PESTICIDE RESIDUES IN FOOD - 1980
Sponsored jointly by FAO and WHO
EVALUATIONS 1980
Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Expert Group on Pesticide Residues
Rome, 6-15 October 1980
CARBOFURAN
Explanation
Carbofuran was evaluated for an acceptable daily intake in 1978 and
1979 and a temporary ADI for man was estimated to be 0-0.003 mg/kg
body weight (FAO/WHO 1977; FAO 1980). The available data reflected
that carbofuran is a highly toxic carbamate ester whose metabolic
profile has been well defined. Carbofuran is a potent, reversible
cholinesterase inhibitor. Cholinesterase inhibition and acute toxic
signs of poisoning are subject to rapid spontaneous reversal and
recovery. The measurement and evaluation of cholinesterase depression
induced by carbofuran, because of the rapid reversibility, is
difficult and requires substantial care.
In a variety of short-term and long-term studies, in several animal
species, cholinesterase depression was the principal effect noted.
The 1976 Meeting expressed concern over the lack of appropriate data
evaluating the reversible cholinesterase depression in dietary studies
and on the apparent sensitivity of brain rather than erythrocyte or
plasma cholinesterase. No-effect levels were based on data from a rat
reproduction study (short-term) and a short-term dietary study in
dogs.
Further studies on cholinesterase depression, induced by dietary
administration of carbofuran, were requested. It was suggested that a
definition be made of the sensitivity of juveniles, when compared with
adults on acute toxicity and cholinesterase depression levels, as
observed in preliminary studies and that an additional reproduction
study define the highest no-effect level.
Additional data were received with respect to all of the requested
information. In addition, further long-term studies in several rodent
species were made available. These new data are reviewed in this
monograph addendum.
DATA CONSIDERED FOR DERIVATION OF ACCEPTABLE DAILY
INTAKE
BIOCHEMICAL ASPECTS
Effects on enzymes and other biochemical parameters
An evaluation was made on the possible differences in brain and
erythrocyte cholinesterase activity in adult and juvenile rats of both
sexes following acute exposure to carbofuran.
Groups of rate (five rats per sex, using either juvenile or adult
animals) were sacrificed and cholinesterase activity measured by a
colorimetric procedure suitable for evaluating a rapidly reversible,
carbamate-induced inhibition. These animals were untreated and served
as baseline values of cholinesterase activity. A further group of
eight animals per sex, both juvenile and adult, were treated with
carbofuran and sacrificed at various times up to 24 hours after
treatment to evaluate the optimal time for depression of both brain
and erythrocyte cholinesterase. In all cases, sample preparation was
rapid following sacrifice and dilution of tissues was kept to a
minimum. Baseline cholinesterase data in brains of juveniles were
slightly higher than those noted for adults in both males and females.
With erythrocyte cholinesterase, adult values were found to be higher
than juvenile values in both males and females. Examination of the
optimal sampling time for evaluating maximum erythrocyte
cholinesterase depression showed that approximately 30 minutes
following acute administration was the optimal in both sexes in adults
and juveniles. The optimal sampling time for brain cholinesterase
depression was approximately one hour following acute poisoning. The
data showed recovery was almost complete four hours following acute
carbofuran treatment. Complete recovery was noted at the 24-hour
interval. While the cholinesterase depression and recovery studies
were difficult to fully evaluate because of large standard deviations
of the mean cholinesterase activities, it was quite evident that
complete recovery of all enzyme depression was attained within one
day. There were no significant differences in juvenile and adult
recovery studies in either brain or erythrocyte cholinesterase.
Further, the data did not suggest significant differences in
sensitivity to carbofuran-induced inhibition in either adult or
juvenile rats (Case, 1980).
Toxicological studies
Special studies on reproduction
Groups of rats (10 male and 20 female rats per group, 12 male and 24
females per group were used in the third generation) were fed
carbofuran in the diet at dosage levels of 0, 20, or 100 mg/kg and
subjected to a standard 2-litter per generation, 3-generation
reproduction study.
Growth, as evidenced by differences in body weight and food
consumption, was reduced in parental rats administered 100 mg/kg in
the diet. This was consistent throughout three generations at the 100
mg/kg dosage level. No treatment-related growth or food consumption
depression was noted with any of the animals administered 20 mg/kg in
the diet.
There was no effect associated with carbofuran with respect to the
standard reproduction parameters (fertility, gestation, lactation, and
viability) with the exception of a slight reduction in viability at
day 4 at 100 mg/kg. This was observed in the first litter of all
generations. These differences were not noted in the second litters
of the 100 mg/kg group and the significance of this reduction is
questionable. Mean pup body weights in the 100 mg/kg group were
consistently lower than control values. This growth reduction, noted
at day 21, was statistically significant in both males and females at
the 100 mg/kg dosage level throughout the study.
At the conclusion of the study, parental animals and pups from the
second litters of the second and third generations were sacrificed and
subjected to gross examination. No pathological lesions or
abnormalities, which were considered to be compound-related, were
noted at the time of gross examination. Statistically significant
mean weight variations occurred in various organs among the F2 and
F3b weanling rats. The significance of these weight variations,
which appear to occur frequently in toxicity studies, is questionable
in light of the microscopic examination of tissues and organs which
did not appear to show substantial effects attributable to the
presence in the diet of carbofuran. Microscopic examination of
tissues and organs of the parental F2b and F3b showed a variety of
morphologic changes, none of which were considered to be related to
the presence of carbofuran. All histologic lesions were considered to
represent spontaneous occurrences. Based upon the slight, but
significant, growth reduction at 100 mg/kg, it may be concluded that
20 mg/kg represents a dietary level that would induce no effect on
reproduction (Goldenthal, 1979b; 1980b).
Special studies on teratogenicity
Groups of rate (24 mated females per group) were administered
carbofuran (suspended in 0.25% methylcellulose) at dosage levels of 0,
0.1, 0.3, or 1.0 mg/kg body weight/day throughout gestation (days 6-15
of pregnancy). On day 20 of gestation, animals were sacrificed and
parents and foetuses were subject to complete internal and external
examination to evaluate effects indicative of a teratogenic response
of carbofuran.
Toxicological signs of poisoning were evidenced in females at the two
higher dose levels. There was one death at the highest dose level.
The signs of poisonings were of the typical cholinergic response
associated with carbofuran. There were no differences in growth
within any treatment group over the 20-day gestation period. At the
conclusion of the study, examination of all animals showed a larger
number of resorptions in the control group than in any of the
treatment groups. There were no differences within the groups
administered carbofuran and the high incidence of resorptions in the
control group could not be explained. Somatic and skeletal
examinations failed to show teratogenic effects of the administration
of carbofuran. Carbofuran, while inducing toxicological signs of
poisoning in maternal rats, induced no teratogenic response in rats
(Barron, et al., 1978).
Groups of pregnant rabbits (17 rabbits/group) were administered
carbofuran ( in 0.25% methylcellulose) at dosage levels of 01 0.21
0.6l or 2.6 mg/kg body weight daily from day 6-18 of gestation.
On day 30, all animals were sacrificed and, following laparotomy, an
examination of the parents and foetuses was performed to evaluate a
potential teratogenic effect. For the parents, this included an
examination for early and late resorptions, implantation sites,
abnormal placental sites, and for any other abnormalities. Corpora
lutea were counted and the placenta was weighed. Gross examinations
were made of the parents and somatic and skeletal examinations of
foetuses were performed.
Toxic signs of poisoning were observed in rabbits administered the
high dose level of carbofuran. Toxic cholinergic signs of poisoning
were evident and several animals died during the course of the study.
There ware no toxicological signs of poisoning evident at the lower
dose levels. Growth, over the period of gestation, did not appear to
be affected by the administration of carbofuran. There were no
significant gross somatic effects observed in either parents or
foetuses at any dose level in the study. Pregnancy and viability was
unaffected by carbofuran. Foetal body weights, placental weights, and
development of foetuses were not affected. Soft tissue and skeletal
examinations showed no adverse effect as a result of carbofuran
treatment. Under the conditions of this bioassay, carbofuran did not
induce a teratogenic effect in rabbits at dose levels up to and
including that which induced maternal toxicity (Felton, et al.,
1978).
Long-term studies
Mouse
Groups of mice (100 male and 100 female, Charles River CD-1
mice/group) were fed carbofuran in the diet at dosage levels of 0, 20,
125 or 500 mg/kg for two years in an effort to evaluate the
carcinogenic potential of carbofuran. Groups of animals were examined
at periodic intervals (6, 12, 18, and 24 months) for haematologic
parameters, biochemical studies, and urinalyses. At similar intervals
over the course of the study, groups of animals were sacrificed and
subjected to gross and microscopic examination of tissues and organs
for abnormalities, particularly to assess the carcinogenic potential.
There was no mortality in the course of the study that could be
attributed to the presence of carbofuran. Additionally, no changes in
appearance or behaviour were noted. A localized hair loss and
reddening of the ear(s) frequently followed by scabbing or sloughing
of portions of the ears was noted with greater frequency in the
treated mice. There was no mortality attributable to carbofuran. A
slight decrease in body weight was evidenced through the first year of
the study predominantly at the high dose level. During this period
where growth reduction was noted, food consumption was also reduced
slightly at the high dose level. With the exception of brain
cholinesterase depression, there were no specific differences with
respect to haematological parameters, clinical chemistry parameters,
and urinalysis determinations that could be attributable to
carbofuran. Brain cholinesterase was depressed in both males and
females at the two highest dose levels and all time intervals
recorded.
At the conclusion of the study, gross and microscopic examination of
tissues and organs showed several statistically significant weight
variations occurring periodically throughout the study. These changes
in organ weights were not accompanied by changes observed
microscopically which would be indicative of significant morphologic
effects. There were no differences with respect to the occurrence of
neoplastic or non-neoplastic changes in any of the carbofuran-treated
mice. It was concluded that carbofuran was not tumorigenic nor
carcinogenic in this strain of mice (Goldenthal, 1980; Brown, 1980).
Rat
Groups of rats (90 male and 90 female/group) were fed carbofuran in
the diet at dosage levels of O, 10, 20, or 100 mg/kg for 2 years.
Carbofuran was dissolved in acetone and mixed with a small amount of
laboratory diet to provide a premix, which was then diluted to provide
larger quantities of the appropriate concentration for the test diet.
Analytical evaluations of the test diets at periodic intervals
verified the presence and concentration of carbofuran.
Rats were observed daily for overt toxicity, mortality, and general
behaviour and appearance. Body weights were measured weekly, as was
food consumption. At 6, 12, 18 and 24 months, groups of animals were
sacrificed for a variety of clinical chemistry studies, haematologic
examinations, and urinalysis. Ophthalmologic examinations were
performed at 0, 12, and 24 months. Groups of 10 animals of each sex
were sacrificed at 6, 12, and 18 months and a final sacrifice of all
survivors was made at the conclusion of the study. Gross and
microscopic examinations of tissues and organs were performed on all
animals that died during the study or were sacrificed at periodic
intervals.
There was no excessive mortality over the course of the study that
could be attributed to the presence of carbofuran. Growth, as
evidenced by body weight, was depressed significantly at the high dose
level, although food consumption was unaffected.
With the exception of cholinesterase activity depression, there were
no significant findings in any of the clinical chemistry, haematology,
urinalysis, or ophthalmological examinations. At 100 mg/kg
cholinesterase depression was observed in plasma, red blood cell, and
brain of both male and female rats. There was no significant
cholinesterase depression observed at 20 mg/kg. (Cholinesterase
depression was evaluated in the study using specific techniques and
procedures that would be applicable for assessing the reversible
nature of the inhibition.)
Analysis of the gross and microscopic pathology data did not suggest a
compound-related pattern in any of the lesions observed. While there
were sporadic lesions observed in a variety of tissues at various
sacrifice times with respect to gross changes in weight, these were
not accompanied by histological evidence of adverse morphologic
effects. It was considered that both neoplastic and non-neoplastic
histologic changes represented spontaneous lesions and were unrelated
to the presence of carbofuran in the diet. Gross lesions without
definitive morphologic change were assumed to have represented
physiological variations, artifacts, or other causes that did not
induce detectable morphologic alterations on histological
examinations.
A no-effect level in this study is 20 mg/kg in the diet equivalent to
1.0 mg/kg body weight. This is based on changes in growth and on
depression of cholinesterase activity level observed at the highest
dose level (Goldenthal, 1979a; Rapp, 1980a).
EVALUATION
COMMENTS
Carbofuran, an anticholinesterase carbamate ester currently in
extensive use as an insecticide, has been reviewed at two previous
meetings. Additional studies made available to this meeting were
reviewed.
There were no differences between juvenile and adult rats with respect
to cholinesterase inhibition. Studies on reproduction, including an
evaluation of the teratogenic potential of carbofuran, showed that
carbofuran was not teratogenic and did not affect reproduction in
mammals.
Long-term dietary studies on both rat and mouse failed to demonstrate
a carcinogenic potential for carbofuran. In two new long-term
studies, a dietary no-effect level was observed. Cholinesterase
depression was the most sensitive effect noted in these studies.
The data reviewed met the requirements of previous meetings and
allowed the estimation of no-effect levels in two mammalian species
(mouse and rat). The rat and the mouse were used in an evaluation of
the no-effect levels. Data with the dog (1976 report) showing a
no-effect level of 50 mg/kg were based on clinical signs of poisoning
where cholinesterase assays were not performed. The available studies
with the dog are of limited value in the toxicological evaluation as
cholinesterase data were not included.
Level causing no toxicological effect
Rat: 20 mg/kg in the diet equivalent to 1.0 mg/kg bw/day.
Mouse: 20 mg/kg in the diet equivalent to 2.5 mg/kg bw/day.
Estimate of acceptable daily intake for man
0-0.01 mg/kg bw/day.
REFERENCES
Barron, P,. Giesler P., and Rao, G.N. Teratogenicity of Carbofuran in
Rats. (1978) Unpublished report from WARF Institute submitted by the
FMC Corporation to the World Health Organization.
Brown, W.R. 2-Year Dietary Toxicity and Carcinogenicity Study in Mice.
Technical Carbofuran Histopathology. (1980) Unpublished report from
Research Pathology Services, Inc. submitted by the FMC Corporation to
the World Health Organization.
Case, R.S. A Comparative Evaluation of Cholinesterase Activity of
Juvenile and Adult Rats Treated with Carbofuran. (1980) Unpublished
report from the FMC Corporation submitted by the FMC Corporation to
the World Health Organization.
Felton, S., Gielsler, P., and Rao, G.M. Teratology Study of Carbofuran
in Rabbits. (1978) Unpublished report from WARF Institute submitted by
the FMC Corporation to the World Health Organization.
Goldenthal, E.I. A Two-year Dietary Toxicity and Carcinogenicity Study
in Rats. (1979a) Unpublished report from International Research and
Development Corporation submitted by the FMC Corporation to the World
Health Organization.
Goldenthal, E.I. Three-generation Reproduction Study in Rats. (1979b)
Unpublished report from International Research and Development
Corporation submitted by the FMC Corporation to the World Health
Organization.
Goldenthal, E.I. Two-year Dietary Toxicity and Carcinogenicity Study
in Mice. (1980) Unpublished report from International Research and
Development Corporation submitted by the FMC Corporation to the World
Health Organization.
Rapp, W.R. A 24-month Dietary Toxicity/Carcinogenicity Study of
Carbofuran in Rats - Pathology report (terminal phase). (1980a)
Unpublished report from Wm. R. Rapp and Company submitted by the FMC
Corporation to the World Health Organization.
Rapp, W.R. A Three-generation Reproduction Study of Carbofuran in
Rats. Pathology report. (1980b) Unpublished report from Wm R. Rapp.
and Company submitted by the FMC Corporation to the World Health
Organization.
See Also:
Toxicological Abbreviations
Carbofuran (ICSC)
Carbofuran (Pesticide residues in food: 1979 evaluations)
Carbofuran (Pesticide residues in food: 1996 evaluations Part II Toxicological)
Carbofuran (JMPR Evaluations 2002 Part II Toxicological)