 17/3 500, respectively; respective control values were
5/3 500 and 6/5 000) (Kirkhart 1980).

Ovary cells in vitro

Chinese hamster ovary cells in cultures were exposed to varying
concentrations of benomyl without metabolic activation (0, 0.625,
1.25, 2.5, 5, or 10 µg/ml) and with metabolic activation (0, 9.375,
18.75, 37.5, 75 or 150 µg/ml). EMS and dimethylnitrosamine (DMN) were
used as positive controls and a vehicle control (0.95 percent ethanol
in culture medium) was included. Two samples of 25 cells each were

Table 2 Results of Mutagenicity Assays of Benomyl/Benlate

Test Organism Test Substance Results Reference

Gene Mutation Studies

BACTERIA

Salmonella typhimurium Benlate or Positive Kappas et al. 1976
similar formulation

Negative DuPont 1977

Series of tests: spot,
liquid culture and host-mediated
assays using strains his G46,
TA1530, TA1535, TA1950 using
benomyl. No mutagenic activity
was noted in any of these tests

Benomyl Bacterial assays with Ercegovich & Rashid 1977;
Benomyl. Strains TA98, Rashid & Ercegovich 1976
TA100, TA1535, TA1537,
and TA1538. Doubtful
mutagenic activity was
reported for benomyl
both with and without
metabolic activation.

Positive Kappas et al. 1976

Negative Shirasu et al. 1978

Negative Ficsor et al. 1978

Table 2 (con't)

Test Organism Test Substance Results Reference

Benomyl was not mutagenic Donovan & Krahn 1981
at doses as high
as 250 µg per plate in
strains TA1535, 1537,
98 and 100

Positive/Negative Russel 1978; Donovan & Krahn 1981

S. typhimurium and Spot tests on plates Carere et al. 1978
Streptomyces coelicolor with Salmonella strains
TA1535, 1536, 1537 and
1538 and with
Streptomyces. No
mutagenic activity was
reported for benomyl in
either organism.

S. typhimurium Benomyl Negative Shirasu et al. 1978
(host-mediated
assay)
Escherichia coli Benlate Positive Kappas et al. 1976

Benomyl Positive Kappas et al. 1976

Negative Shirasu et al. 1978

Table 2 (con't)

Test Organism Test Substance Results Reference

YEAST AND FUNGI

Fusarium oxysporum Benomyl Inconclusive Dassenoy & Meyer 1973

Aspergillus nidulans Benlate Negative Hastie 1970

A. nidulans Benomyl Benomyl tested in an excision Kappas & Bridges 1981
repair deficient
strain. A dose-dependent
effect was reported.

Saccharomyces Benomyl Mitotic gene conversion De Bertoldi et al.
cerevisiae and A. study was negative on 1980
nidulans testing benomyl with
metabolic activation.

A. nidulans Benomyl Negative for nondisjunction De Bertoldi &
or crossing over. Griselli 1980
Increased frequency of
segregants due to spindle
inhibition reported.

INSECTS

Drosophila melanogaster Benomyl/Benlate Genetic toxicity tested Lamb & Lilly 1980
in Drosophila. Noted
sterility in some broods.
This was considered to be
consistent with spindle
effects of benomyl.

Table 2 (con't)

Test Organism Test Substance Results Reference

Chromosomal Effects
Cytogenetics - in vitro

Chinese hamster Benomyl Weakly positive results Evans & Mitchell
ovary cells in vitro for sister chromatid exchange 1980
were reported, with
and without metabolic
activation.

Chinese hamster Benomyl Looked for mutations at Fitzpatrick & Krahn
ovary cells in vitro the HGPRT locus. Benomyl 1980
was not mutagenic under
these test conditions.

Human leukocyte cell Benlate Primary cultures were Gupta & Legator
cultures treated with benomyl. 1975
Cells examined in the high
dose cultures did not show
a statistically significant
increase in incidence of
chromosomal aberrations.

Table 2 (con't)

Test Organism Test Substance Results Reference

Cytogenetics - In Vivo

Rats Benomyl/Fundazol Bone marrow and cultures of Ruzicska et al 1976
50WP embryo cells of pregnant
rats were examined for
chromosomal aberrations
after treatment with
benomyl on days 7 to 14
of gestation. No increase
in frequency of cells with
chromosomal aberrations
were reported in bone
marrow cells. Embryonic
cells had 5 times more
chromosomal damage at the
two highest doses (200,
500 mg/kg) than controls.

Human Benomyl/Fundazol Peripheral blood cells Ruzicska et al 1976
50WP from workers at a benomyl
(Fundazol 50WP) plant were
examined. No effects attributable
to benomyl reported.

Dominant Lethal

Rat Benomyl Negative Sherman et al 1975

Table 2 (con't)

Test Organism Test Substance Results Reference

Micronucleus Test

Mice Benomyl Dosed by gavage at 250, Kirkhart 1980
500 or 1 000 mg benomyl/kg
on two consecutive days.
Reported a statistically
significant increase in
number of micronuclei in
bone marrow from femur
bones at 48 h in 250 and
500 mg/kg dose groups and
1 000 mg group at 24 and
48 h.

DNA Damage and Repair

B6C3F1 Mice and Benomyl Benomyl was tested for DNA Tong 1981a,b
F344 Rats repair using primary
hepatocyte cultures.
Benomyl did not induce DNA
repair in either rat or
mouse.

Mitotic Gene Conversion

S. cerevisiae Benlate Negative Siebert et al. 1970
Benomyl Negative De Bertoldi et al 1980

A. nidulans Benomyl Negative De Bertoldi et al 1980

Table 2 (con't)

Test Organism Test Substance Results Reference

Mitotic Crossing-Over

Differential Toxicity
(Bacteria)

Bacillus subtilis Benomyl Negative Shirasu et al. 1978

Plant Studies

Allium cepa Benomyl Negative Dassenay & Meyer 1973

Positive Richmond & Phillips 1975
Chromosome Nondisjunction
Yeast and Fungi

A. nidulans Benlate Positive Hastie 1970

Benomyl Positive Kappas et al. 1974

Positive De Bertoldi &
Griselli 1980
Mammals

Microtus oeconomus Benlate Inconclusive Tates 1979
Benomyl Inconclusive Tates 1979

scored for the number of sister chromatid exchanges (SCE) and number
of chromosomes. A total of 50 cells were scored for each group. The 5
and 10 µg/ml dosage did not allow sufficient numbers of second
division metaphases to occur for an evaluation.

The number of SCE in the ethyl methane-sulphonate group was
triple that of the negative controls, while the three non-activated
benomyl groups had one-third more SCE than controls. In the
experiments with activated benomyl, the DMN positive controls had
approximately twice the number of SCE found in negative controls. The
benomyl groups had increased numbers of SCE (by approximately 25 to
100 percent above controls). The number of SCE per cell was increased
by one sixth to one half above that for negative controls for non-
activated benomyl. The activated fungicide increased the number of SCE
by approximately 15 to 100 percent over that seen in controls. Benomyl
proved weakly positive in this study (Evans & Mitchell 1980).

A mutagenicity assay with a Chinese hamster ovary cell line,
which can demonstrate mutations at the gene locus coding for
hypoxanthine-guanine phosphoribosyl transferase (HGPRT) was conducted
using benzo(a)pyrene and ethyl methane sulphonate as positive
controls, as well as a vehicle control (DMSO) Benomyl was added to
test cultures with or without metabolic activation. Resistance of
cells to 6-thioguanine was used as the indicator of mutagenic effects.

A dose-related cytotoxic response was more evident in cultures
exposed to the chemical without activation. No statistically
significant differences in mutation frequency were noted in cultures
treated with activated or nonactivated benomyl at concentrations
ranging from 17 to 172 µM. Positive controls demonstrated that the
test system was sensitive and cell survival was greater than 10
percent at most concentrations used. Benomyl was not mutagenic under
these test conditions (Fitzpatrick & Krahn 1980).

DNA repair

DNA repair assays in rat or mouse hepatocyte primary cultures
(HPC) were evaluated for benomyl along with dimethylnitrosamine,
dimethylformamide, fluorene and 2-aminofluorene, which were used as
positive controls. The liver was removed and primary cultures were
initiated with hepatocytes from B6C3F1 mice or 344 rats. Benomyl and
tritiated thymidine (10 µCi) were added to the culture medium. After
18 to 20 h incubation they were fixed and examined microscopically for
morphological changes and absence of s-phase nuclei indicative of
cytotoxicity. Autoradiographic techniques are used to determine the
number of nuclear grains induced by test chemicals. Background counts
were obtained by evaluating three nuclear-sized areas in the
cytoplasm; these values were averaged and subtracted from the number
counted in the nucleus to obtain a net value for each nucleus.

Benomyl did not induce DNA repair in rat or mouse hepatocytes.
The dimethylnitrosamine and 2-amine fluorene increased the number of
nuclear grains from 7 to 15 times the level set as the criterion for a
positive response (Tong 1981a,b).

Bacterial tests

Benomyl and Benlate were tested for mutagenic activity according
to the plate incorporation procedure, essentially as described by Ames
et al. (1975). Salmonella typhimurium strains used included
TA1535, TA1537, TA98 and TA100. DMSO was the solvent used for benomyl
and water was the solvent for Benlate. One Benlate sample, tested at
concentrations up to 1 200 µg/plate, was not mutagenic under the test
conditions (DuPont 1977). At concentrations up to 500 µg/plate benomyl
(technical grade) exhibited slight mutagenic activity in TA1537, but
only in the presence of the activation system. The induced reversion
frequency was 3.8 times the control value and the average revertants/n
mole value was 0.06 (Russel 1978). Benomyl was tested with and without
activation by three different liver microsomal enzyme preparations
(mouse liver S-9 mixes containing 0.8 or 2.5 mg of protein per plate,
and rat liver S-9 mix containing 3.5 mg of protein/plate). Only one of
two trials with strain TA1535 showed a statistically significant dose-
related trend in the induction of mutations. Dose group differences
were not significantly greater than controls. Benomyl at dosages as
high as 250 µg/plate was not mutagenic under the test conditions
(Donovan and Krahn 1981). An analytical grade sample of benomyl was
not mutagenic at concentrations up to 500 µg/plate (Russell 1978).

A series of spot tests was conducted with paper disks containing
benomyl. The disks were placed on media, which were streaked with
S. typhimurium (strains TA1535, TA1536, TA1537, or TA1538)
or Streptomyces coelicolor. Benomyl was used alone or
combined with liver microsomal enzymes for activation and
methylnitrosonitroguanidine was used as a reference mutagen. Disks
contained 20 µg or 500 µg benomyl for the S. typhimurium and
S. coelicolor tests, respectively. No mutagenic activity was noted
for benomyl in these tests (Carere et al. 1978).

The mutagenic activity of benomyl, Benlate^R and Fundazol 50 WP
was investigated in a series of spot, liquid culture and host
mediated assays in strains his G46, TA1530, TA1535 and TA1950 of
S. typhimurium. Doses of 0.25 to 10 000 µg/ml in overlay spot
test and liquid culture treatments were negative. Nice given
subcutaneous injections of 500 mg/kg did not produce mutations in
S. typhimurium strain his G46. No mutagenic activity was
observed in S. typhimurium TA1950 when rats and mice were orally
dosed with 4 000 mg/kg benomyl (Ficsor et al. 1978).

In similar bacterial assays with benomyl in S. typhimurium
TA98, TA100, TA1535, TA1537 and TA1538, there was doubtful mutagenic
activity in the base substitution sensitive strains (TA100 or TA1535)
for benomyl (1 to 325 µg/plate) with and without liver microsomal
activation. Responses were defined in terms of the ratio of the number
of revertants observed on treated plates to that found on untreated
control plates and a doubtful response was defined as a ratio of 1.5
to 2 (Ercegovich & Rashid 1977; Rashid & Ercegovich 1976).

Benlate^R was tested for mutagenic activity in
S. typhimurium and Escherichia coli, using a simplified
fluctuation assay. Benlate^R was mutagenic in the base pair
substitution specific strain, TA1535, but not in the frameshift
specific strain, TA1538. It was also mutagenic for E. coli strain
WP2 uvrA, which lacks excision repair, but not for E. coli
strain WP2 (DNA repair proficient) and CM611 (misrepair and excision
repair deficient). With both S. typhimurium and E. coli, the
degree of mutagenic activity observed at concentrations between 0.125
to 1 µg/ml was similar when tested at concentrations greater than
1 mg/ml Benlate^R was not mutagenic (Kappas et al. 1976).

Benomyl was non-mutagenic in S. typhimurium TA1535, TA1537,
TA1538, TA98 and TA100, and E. coli WP2 hcr. Concentrations
between 5 and 1 000 µg/plate were tested in a plate incorporation
assay with DMSO as the solvent both in the presence and absence of an
activation system, which included a 9 000 x g supernatant fraction of
homogenized livers from Aroclor 1254-treated Sprague Dawley rats
(Shirasu et al. 1978).

Tests with yeast and fungi

Benomyl added to cultures of an Aspergillus nidulans strain,
which is excision repair deficient, induced back mutations to biotin
and pyridoxin-requiring strains. Mutagenic activity was observed at
0.25, 0.3 and 0.4 µg/ml but there was a plateau of positive response
rather than a linear dose-response through zero concentration. Benomyl
dissolved in ethanol was not mutagenic in either of the repair-
proficient strains (Kappas & Bridges 1981).

No mitotic gene conversion was noted in diploid strains of
Saccharomyces cereviciae or A. nidulans exposed to as much as
3 200 ppm or 200 ppm benomyl, respectively. Benomyl was activated with
a mouse liver microsomal fraction; survival at the highest level
tested was equal to 83 percent and 51 percent for S. cereviciae
and A. nidulans, respectively (de Bertoldi et al. 1980).

In another study with A. nidulans exposed to benomyl, no
non-disjunction or crossing over were reported. An increased frequency
of segregants occurred, probably due to benomyl's spindle-inhibiting
effects (de Bertoldi et al. 1980).

Insect tests

Gamma radiation was used as the reference mutagen. Treated and
untreated adult male Drosophila melanogaster were mated with
virgin females so that at least nine broods of offspring were produced
from each male. Each male was mated with two untreated females for two
to three days following treatment. At that time, a subsequent mating
was conducted. The offspring were classified by sex and phenotype
(regular or exceptional). Some of the exceptional offspring could have
resulted from exchanges between the X and Y chromosomes in the parent
male rather than from whole or partial chromosome loss. Exceptional
offspring resulting from chromosome loss, breakage or non-disjunction
could, therefore, not be clearly distinguished from those resulting
from the sex chromosome exchanges.

No deaths resulted from the treatments. However, there was an
increased incidence of sterility in the later broods from treated
Oregon-R males, but no effect in treated yw^{+B^sY⁺ males. The}
first and second broods result from germ cells which were post-meiotic
at the time of treatment, those in the third and fourth broods were
from meiotic germ cells and those in the last broods were from germ
cells that were premitotic at the time of treatment. No compound-
related effects were noted when chromosomes were examined for breakage
in a second set of experiments and the overall incidence of recessive
lethal mutation reported was 5/4 807 (0.1 percent). The sterility
observed in broods from matings involving mitotic spermatogonial cells
is probably consistent with the suspected spindle effects of the
chemical (Lamb & Lilly 1980).

Cytogenetics

Primary cultures of human leukocytes were treated with Benlate^R
in DMSO at 0, 200, 2 000 or 20 000 ppm. Each test solution (0.1 ml)
was added to 5 ml cultures, which were examined for cells with
chromosome breaks, deletions and small fragments after exposure. In
cultures exposed to 0, 200 or 2 000 ppm benomyl, 1.3, 5.8, and 3.8
percent of the cells examined had chromosomal aberrations. Cultures
exposed to the highest dose contained fewer dividing cells, owing to
toxicity. Those cells examined in high-dosed cultures did not show a
significantly increased incidence of chromosomal aberrations above
that in control cultures. The mitotic index was not measured nor were
replicates performed. Thus, the results are difficult to assess,
particularly regarding toxicity (Gupta & Legator 1975).

Bone marrow and cultures of the embryo cells of pregnant rats
were examined for chromosomal aberrations. The rats were given daily
doses of 0, 25, 50, 200 or 500 mg Fundazol 50WP per kg b.w. by gavage
on days 7 through 14 of gestation. No increases in frequency of cells
with chromosomal aberrations were reported in bone marrow. However,

embryonic cells from rats given 200 or 500 mg/kg/day had five times
the frequency of cells with damaged chromosomes. The aberrations were
described as rings, acentric chromatids and translocations (Ruzicska
et al. 1976).

COMMENTS

Available information demonstrates that benomyl and carbendazim
have a similar metabolism. Benomyl is rapidly metabolized to
carbendazim in mammals and is eliminated preferentially in urine as
methyl 5-hydroxy-2-benzimidazolecarbamate (5-hydroxycarbendazim,
5-HBC). In rats following intubation, inhalation and dietary
exposures, benomyl and carbendazim were present in the blood within
the first six hours, together with comparable levels of 5-HBC. Within
18 hours after exposure, only 5-HBC was identified in blood.
Metabolism proceeds via hydroxylation and ester hydrolysis in the
liver followed by elimination, primarily in the urine (41-71 percent)
and to a lesser extent in faeces (21-46 percent). There is no
retention of ¹⁴C activity in muscle tissue or fat; only liver and
kidney demonstrate bound residues identified as 5-HBC.

Benomyl is not acutely toxic to mammals. It has an acute oral
LD₅₀ in rats and an acute dermal LD₅₀ in rabbits above 10 000 mg/kg.
Gross and histopathological examination of many of these animals
demonstrated a measurable compound-related effect on the male gonads
at high doses (testicular degeneration, necrosis of germinal
epithelium and aspermatogenesis).

Spermatogenesis in pre-pubertal rats was not affected at dose
levels affecting adult rats. Previous JMPR evaluations have identified
variations in teratogenic response dependent on the mode of oral
administration (diet or gavage). The present Meeting reviewed
additional data, which confirmed the difference in sensitivity
according to the route of administration in the rat, the dietary NOEL
for teratogenicity being 6 760 ppm. This dose did, however, induce
embryotoxicity. In the mouse, gavage studies resulted in terata
induction at levels above 50 mg/kg. In the rabbit, a limited study did
not result in induction of terata at 500 ppm in the diet.

Short- and long-term dietary studies in rats did not demonstrate
compound-related effects at doses up to and including 2 500 ppm.
Ninety-day and two-year dietary studies in dogs demonstrated adverse
effects on the liver at 2 500 ppm, but not at 500 ppm, evidenced by
increased cholesterol, alkaline phosphatase and GPT levels, by
decreased total protein and albumin/globulin ratio, and by bile duct
proliferation and haemosiderosis (see also carbendazim).

A carcinogenicity study in CD-1 mice at 500, 1 500 and 5 000 ppm
in the diet showed oncogenicity at all levels. Neoplastic changes
included lung carcinomas in males, but not in females, at 500 and
5 000 ppm. There was an increased incidence of hepatocellular
carcinomas in males at 500 and 1 500 ppm and in females at 500 and
5 000 ppm. There was no measurable effect on latency period. It was
concluded that benomyl was hepatocarcinogenic to mice (see also
carbendazim).

Mutagenicity studies with benomyl gave both positive and negative
results. Benomyl was positive in the micronucleus, yeast, fungi and
drosophila tests. Conflicting negative and positive results in other
tests prevented evaluation of the mutagenic potential. The potential
impact of these results on human health cannot be adequately assessed
at this time.

The monographs on benomyl and carbendazim have stated that the
metabolism of the two compounds is essentially the same, with benomyl
being converted rapidly to carbendazim in mammals. Accordingly, the
available data for benomyl and carbendazim should be considered
collectively for the evaluation of specific studies such as
teratology, reproduction, chronic toxicity and oncogenicity, taking
into account the different molecular weights of the two compounds.

Previous Meetings in 1970, 1973 and 1976, have discussed the
etiology and pathogenesis of liver tumours in certain strains of mice,
with particular emphasis on organochlorine pesticides. It was
recognized that liver tumours are known to develop spontaneously in
many strains of mice, at relatively high incidence without intentional
exposure to chemicals. Evidence of such tumours in several strains of
mice has been found in many of the oncogenicity studies performed with
benomyl and carbendazim. Furthermore, one strain of mouse used (HOE
NMR) is known to have a low background incidence of liver tumours
(1-2 percent) and did not provide evidence of oncogenicity when
exposed to carbendazim at doses up to and including 5 000 ppm. Two
additional studies have been carried out in rats using both benomyl
and carbendazim. Both studies were negative for oncogenicity at doses
up to and including 2 500 and 10 000 ppm, respectively. The hepatic
tumours produced in mice, therefore, appear to be a species-related
phenomenon.

The Meeting expressed concern at the equivocal nature of the
results of a wide range of mutagenicity studies. The possibility that
conflicting results were due to variations in the type and amount of
impurities was considered. However, the Meeting received information
that current levels of the relevant impurities are very low in
technical materials.

In view of established no-observed-effect levels determined in
several studies, including teratology, reproduction and chronic
feeding, an ADI for both benomyl and carbendazim could be estimated.
However, a safety factor of 200 was used to reflect the Meeting's
concern for the paucity of individual animal data for many studies on
carbendazim, which reflect the toxicity of benomyl.

TOXICOLOGICAL EVALUATION

Level causing no toxicological effect

Rat: 2 500 ppm in the diet, equivalent to 125 mg/kg b.w.

Dog: (see carbendazim)

Rat: 30 mg/kg b.w./day (teratology)

Estimate of acceptable daily intake for man

0-0.02 mg/kg b.w.

FURTHER WORK OR INFORMATION

Desirable

1. Data on individual animals in studies on carbendazim that have
been identified in the monograph.

2. Additional data to elucidate the mechanism of degenerative
testicular effects on mammals.

3. Elucidation of the variability of the mutagenicity data.

REFERENCES - TOXICOLOGY

Ames, B.N., McCann, J. & Yamasaki, E. Methods for detecting
1975 carcinogens and mutagens with the Salmonella mammalian-
microsome mutagenicity test. Mutat. Res., 31: 347-364.

Belasco, I.J. Study showing the absence of acetylcholinesterase
(undated) inhibition with a wettable powder formulation (50% benomyl).
Reported submitted to WHO by DuPont. (Unpublished)

Busey, W.M. Acute dermal LD₅₀ test and dermal irritation test on
1968a rabbits using a wettable powder formulation (50% Benomyl)
with histological addendum. MRO 581-239. Report dd. 6/21/68
by Hazleton Laboratories, Inc. submitted to WHO by DuPont.
(Unpublished)

Busey, W.M. Acute inhalation exposure test in rats using a wettable
1968b powder formulation (50% benomyl). MRO 1126. Report dd.
10/18/68 by Hazleton Laboratories, Inc. submitted to WHO by
DuPont. (Unpublished)

Busey, W.M. Teratology study in rabbits using a wettable powder
1968c formulation (50% benomyl) MRO 1079. Report dd. 7/15/68 by
Hazleton Laboratories, Inc. submitted to WHO by DuPont.
(Unpublished)

Busey, W.M. Repeated dermal application test on rabbits using a
1968d wettable powder formulation (50% benomyl). HLO No. 298-68.
Report, dd. 12/13/68 submitted to WHO by DuPont.
(Unpublished)

Carere, A., Ortali, V.A., Cardamone, G., Torracca, A.M. & Raschetti,
1978 R. Microbiological mutagenicity studies of pesticides in
vitro. Mutat. Res., 57:277-286.

Carter, S.D. Effect of benomyl on the reproductive development in the
1982 prepubertal male rat. From Thesis, North Carolina State
University. Submitted to WHO by DuPont.

Carter, S.D. & Laskey, J.W. Effect of benomyl on reproduction in the
1982 male rat. Toxicology Letters II: 87-94.

Colburn, C.W. Skin irritation and sensitization tests on guinea pigs
1967 using technical benomyl (> 95% benomyl). HLR No. 84-69.
Report dd. 4/18/69 submitted to WHO by DuPont. (Unpublished)

Culik, R. at al. Determination of benomyl/methyl-2-benzimidazole
1981 carbamate (MBC) concentrations in maternal blood and in the
concepti of rats exposed to benomyl and Benlate by diet.
Report submitted to WHO by DuPont. (Unpublished)

Dessenay, B. & Meyer, J.A. Mutagenic effect of benomyl on Fusarium
1973 oxysporum. Mutat. Res. 21:119-120.

De Bertoldi, M. et al. Mutagenicity of pesticides evaluated by means
1980 of gene-conversion in Saccharomyces cerevisiae and in
Aspergillus nidulans. Environ. Mutagen., 2: 359-370.

De Bertoldi, M. & Griselli, M. Different test systems in Aspergillus
1980 nidulans for the evaluation of mitotic gene conversion,
crossing-over and nondisfunction. Mutat. Res., 74:303-324.

Donovan, S.M. & Krahn, D.F. Mutagenicity evaluation in Salmonella
1981 typhimurium using technical benomyl (> 95% benomyl), HLR
No. 434-81. Report dd. 8/26/81 submitted to WHO by DuPont.
(Unpublished)

DuPont. Mutagenic activity of benomyl in the Salmonella/microsome
1977 assay (50% benomyl as a wettable powder formulation) HLR No.
819-77. Report dd. 10/14/77 submitted to WHO by DuPont.
(Unpublished)

DuPont. Benlate dust exposure survey - blood profile analysis - 23
1979a February 1979, with supplement of 15 March 1979. Report
submitted to WHO by DuPont. (Unpublished)

DuPont. Benlate dust exposure survey. Report submitted to WHO by
1979b DuPont. (Unpublished)

DuPont. Applicator exposure during filling and spraying of Benlate
undated benomyl fungicide - orchard crops. Report submitted to WHO
by DuPont (Unpublished)

Ercegovich, C.D. & Rashid, K.A. Mutagenesis induced in mutant strains
1977 of Salmonella typhimurium by pesticides, 174th American
Chemical Society National Meeting. (Text of oral
presentation)

Evans, E.L. & Mitchell, A.D. An evaluation of the effect of benomyl on
1980 sister chromatic exchange frequencies in cultured Chinese
hamster ovary cells, SRI International, August 1980. Report
submitted to WHO by DuPont (Unpublished)

Everhart, L.P. & Holt, R.F. Potential Benlate fungicide exposure
1982 during mixer/loader operations, crop harvest and home use.
I. Agric. Food Chem. 30:222-227.

Ficsor, G., Bordas, S. & Stewart, S.J. Mutagenicity testing of
1978 benomyl, methyl 2-benzimidazolecarbamate, streptozotocin and
N-methyl-N¹-nitro-N-nitro-soguanidine in Salmonella
typhimurium in vitro and in rodent host-mediated
assays. Mutat. res., 51: 151-164.

Fisher, R.L. et al. Dermal absorption and fate of intravenously
1981 injected (2-¹⁴C)-benomyl in the rat. Report submitted to
WHO by DuPont. (Unpublished)

Fitzpatrick, K. & Krahn, D.F. Chinese hamster ovary cell assay for
1980 mutagenicity using technical benomyl (> 95% benomyl), HLR
No. 438-80. Report dd. 7/15/83 (revised) submitted to WHO by
DuPont. (Unpublished)

Frank K.M. Eye irritation tests in rabbits using technical benomyl
1968 (95% benomyl) and a wettable powder formulation (50%
benomyl) HLR No. 255-68. Report dd. 11/14/68 submitted to
WHO by DuPont. (Unpublished)

Frank, K.M. Skin irritation and sensitization tests on guinea pigs
1969 using a wettable powder formulation (50% benomyl) HLR No.
85-69. Report dd. 4/18/69 submitted to WHO by DuPont.
(Unpublished)

Frank, K.M. Eye irritation test in rabbits using a wettable powder
1972 formulation (50% benomyl) HLR No. 233-72. Report dd. 6/22/72
submitted to WHO by DuPont. (Unpublished)

Fritz, S.B. Acute oral ALD test in rabbits using a wettable powder
1969 formulation (50% benomyl) HLR No. 109-69. Report dd. 5/I/69
submitted to WHO by DuPont. (Unpublished)

Goldenthal, E.I. et al. Neurotoxicity study in hens using technical
1978 benomyl (>95% benomyl), with addendum. Report No. 125-028
dd. 3/3/78 by International Research and Development
Corporation submitted to WHO by DuPont. (Unpublished)

Gooch, J.J. Fertility of workers potentially exposed to benomyl.
1978 Report dd. October 1978 submitted to WHO by DuPont.
(Unpublished)

Goodman, N.C. Intraperitoneal LD₅₀ test in rats using technical
1975 carbendazim (less than 98%). Report from Haskell
Laboratories submitted to the WHO by DuPont. (Unpublished)

Gupta, A.K. & Legator, M.S. Chromosome aberrations in cultured human
1975 leukocytes after treatment with fungicide Benlate. In
Proc. Symp. Mutagenicity, Carcinogenicity and Teratogenicity
of Chemicals, Dept. of Atomic Energy, India, pp. 95-103.

Han, J. C-Y. Metabolism of ¹⁴C-labelled benomyl in the mouse and
1974 hamster. Report submitted to WHO by DuPont. (Unpublished)

Han, J. C-Y. Metabolism of 2-¹⁴C-benomyl in the lactating nanny goat
1980 (including supplements I and II). Report submitted to WHO by
DuPont. (Unpublished)

Hardesty, P.T. Attempts to characterize liver residues from ¹⁴C-
1982 benomyl dosed goat. Report submitted to WHO by DuPont.
(Unpublished)

Hastie, A.C. Benlate-induced instability of Aspergillus Diploids.
1970 Nature, 226: 771.

Hood, D.B. Fifteen exposure dermal test on rabbits using a wettable
1969 powder formulation (50% benomyl) HLR No. 211-69. Report No.
7/30/69) submitted to WHO by DuPont. (Unpublished)

Hornberger, C.S. Acute dust inhalation test in rats using a wettable
1969 powder formulation (50% benomyl) with report on
spermatogenesis effects HLR No. 95-69. Report dd. 4/24/69
submitted to WHO by DuPont. (Unpublished)

Jessep, D.C. & Dean, W. Acute delayed neurotoxicity study in chickens
1979 using technical benomyl (less than 95% benomyl). Report from
International Research and Development Corporation submitted
to WHO by DuPont. (Unpublished)

Jessup, C.D. Acute delayed neurotoxicity study in chickens using
1979 technical benomyl (>95% benomyl) HLO 674-79. Report dd.
12/7/69 by International Research and Development
Corporation submitted to WHO by DuPont. (Unpublished)

Kappas, A. et al. On the genetic activity of benzimidazole and
1974 thiophanate fungicides on diploid Aspergillus nidulans.
Mutat. Res., 26: 17-27.

Kappas, A. et al. Benomyl - A novel type of base analogue mutagen?
1976 Mutat. Res., 40: 379-382.

Kappas, A. & Bridges, B.A. Induction of point mutations by benomyl in
1981 DNA-repair-deficient Aspergillus nidulans. Mutat. Res.,
91: 115-118.

Kavlock, R.J. et al. Teratogenic effects of benomyl in the Wistar rat
1982 and CD-1 mouse with emphasis on the route of administration.
Toxicol. Appl. Pharmacol. 62: 44-54.

Kirkhart, B. Micronucleus test on benomyl (> 9% benomyl), SRI
1980 International, 12 Feb. 1980. Report submitted to WHO by
DuPont. (Unpublished)

Lamb, M.J. & Lilly, L.J. An investigation of some genetic
1980 toxicological effects of the fungicide benomyl. Toxicology,
17: 83-95.

Lee, K.P. The two-year feeding study in dogs with benomyl. Pathology
1970 Report No. 129-69 dd. 1/6/70 submitted to WHO by DuPont
(Unpublished)

Lee, K.P. The two-year feeding study in dogs with benomyl.
1971a Supplementary Pathology Report No. 53-71 dd. 7/28/71
submitted to WHO by DuPont (Unpublished)

Lee, K.P. The two year feeding study in dogs with benomyl.
1971b Supplementary Pathology Report No. 54-71 dd. 7/27/71
submitted to WHO by Du Pont. (Unpublished)

Lee, K.P. Testicular changes in control Beagle Dogs. Pathology Report
1971c No. 23-71 dd. 4/7/71 submitted to WHO by Du Pont.
(Unpublished)

Lee, K.P. The two year feeding study in rats with benomyl.
1977 Pathology Report No. 66-77 dd. 11/30/77 submitted to WHO by
Du Pont. (Unpublished)

Littlefield N.A. Four-hour acute inhalation exposure test in dogs
1969 using a wettable powder formulation (50% benomyl) HLR
192-69. Report dd. 7/14/69 by Hazleton Laboratories, Inc.
submitted to WHO by DuPont. (Unpublished)

Majkut, J.C. Skin irritation and sensitization tests on guinea pigs
1966 using technical benomyl (>95% benomyl) HLR No. 174-66.
Report dd. 9/28/66 submitted to WHO by DuPont (Unpublished)

Rashid, K.A. & Ercegovich, C.D. New laboratory tests evaluate
1976 chemicals for cancer or gene damage. Sci. Agric., 23:7.

Reinke, R.E. Eye irritation test in rabbits using technical benomyl
1966 (>95% benomyl) HLR No. 81-66. Report dd. 5/26/66 submitted
to WHO by DuPont. (Unpublished)

Richmond, D.V. & Phillips, A. The effect of benomyl and carbendazim on
1975 mitosis in hyphae of Botrytis cinerea Pers. ex Fr. and
roots of Allium cepa L. Pestic. Biochem. Physiol. 5:
367-379.

Russell J.F. Mutagenic activity of technical benomyl (> 95% benomyl)
1978 in the Salmonella/microsome assay HLR No. 18-78. Report
dd. 1/20/78 submitted to WHO by DuPont. (Unpublished)

Ruzicska, P. et al. Study on the chromosome mutagenicity of fundazol
1976 50WP (50% benomyl) Egeszsegtudomany (Budapest), 20: 74-83.

Sherman, H. Three-month feeding study in dogs using a wettable powder
1968 formulation (50% benomyl) HLR No. 269-68. Report dd.
11/20/68 submitted to WHO by DuPont. (Unpublished)

Sherman, H. Acute oral LD₅₀ test in rats using technical benomyl (>95%
1969a benomyl) and a wettable powder formulation (50% benomyl) HLR
No. 17-6. Report dd. 1/22/69 submitted to WHO by DuPont.
(Unpublished)

Sherman, H. Acute oral ALD test in a dog using technical benomyl (>95%
1969b benomyl) HLR No. 168-69. Report dd. 7/3/69 submitted to WHO
by DuPont. (Unpublished)

Sherman, H. Long-term feeding study in dogs with 1-butylcarbamoyl-2
1970 benzimidazole-carbamic acid, methyl ester (INT-1991). Final
report HLR No. 48-70 dd. 3/17/70 submitted to WHO by DuPont.
(Unpublished)

Sherman, H., Culik, R. & Zapp, J.A. Teratogenic study in rats with
1970 1-butylcarbamoyl-2-benzimidazolecarbamic acid, methyl ester
(INT-1991), Benlate; benomyl). Report submitted to WHO by
DuPont. (Unpublished)

Sherman, H. et al. Ninety-day feeding study in rats using a wettable
1967 powder formulation (70% benomyl) HLR No. 11-67. Report dd.
1/31/67 submitted to WHO by DuPont. (Unpublished)

Sherman, H. et al. Long-term feeding study in rats with
1969 1-Butyl-carbamoyl-2-benzimidazolecarbamic acid, methyl ester
(INT-1991). Final Report HLR No. 232-69 dd. 8/15/69 and
Addendum submitted to WHO by DuPont. (Unpublished)

Sherman, H., Culik, R. & Jackson, R.A. Reproduction, teratogenic and
1975 mutagenic studies with benomyl. Toxicol. Appl.
Pharmacol. 32: 305-315.

Sherman, H. & Fritz, S.B. Acute oral ALD test in rats using technical
1969 2-AB (>95% 2-AB) HLR No. 51-69. Report dd. 3/11/69
submitted to WHO by DuPont. (Unpublished)

Sherman, H. & Krauss, W.C. Acute oral ALD test and ten-dose subacute
1966 oral test in rats using technical benomyl (>95% benomyl)
HLR No. 100-66. Report dd. 7/15/66 submitted to WHO by
DuPont. (Unpublished)

Shirasu, Y. et al. Mutagenicity testing in fungicide 1991 (>95%
1978 benomyl) in microbial systems. Report by the Institute of
Environmental Toxicology submitted to WHO by DuPont.
(Unpublished)

Siebert, D., Zimmerman, F.K. & Lemperle, E. Genetic effects of
1970 fungicides, Mutat. Res., 10: 533-543.

Snee, D.A. Acute oral ALD test in rats and ten-dose subacute oral test
1969 in rats using technical 5-HBC (> 95% 5-HBC) HLR No. 134-69
with pathology on the acute oral ALD test described in HLR
No. 43-69. Report dd. 5/27/69 submitted to WHO by DuPont.
(Unpublished)

Staples, R.E. Teratogenicity study in the rat using technical benomyl
1982 (>95% benomyl) administered by gavage and supplement with
individual animal data HLR No. 582-82. Report dd. 10/1/82
submitted to WHO by DuPont. (Unpublished)

Staples, R.E. & Culik, R. Teratogenicity study in the rat after
1980 administration by gavage of technical benomyl (>95%
benomyl), Parts I, II and III. Reports submitted to WHO by
DuPont. (Unpublished)

Tates, A.D. Microtus oeconomus (Rodentia), a useful mammal for
studying the induction of sex-chromosome nondisjunction and
diploid gametes in male germ cells. Environ. Health
Perspect,, 31: 151-159.

Tong, C. Hepatocyte primary culture/DNA repair assay on compound 10,
1981a 962-02 (>95% benomyl) using mouse hepatoctytes in culture,
HLO 741-81. Report by the Naylor Dana Institute dd. 10/20/81
submitted to WHO by DuPont. (Unpublished)

Tong C. Hepatocyte primary culture/DNA repair assay on compound
1981b 10-962-02 (>95% benomyl) using rat hepatocytes in culture,
HLO 742-81. Report dd. 10/20/81 by the Naylor Dana Institute
submitted to WHO by DuPont. (Unpublished)

Wiechman, B.E. et al. Long-term feeding study with methyl
1982 1-(butylcarbamoyl)-2-benzimidazolecarbamate in mice
(INT-1991) - HLR No. 20-82 Parts I, II and III (>95%
benomyl). Report dd. 1/26/82 submitted to WHO by DuPont.
(Unpublished)

Zwicker, G.M. Acute oral ALD test in rats using technical benomyl
1965 (>95% benomyl) HLR No. 174-65. Report dd. 12/15/65
submitted to WHO by DuPont. (Unpublished)

RESIDUES

RESIDUES IN FOOD AND THEIR EVALUATION

USE PATTERNS

Information on approved uses of benomyl was available from the
United States, Algeria, Austria, Bulgaria, Denmark, France, Kenya, the
Federal Republic of Germany, the Netherlands, Spain, Switzerland, the
United Kingdom, Turkey, South Africa, Yugoslavia, Australia, Japan,
China (Taiwan), the Philippines, Indonesia, Chile and Colombia.
Because of its large volume, it was not practical to tabulate all
available information, but current registered uses in the United
States are presented in Table 1 as typical examples (DuPont 1983a).
Many other fruits, vegetables and commodities are included among the
registered uses of other countries where they are grown.

RESIDUES RESULTING FROM SUPERVISED TRIALS

Data on benomyl residues in crops following seasonal treatments
with Benlate WP applied according to US good agricultural practice
(GAP) are summarized in Table 2 (DuPont 1983b).

The results of supervised trials in the United States on head
lettuce are summarized in Table 3 (DuPont 1983c).

Supervised trials of foliar treatments (1 or 2 kg/ha) of wheat at
15 locations in the United States showed no detectable residues
(<0.05 mg/kg) in grain or in milled fractions (street grade flour,
red dog flour, bran, shorts, germ). A maximum of 6.8 mg/kg was found
in wheat straw. The results of extensive seed treatment trials
involving wheat, oats and barley showed that residues in either grain
or straw would not be likely to exceed 0.1 mg/kg (DuPont 1983d).

Data available from Kenya on residues in dried coffee beans 1 and
12 days after treatment at 1-1.5 kg/ha showed no measurable residues
(<0.1 mg/kg) (DuPont 1983e).

Residues of benomyl in hope treated in the Federal Republic of
Germany with two applications at 0.05 percent were 0.3 mg/kg in green
hope and 1.08 mg/kg in dried hope after a 29-day preharvest interval
and 0.51 mg/kg in green hope and 0.42 mg/kg in dried hope after a
25-day interval (DuPont 1983f).

Supervised trials in the Federal Republic of Germany at
recommended levels gave residues of 0.06-0.25 mg/kg for winter
wheat, 0.03-0.19 mg/kg for summer wheat, 0.06-0.25 mg/kg for oats,
0.03-0.13 mg/kg for rye and 0.03-2.53 mg/kg for grapes. (DuPont
1983g).

Supervised trials in England on tomatoes and cucumbers treated
at 50-100 g/100 1 and 38 g/100 1 respectively, resulted in residues
ranging from <0.02 to 1.17 mg/kg in tomatoes and from <0.02 to
0.5 mg/kg in cucumbers (DuPont 1983h).

Some test data from England on residues in the grain and straw of
winter wheat could not be interpreted owing to some anomalous results
(DuPont 1983i).

The results of supervised trials in Australia and Japan on a
variety of crops are summarized in Tables 4 and 5, respectively
(DuPont Australia 1983; DuPont 1983j).

Table 1 Registered Uses of Benomyl in the United States

Crop Application Pre-harvest
interval Remarks
Rate (kg a.i./ha or Number (days)
specified concentration

Almonds 1-1.5 1-2

Apples 0.1-0.5 at 7-14 day 30 also post-harvest
intervals uses

Avocados 1-2 3-4 wk 30
intervals

Beans 1.5-2 2 14 28 days for lima
beans

Blueberries 1 4 21

Cabbage 2 1-3 -- seed crop only

Cranberries 0.75 2-5 3

Celery 0.25-0.5 7-10 day
intervals 7

Citrus 1.5-3 1-2 0 also post-harvest
uses

Cucurbits 0.25-0.5 7-14 day --
intervals

Grapes 0.75-1.5 14-21 day 7 east of Rocky Mt.
intervals

1-1.5 2-4 7 west of Rocky Mt.

Table 1 (con't)

Crop Application Pre-harvest
interval Remarks
Rate (kg a.i./ha or Number (days)
specified concentration

Macadamia nuts 1.75 7-14 day intervals
through bloom period

Mangoes 1-2 weekly 14

Mushrooms 60 g/100 l 2 2

Peanuts 0.25 7-14 day 14
intervals

Pears 0.2-1.0 7-14 day -- also post-harvest
intervals uses

Pecans 0.5-1 3-4 wk -- do not apply after
intervals shucks split

Pineapple 240-480 g/100 l -- -- post-harvest

Rice 1-2 2 21

Soybeans 0.5-1 2 at 14-21 35
day int.

Stone fruits 0.75-2.0 1-2 none east of Rocky Mt.
1.5 -2.0 1-2 none west of Rocky Mt.
also post-harvest
uses

Table 1 (con't)

Crop Application Pre-harvest
interval Remarks
Rate (kg a.i./ha or Number (days)
specified concentration

Strawberries 1 7-14 day --
intervals

Sugarbeets 0.4-0.5 14-21 day 21
intervals

Sugarcane 30-60 g/100 l dip for seed -- Hawaii
pieces

Tomatoes 0.5-1 7-14 day --
intervals

Table 2 Benomyl Residues in Crops Following Seasonal Treatments

Application Residue (mg/kg) calculated
as benomyl¹ Control
Crop Number per
Rate season Avg. Range

Almonds
nutmeats 1.1-2.2 kg/ha 1 to 3 <0.1 - 0.1
hulls 1.1-2.2 kg/ha 1 to 3 0.34 0.2 - 0.74 0.2
Apples2 45-120 g/100 l 6 to 12 1.7 0.5 - 4.8 0.1
Post Dip 0.5 0.12 - 0.88

Apricots 30-60 g/100 l 2 to 3 4.5 2.6 - 6.5 0.1
Post Dip 2.9 0.9 - 5.1

Avocadoes 1.1-2.2 kg/ha 3 to 6 0.17 0.11 - 0.18 0.2

Bananas 0.2-0.3 kg/ha 6 to 9 <0.1²
30-60 g/100 l Post Dip 0.28 0.15 - 0.43 0.1

Beans 1.7-2.2 kg/ha 1 to 2 0.3 0.11 - 0.94 0.1

Bean vine forage 0.6-2.2 kg/ha 1 to 2 12.66 0.1 - 46 0.2

Blueberries IR-4 petition - not available.

Cranberries 0.6-2.2 kg/ha 2 to 5 2.8 0.75 - 6.0 0.1

Carrots 0.1-0.6 kg/ha 6 to 11 0.05 <0.05 - <0.05 0.05⁴

Celery 0.2-0.6 kg/ha 4 to 11 0.59 0.01 - 2.6 0.1

Cherries 30-60 g/100 l 2 to 3 3.2 0.2 - 12.6 0.1
30 g/100 l Post Dip 1.3 0.5 - 2.3 0.1

Table 2 (con't)

Application Residue (mg/kg) calculated
as benomyl¹ Control
Crop Number per
Rate season Avg. Range

Citrus³ 60-105 g/100 l 1 to 5 0.6 0.2 - 1.3 0.4
500 mg/l Post Dip 0.73 0.54 - 1.26
1000 mg/l Post Dip 1.1 0.39 - 2.35
1250 mg/l Post Dip 1.5 1.3 - 1.8
2500 mg/l Post Dip 3.3 2.7 - 3.9
5000 mg/l Post Dip 5.0 4.5 - 5.4

Cucumber 0.2-0.6 kg/ha 2 to 6 0.39 0.13 - 0.55 0.1

Grapes 1.1-1.7 kg/ha 3 to 5 3.2 0.14 - 10.3 0.1

Mangoes 1.1-2.2 kg/ha 5 to 17 0.7 0.15 - 3.0 0.1
Melons 0.2-0.8 kg/ha 2 to 7 0.29 0.27 - 0.32 0.1

Mushrooms IR-4 petition not available.

Nectarines 0.6 kg/ha 1 to 3 1.8 1.6 - 2.2 0.1

Macadamia Nuts 2.2-2.8 kg/ha 4 to 10 <0.1 - 0.1

Peaches 11-60 g/100 l 1 to 15 2.4 0.2 - 8.2 0.2
30 g/100 l Post Dip 4.3 3.9 - 4.7 0.2

Peanuts 0.2 - 0.6 kg/ha 2 to 13 0.1 - 0.1

Peanut hay & forage 0.4-1.1 1.1 kg/ha 3 to 13 7.7 1.4 - 1.6 0.4
0.4-1.1 kg/ha 3 to 13 7.7 1.4 - 1.6 0.4

Peanut hulls 0.2-1.1 kg/ha 1 to 13 0.58 0.16 - 0.98 0.4

Table 2 (con't)

Application Residue (mg/kg) calculated
as benomyl¹ Control
Crop Number per
Rate season Avg. Range

Pears³ 60-120 g/100 l 3 to 5 2.3 1.7 - 3.2 0.2
Post dip 0.38 0.19- 0.55

Pecans 1.1-1.4 kg/ha 3 to 6 <0.1 0.1

Pineapple⁵ 500 mg/l Post dip 6.8 0.1
1250 mg/l Post dip 8.9 0.1
2500 mg/l Post dip 26.7 0.1

Plums (fresh prunes) 30-60 g/100 l 2 to 4 0.8 0.4 - 1.4 0.2
30 g/100 l Post dip 1.2 0.5 - 1.9 0.2

Rice 0.3-1.1 kg/ha 1 to 2 0.49 0.05 - 2.8 <0.05⁴

Rice Straw 0.3-1.1 kg/ha 1 to 2 3.52 <0.05 - 9.0 <0.05⁴

Soybeans O.3-1.1 kg/ha 1 to 2 0.05 0.08- 0.05 <0.05⁴

Squash 0.4-0.8 kg/ha 2 to 5 0.3 0.10 - 0.50 0.1

Strawberries 30-60 g/100 l 3 to 7 1.1 0.43 - 2.6 0.4

Sugarbeet roots 0.2-0.6 kg/ha 3 to 6 <0.1 - 0.1
Sugarbeet tops 0.4-1.1 kg/ha 1 to 6 1.3 0.15 - 4.7 0.1

Tomatoes 15-120 g/100 l 1 to 9 1.1 0.1 - 4.8 0.4

Table 2 (con't)

Application Residue (mg/kg) calculated
as benomyl¹ Control
Crop Number per
Rate season Avg. Range

Wheat grain 0.3-1.4 kg/ha 1 to 2 0.06 <0.05 - 0.23 <0.05⁴

Wheat straw 0.3-1.4 kg/ha 1 to 2 1.5 <0.05 - 6.8 <0.05⁴

Lettuce 1.1-3.4 kg/ha 1 to 6 2.2⁶ <0.05 - 7.5 <0.05⁴

¹ Residue data is based on fluorometric/colorimetric procedure by Pease and Holt, unless otherwise indicated;

² Bananas - 0.1 mg/kg found in pulp from pre- or post-applications or a combination of these;

³ Pre and postharvest applications;

⁴ Residue analysis by liquid chromatographic method of Kirkland;

⁵ Postharvest application;

⁶ Average of both trimmed and untrimmed lettuce.

Table 3 Benomyl Residues in Lettuce from Supervised Trials
in the United States

Application Residue (mg/kg)¹
Interval
Location Rate (kg/ha) Number (days) Untrimmed Trimmed

Arizona
Scottsdale 1 5 9, 14, 22 0.67-3.0 <0.05-1.2
2 5 9 1.1-4.0 <0.05-0.72
1 5 9
2 5 9

Tacno 1 5 7 5.8-7.6 2.7-5.3
5 14,22 0.98-7.5 0.52-4.7
2 5 14,21 10-19 0.69-7.2

New York
Fulton 2 3 4-22 <0.05-2.7 <0.05-0.17
1.5 3 7 -- <0.05
3.0 3 15 -- <0.05-0.06
1.5 4 7 0.08
2.0 4 7 <0.05
3.0 4 7 0.09

Hannibal 2 4 7 <0.05

California
Salinas 1 2-4 9,22 <0.05-0.36²
2 3 9,22 <0.05-0.75²

New Jersey
Bridgeton 0.5 3 14
1.0 1 14 3.7²

Table 3 (con't)

Application Residue (mg/kg)¹
Interval
Location Rate (kg/ha) Number (days) Untrimmed Trimmed

Buena 1 3 1-13 0.09-18²
2 3 1-13 0.70-18²

¹ Measured as carbendazim by high performance liquid chromatography (HPLC).

² As received - number of wrapper leaves undefined.
Table 4 Residues of Benomyl from Supervised Trials on Crops in
Australia

Application
Residues Pre-harvest
Crop Rate and type Number (mg/kg) interval
(days)

Mangoes 25 g a.i./100 l 6 0.13 0
11 0.17 0
25 g+0.5 g/l dip 6 1.13 0
25 g+0.5 g/l dip 11 2.1 0
0.5 g/l dip 1 2.4¹ 0
1 1.8² 0
1 1.5³ 0

Rockmelons 525 mg/kg dip 1 1.24⁴
940 mg/kg dip 1 1.84⁴
210 mg/kg dip 1 0.66
405 mg/kg dip 1 0.97
615 mg/kg dip 1 1.23
790 mg/kg dip 1 1.57
985 mg/kg dip 1 1.90
397 mg/kg dip 1 1.60

Strawberries 650-1 300 g/ha 9 at 14-day 2.8-9.8 1-7
intervals

Citrus 0-1 000 mg/l dip 1 n.d.-1.7

Grapes 0.5-1 kg/ha 4 0.3-1.8 0-5

Wheat
grain 250-1 000 g/ha 4 <0.05 0-28
foliage 250-1 000 g/ha 4 4-31 0-28
straw 250-1 000 g/ha 4 0.6-5.6 0-28

¹ 36 hours after dip.
² 5 days after dip.
³ 7 days after dip.
⁴ <0.1 mg/kg in edible part.

Table 5 Benomyl Residues from Supervised Trials on Crops in Japan

Crop Application Interval after
last application Residue range
Rate & type Number (days) (mg/ha)

Rice (brown) 200 g/100 l soak 1 146 <0.03-0.03
0.5% seed coat 1 146 <0.03-0.04

Rice straw 200 g/100 l soak 1 146-180 0.06-0.07
0.5% seed coat 1 146-180 0.06-0.07
0.2-1 kg/100 l 3 122-138 <0.04-0.07

Cabbage 50 g/100 l 6 7-21 <0.05-0.06

Lettuce 50 g/100 l 2 3-14 0.02-1.10
50 g/100 l 4 3-14 0.03-1.58
50 g/100 l 6 3-14 0.03-0.77

Cucumber 50 g/100 l 1-3 1-7 0.10-0.75
200 g/100 l 2-3 14-45 0.07-0.38

Strawberry 200 g/100 l 1-2 156-268 0.02-0.16

Tomato 50 g/100 l 3-5 1-14 0.20-0.45
50 g/100 l 2-3 14-43 <0.02-0.38

Watermelon 50 g/100 l 5 1-19 0.03-0.05

Asparagus 50 g/100 l 5-6 259-315 <0.05

Onion 50 g/100 l + dip 7 1-7 <0.04-0.025
5 kg/100 l dip 1 112-142 0.02-<0.04

Sugarbeet 50 g/100 l 2-4 14-21 <0.008-2.02

Grape 50 g/100 l 1-5 30-122 <0.02-3.67

Citrus 25-50 g/100 l 1-3 1-14 0.004-1.31

Citrus juice 25-50 g/100 l 1-3 1 0.004-0.03

Peach 50 g/100 l 3-5 1-3 0.01-1.13

Pear 50 g/100 l 6 1-7 0.03-0.15

Table 5 (continued)

Crop Application Interval after
last application Residue range
Rate & type Number (days) (mg/ha)

Cherry 33 g/100 l 2 14 0.30-0.58

Chestnut 50 g/100 l 6 1-7 0.01-0.25

Kidney bean 100 g/100 l 2-4 7-21 0.04-0.37

Tea 50 g/100 l 1-2 7-28 0.50-35.2

Wheat 50 g/100 l 1 250-259 <0.02-<0.05

Potato 0.4% seed coat 1 142-152 <0.02-<0.03

Sweet potato 200 g/100 l 1 3-6 mo. 0.99-1.69
5 kg/100 l dip 1 117-141 <0.02-<0.04
0.4% seed coat 1 117-141 <0.02-<0.04

Taro 5 kg/100 l dip 1 162 0.06-0.08

Konnyaku 1-2 kg/100 l 1 150-176 <0.05-0.05

Shiitake 100 g/100 l 6 27-28 <0.04-0.05

Enokitake 100-250 mg/l 1 45-55 <0.04-0.28
nursery dip

Namekotake 0.02-0.04% 1 104-131 <0.05-<0.06
medium

FATE OF RESIDUES

In soil

Under anaerobic conditions in two soil types, ¹⁴C-labelled
benomyl was degraded only to carbendazim (methyl
benzimidazolecarbamate, MBC) and a small amount of
2-amino-benzimidazole (2-AB). Incorporation of the label into
soil humus was shown by fractionation studies (Han 1983a).

When benomyl was applied to flooded rice fields at recommended
rates water samples taken at various times showed residues (as
benomyl) from <0.02 to 0.05 mg/kg. Discharge water samples were in
the same range. Total residues (benomyl, MBC, and 2-AB) in muds ranged
as high as 3.8 mg/kg, indicating adsorption on soil particles (DuPont
1983k).

The stability of benomyl (¹⁴C-labelled) suspensions in water
was studied as a function of Ph, time and temperature. At pH 7.3 and
25°C, no STB (3-butyl-s-triazino(1,2,a-benzimidazole-2,4(1H,
3H)dione), BUB 2-(3-butylureido)benzimidazole or 2-AB (<0.1
percent) was formed during 6 h. After 49 h 0.9 percent STB was
present. Similar results were obtained at pH 9. At 50°C, 4 to 6
percent conversion to STB was observed in 1 h and 50 percent by
24-49 h. Less than 1 percent BUB was found after 49 h (Baude 1983a,b).

Benomyl was shown to inhibit drastically nitrification in soil at
high levels (1 000 mg/kg) as measured by nitrate production, even
after incubation for 30 days (Ramakrishna et al. 1979).

Photodegration

Apples were dipped into a suspension of ¹⁴C-benomyl (WP), air
dried and exposed on a window sill. Aliquots of a dried suspension on
glass plates were similarly exposed. After 16 days, 34 percent and 17
percent of the residue on apples and glass, respectively, was parent
compound and the remainder was MBC (Gardiner 1983). In a similar
experiment using oranges and glass plates with outdoor exposure, 61
percent and 19 percent of the parent compound remained on oranges and
glass, respectively after 15 days. The remainder was again MBC. Juice
from oranges exposed for 15 days contained only 0.05 mg/kg (Baude
1983c). Oranges and apples were treated with a wax coating containing
1 400 mg/kg ¹⁴C-Benlate, giving 10 mg/kg benomyl on oranges and
8 mg/kg on apples. After one week, the surface residue consisted of
only benomyl or MBC; no STB or BUB was formed (Baude 1983d).

In animals

A lactating female goat was given five consecutive daily doses of
2-¹⁴C-benomyl by capsule at a rate equivalent to 36 mg/kg in the
total daily diet. At sacrifice, 24 h after the last dose, small
amounts of radioactivity were detected in the liver (3.8 mg/kg),
bladder (4.5 mg/kg) and kidney (0.09 mg/kg). Activity in muscle and
fat was equivalent to <0.01 mg/kg. Most of the recovered
radioactivity (96 percent) was eliminated in the urine and faeces (Han
1983b). Incubation of liver tissue with either protease or amylase
failed to release any benomyl metabolites other than methyl
5-hydroxy-2-benzimidazolecarbamate. Further attempts to characterize
the liver residues were not successful, indicating that the
¹⁴C-residues were strongly bound or incorporated into the tissue
(Hardesty 1983).

In processing and storage

In simulated cooking experiments, fresh green beans were
fortified at the 2 mg/kg level with 2-¹⁴C-benomyl and boiled in water
for 30 min. Control water without beans was also boiled. Slightly more
than half of the residual was found in the water (as MBC) with the
remainder in the beans. This residue was identified as MBC
(99 percent) and a trace of 2-AB (Holt 1983).

Residue studies were conducted on soybean-based products such as
infant formulas, vegetable oil, salad dressing, etc. No residue
(<0.05 mg/kg as benomyl) was detected in any of the samples (DuPont
1983 1).

METHODS OF RESIDUE ANALYSIS

The residue data from supervised trials were mainly based on
the method of Pease and Holt (1971). A high performance liquid
chromatographic (HPLC) method has since been developed that can
distinguish between benomyl, MBC and 2-AB on apple foliage without
clean-up. This procedure seems promising for further development and
application (Chiba & Veres 1980).

RESIDUES IN FOOD IN COMMERCE OR AT CONSUMPTION

A market-basket analysis programme was conducted on fresh, frozen
and canned foods derived from crops normally treated with benomyl pre-
or postharvest. Eighteen food items from retail outlets in ten cities
in the United States were analysed for benomyl plus MBC as MBC using a
procedure with a limit of determination of 0.05 mg/kg. The foods
analysed were cucumbers, pickles, apples, applesauce, canned and fresh
pears, celery, canned grape juice, tomatoes, canned tomato paste,
puree and sauce, canned peaches, canned and frozen green beans,
strawberries, frozen orange juice concentrate, dried prunes, dried

apricots and rice. Of 227 food items analysed, 173 (76 percent) had no
detectable residues. Of the 54 samples having detectable residues, 26
had 0.1 mg/kg or less and three had more than 0.5 mg/kg. The highest
level found was 0.72 mg/kg on one sample of dried apricots (Baude
1983e).

NATIONAL MAXIMUM RESIDUE LEVELS REPORTED TO THE MEETING

The following MRLs were reported to the Meeting (DuPont 1981,
1983m,n).

Country Crop MRL (mg/kg)

Australia mushrooms, ginger, litchi, citrus 10
(postharvest dip)
strawberries 6
pome and stone fruits, mangoes 5
(postharvest dip)
avocados and vegetables 3
grapes, mangoes (pulp) (post- 2
harvest dip and rockmelons
(postharvest dip)
bananas 1
peanuts and water 0.2
sugarcane (preplanting) 0.1

Belgium cereals 0.5
cucurbits and melons 0.3
pepper 0.3
all other crops except potatoes 2.0
and citrus

Canada apples 5
apricots 5
beans 1
blackberries 4
boysenberries 5
carrots 5
cherries 5
citrus fruits 10
cucumbers 0.5
grapes 5
melons 0.5
mushrooms 5
peaches 10
pears 5

Country Crop MRL (mg/kg)

Canada (con't) pineapples (edible pulp) 1
plums 5
pumpkins 0.5
raspberries 6
strawberries 5
squash 0.5
tomatoes 2.5

Denmark citrus 10

Finland citrus 10

France apples 6
bananas 1
citrus 1.5
pears 6

Federal Republic bananas 0.2
of Germany berries 1.5
(expressed as MBC) citrus 7
cucumber 0.5
grains 0.5
grapes 3
pineapples 2
stone fruit 2
vegetables 1
other crops 0.1

Italy apricots 0.5
grains 0.5
grapes 1
melons 1
peaches 0.5
pears 1
prunes 0.5

Japan beans 0.5
fruits 0.7
materials for sugar 0.7
rice 0.5
tea 5
vegetables 0.8

Country Crop MRL (mg/kg)

The Netherlands apples 2
beans 2
celery 2
cucumbers 2
gherkin 2
melon 2
mushroom 0.5
onion 2
pears 2
peppers 2
scorzonera 2
shallots 2
strawberries 2
tomatoes 2
turnip roots 2
wheat 0.5

Norway citrus 10

Taiwan
(province of China) melon 3
rice 1.5
citrus 3
grape 3
apple 3
pear 3
sugarcane (juice) 1.5
banana 1.5
mushroom 1.5
papaya 1.5
tobacco 3
jujube 3
asparagus 0.3

United Kingdom Does not establish maximum residue limits

United States almonds
nutmeats 0.2
hulls 1
apples² 7
apricots 15
avocadoes 1

Country Crop MRL (mg/kg)

United States (con't) bananas 1
beans 2
bean vine forage 50
blueberries 7
cranberries 7
carrots 0.2
celery 3
cherries 15
citrus² 10
cucumber 1
grapes 10
mangoes 3
melons 1
mushrooms 10
nectarines 15
macadamia nuts 0.2
peaches 15
peanuts 0.2
peanut hay & forage 15
peanut hulls 2
pears² 7
pecans 0.2
pineapple¹ 35
plums (fresh prunes) 15
rice 5
rice straw 15
soybeans 0.2
squash 1
strawberries 5
sugarbeet roots 0.2
sugarbeet tops 15
tomatoes 5
wheat grain 0.2
wheat straw 15
lettuce 10

Yugoslavia fruits 10
vegetables 5

¹ Postharvest application.
² Pre and postharvest applications.

APPRAISAL

New information was received on approved uses of benomyl in 22
countries. Data were available on benomyl residues in crops following
seasonal treatments according to good agricultural practice in the
United States, Kenya, the Federal Republic of Germany, England,
Australia and Japan. The residues in commodities for which carbendazim
guideline levels have been recorded tend to support those levels, with
exceptions to be noted later, and can be used to propose additional
limits for asparagus, chestnuts, pineapples, taro and sweet potatoes.

Information on the fate of benomyl in soil continues to show that
the primary degradation product is carbendazim together with a small
amount of 2-AB. Benomyl is strongly bound to soil and does not enter
the water in flooded rice fields to any significant extent. Water
stability is temperature-dependent, leading to 50 percent conversion
to STB in 48 h at 50°C. High levels (1 000 mg/kg) of benomyl in soil
drastically inhibit nitrification. Photodegradation on surfaces (fruit
or glass) is slow, with the gradual formation of carbendazim over many
weeks.

In feeding studies with a lactating goat given 5 daily doses (by
capsule) of ¹⁴C-labelled benomyl, equivalent to 36 mg/kg in the
total daily diet, most of the fed benomyl (96 percent) was excreted in
the urine and faeces, with radioactivity equivalent to only about
3.8 mg/kg found in the liver, 4.5 mg/kg in the bladder and 0.09 mg/kg
in the kidney. Only methyl 5-hydroxy-2-benzimidazolecarbamate has been
identified as a liver metabolite.

Cooking (boiling) green beans fortified at 2 mg/kg with
radiolabelled benomyl extracted slightly more than half of the residue
into water (as carbendazim) with the remainder, also as carbendazim,
in the beans. Because of the degree of extractability into hot water
thus demonstrated, no maximum residue level for tea could be
estimated.

An analytical survey of soybean-based products such as infant
formulas, vegetable oils, etc. did not show any measurable residues
(<0.05 mg/kg).

The improved method of residue analysis of Pease and Holt (1971)
remains the one of choice for most commodities for regulatory use,
although newer HPLC-based methods are available, which show promise
for future development.

A market-basket type survey of eighteen food items from retail
sources in ten US cities showed that 76 percent had no detectable
residues. The highest residue found was 0.73 mg/kg in dried apricots.

Information on national maximum residue levels was available to
the Meeting for the United States, Belgium, Denmark, Finland, the
Federal Republic of Germany, Italy, Japan, The Netherlands, Norway,
Yugoslavia, Taiwan province of China, Canada and Australia.

In 1978 the Meeting agreed to replace guideline levels for
benomyl by those for carbendazim. Those figures are now converted to
MRLs on the basis of the establishment of an acceptable daily intake
for both compounds. On that basis, additional recommended maximum
residue limits for pineapples of 20 mg/kg, asparagus and taro of
0.1 mg/kg, chestnuts of 0.2 mg/kg and sweet potato of 1 mg/kg can be
estimated for carbendazim, based on benomyl supervised trials. The
data also support increasing the MRLs for carbendazim in wheat straw
from 2 to 5 mg/kg, in melons from 0.5 to 2 mg/kg and in peanut hulls
from 0.2 to 1 mg/kg.

RECOMMENDATIONS

See carbendazim. The following maximum residue limits, amending
or additional to those of 1978, are proposed for carbendazim, based on
supervised trials with benomyl. The figures were derived by dividing
residues expressed as benomyl in the trials data by the conversion
factor of 1.52.

Commodity Maximum residue limits Preharvest interval on
(mg/kg) which levels are based
(days)

*pineapples 20 postharvest dip

wheat straw 5(increased from 2) -

melons 2(increased from 0.5) postharvest dip

*sweet potatoes 1 90

peanut hulls 1(increased from 0.2) -

*chestnuts 0.2 1

*asparagus 0.1 ** 260

*taro 0.1 ** 162

* New MRLs
** At or about the limit of determination

REFERENCES- TOXICOLOGY

Baude, F.J. Benlate benomyl fungicide - stability in aqueous
1983a suspensions. DuPont research report. (Unpublished)

Baude, F.J. The stability of Benlate benomyl fungicide at neutral
1983b and alkaline pH levels. DuPont research report.
(Unpublished)

Baude, F.J. Examination of ¹⁴C-residues on glass and oranges
1983c treated with methyl 1 (butylcarbamoyl)-2-¹⁴C-
benzimidazolecarbamate. DuPont research report.
(Unpublished)

Baude, F.J. Residues on oranges and apples treated with a wax
1983d coating containing Benlate benomyl fungicide. DuPont
research paper. (Unpublished)

Baude, F.J. Benomyl crop residues - A United States market basket
1983e survey. DuPont research report. (Unpublished)

Chiba, M. & Veres, D.F. High performance liquid chromatographic
1980 method for simultaneous determination of residual benomyl
and MBC on apple foliage without cleanup. J. Assoc. Off.
Anal. Chem., 63: 1291-1295.

DuPont. Information table on examples of residue tolerance in
1981 countries outside of the United States, August 1981.

DuPont Table summarizing registered uses of benomyl in the
1983a United States. (Unpublished)

DuPont Table summarizing benomyl residues in crops from
1983b supervised trials in the United States. (Unpublished)

DuPont Benomyl residues in lettuce from supervised trials in the
1983c United States. (Unpublished)

DuPont Residue data on wheat, milled fractions, oats and barley.
1983d (Unpublished)

DuPont Data on residues in coffee beans from Kenya.
1983e (Unpublished)

DuPont Data on residues in hops from the Federal Republic of
1983f Germany. (Unpublished)

DuPont Data on residues in wheat, oats, rye, and grapes from the
1983g Federal Republic of Germany. (Unpublished)

DuPont Data on residues in tomatoes and cucumbers from England.
1983h (Unpublished)

DuPont Data on residues in wheat grain and straw from England.
1983i (Unpublished)

DuPont Data on residues in crops in Australia. (Unpublished)
1983

DuPont Data on residues in crops in Japan. (Unpublished)
1983j

DuPont Monitoring study- benomyl applied to flooded rice fields,
1983k February 1978. DuPont research report. (Unpublished)

DuPont Letter of 12-9-80 on research results - benomyl residue
1983 l data from soy-bean-based products. (Unpublished)

DuPont Information table on maximum residue limits for benomyl
1983m in Canada from the Food and Drugs Act and Regulations,
p. 65c, 3/8/79.

DuPont Information table on maximum residue limits for
1983n benomyl in Australia from the National Health and Medical
Research Council, 93rd Session, June 1982.

Gardiner, Examination of ¹⁴C-residues on glass and apples treated
J.A. with methyl 1-(butylcarbomoyl)-2-¹⁴C-
1983 benzimidazolecarbamate. DuPont research report.
(Unpublished)

Han, J. Anaerobic soil metabolism of 2-¹⁴C-benomyl and methyl
C-Y. 2-¹⁴C-benzimidazolecarbamate. DuPont research report.
1983a (Unpublished)

Han, J. Metabolism of 2-¹⁴C-benomyl in the lactating nanny goat,
C-Y. with supplements I and II. DuPont research reports.
1983b (Unpublished)

Hardesty, DuPont document No. AMR-71-82. (Unpublished)
P.T. 1983

Holt, R.F. Benomyl cooking studies. DuPont research report.
1983 (Unpublished)

Pease, H.L. Improved method for determining benomyl residues. J.
1971 & Holt, R.F. Assoc. Off. Anal. Chem., 54: 1399-1402.

Ramakrishna, C., Gowda, N. Effect of benomyl and its hydrolysis
1979 products, MBC and AB, on nitrification in a flooded soil.
Bull. T.K.S. & Environ. Contam. Toxicol., 21: 328-333.
Sethunathan,

    See Also:
       Toxicological Abbreviations
       Benomyl (EHC 148, 1993)
       Benomyl (HSG 81, 1993)
       Benomyl (ICSC)
       Benomyl (WHO Pesticide Residues Series 3)
       Benomyl (WHO Pesticide Residues Series 5)
       Benomyl (JMPR Evaluation 1995 Part II Toxicological and environmental)