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
CHLOROBENZILATE
Explanation
Chlorobenzilate was evaluated for an Acceptable Daily Intake by the
1965 and 1968 Joint Meetings (FAO/WHO, 1966; 1969). The 1968
Meeting reported that data from adequate short-term and long-term
studies were available upon which an estimation of the ADI could be
made. Additionally, biochemical studies on the metabolism and
biodegradation of chlorobenzilate were available. Based upon a
series of acceptable short-term and long-term studies in dogs and
rats and a three-generation reproduction study in rats, a no-effect
level in rats and dogs was estimated to be 2 mg/kg body weight and
12.5 mg/kg body weight, respectively. Based on these data an
Acceptable Daily Intake for humans was estimated to be 0-0.02 mg/kg
body weight.
The 1968 Meeting indicated its concern over neoplasms observed in
long-term studies in the rat and desired further information in a
second species.
In the interim since the evaluation of chlorobenzilate, two
carcinogenicity studies have been performed and reported. The data
from these newer studies serve as the primary basis for
re-evaluation of this chemical. Additionally, in the U.S. a
Rebutable Presumption Against Registration (RPAR) was issued for
chlorobenzilate suggesting two possible adverse health effects:
carcinogenicity and testicular damage. These factors have been
considered in a review of the new data in this monograph addendum.
DATA CONSIDERED FOR DERIVATION OF ACCEPTABLE DAILY
INTAKE
TOXICOLOGICAL STUDIES
Special studies on mutagenicity
Chlorobenzilate was inactive in inducing a mutagenic effect in two
species of microorganisms used as short-term in vitro test
systems (E. coli and S. cerevisiae) (Fahrig, 1974).
Groups of mice (8 males per group) were administered
chlorobenzilate orally on days 0, 2, 3, 5 and 9 at dosage levels of
0, 61, 122, or 244 mg/kg body weight in an effort to determine any
mammalian mutagenic potential through chromosomal examination of
spermatocytes. Three days after the final administration of either
chlorobenzilate or 2% CMC control solution, mice were administered
an intraperitoneal injection of 10 mg/kg coloemide and were
sacrificed. Preparations of testicular parenchyma were examined.
Primary and secondary spermatocytes of both the control and
treatment groups failed to show any aberrant forms in either the
primary or secondary spermatocytes. Under the conditions of this
trial, no evidence of mutagenic effects was noted with
chlorobenzilate (Hool and Müller, 1978).
Long-term studies
Mice
Groups of two strains of first generation hybrid mice (18 female
and 18 male mice per group) were administered chlorobenzilate for
83 weeks in a carcinogenicity screening study (Innes et al.,
1969). Chlorobenzilate was administered by gavage beginning at day
7 of age at dosage levels of 0 and 215 mg/kg bw. This dosing
regimen was continued daily for 28 consecutive days. After this
initial four week treatment period, chlorobenzilate was
administered ad libitum in the diet for the remainder of the
study at dosage levels of 0 and 603 mg/kg. In this carcinogen
screening study, where 120 compounds were investigated, 11
chemicals showed a relative risk for the development of tumours
that approached or exceeded that of the positive control chemicals.
Chlorobenzilate was among this group of substances, although it was
concluded that chlorobenzilate was not one of the more potent
carcinogens.
The relative risk of development of hepatomas approached that of
the positive control compounds in males of both strains treated
with chlorobenzilate. At the end of 83 weeks, a number of male
mice were reported to have hepatomas (9/17 and 7/18 of the two
strains tested). There were no hepatomas reported in the female
mice sacrificed at the conclusion of the study. This male response
was higher than that noted in control animals. There was no
apparent increase in mice with pulmonary tumours or lymphomas over
that noted in controls (Innes et al., 1969).
Groups of hybrid mice (50 male and 50 female mice, B6C3F1
strain/group; 20 of each sex were used as controls) were
administered chlorobenzilate in the diet for periods ranging up to
78 weeks. The control and treated mice were approximately 6 weeks
of age at the initiation of the test. On a time-weighted average
the dietary concentration for males was 0, 4, 231 or 7,846 mg/kg
and for females, 0, 3, 200 or 5,908 mg/kg. The initial dosage
levels administered to male mice were 0, 6,000, or 12,000 mg/kg.
At week 10, when the male mice ware 16 weeks old, these
concentrations were decreased to 4,000 or 8,000 mg/kg respectively.
This was as a result of a toxic reaction in the high dose level.
Female mice received dosage levels of O, 3,200 or 6,400 mg/kg which
were maintained at these levels throughout the trial. At week 53
of the study, administration of chlorobenzilate to the high dose
group of both males and females was suspended for one week because
of a toxic reaction (the appearance of a hunched posture reflective
of an acute toxic reaction). After this period a modified
treatment schedule for dietary administration of chlorobenzilate
was reinstituted, where the mice were administered chlorobenzilate
in a cycle of 1 week on a control diet and 4 weeks on a
chlorobenzilate diet. This cyclic treatment of male mice continued
until weeks 72 to 78 for the high dose and low dose, respectively.
There was a similar interrupted dosing regimen for female mice
after the initial 53-week dietary period. The pattern of high dose
dietary administration to female mice included a control diet
administered for one week followed
by four weeks of the chlorobenzilate diet. As with males, this
interrupted pattern was also followed for approximately 20 weeks
until the end of the treatment period. Animals were then
maintained on control diets in an observation period for 12-13
weeks until termination of the study.
Growth, food consumption, and behaviour data were recorded
throughout the study. At the conclusion of the study, animals were
sacrificed, and gross and microscopic examination of tissues and
organs were performed.
A dose-related growth depression was observed in both males and
females consistently throughout that part of the study during which
chlorobenzilate was administered. After that period, males rapidly
gained weight to where the final weight values equalled that of the
controls. The females were consistently smaller in the two treated
groups. Clinical signs of poisoning were observed during the first
10 weeks of the study in male mice. These clinical signs of
poisoning were relieved by a decrease in the concentration of
chlorobenzilate administered to mice. At approximately one year,
both males and females administered the high dose level displayed
clinical signs of poisoning. This was alleviated by intermittently
administering a control diet for one week and allowing the animals
to recover followed by reinstituting the treated diet for a period
of four weeks.
There was a substantial number of survivors through the 78-week
dietary administration and on to the conclusion of the study.
Hepatocellular carcinoma was found in both male and female mice
administered chlorobenzilate. The response was significantly
higher in frequency than the occurrence of the lesion in control
animals. Hepatocellular carcinoma appeared in 4/19 control males,
32/48 low dose males, 22/45 high dose males, 0/20 control females,
11/49 low dose females, and 13/50 high dose females. For male
mice, the statistical evaluation of the data indicated a
significantly larger incidence of hepatocellular carcinoma in the
low dose group than in the control. The results for hepatocellular
carcinoma were similar in female mice as both the low and the high
dose groups had significantly higher incidences of tumours than the
control group. There were no other neoplasms for which data were
statistically significant (with the possible exception of a
negative association between chlorobenzilate administration and
malignant lymphomas in female mice). Based on the results of this
study, evidence has been provided under the conditions of this
bioassay for carcinogenicity in hybrid mice treated with
chlorobenzilate in the diet for a substantial part of their
lifetime (NCI, 1978).
Rats
Groups of rats (50 male and 50 female rats, Osborne-Mendel
strain/group) were administered chlorobenzilate in the diet for
periods of time ranging from 73 to 78 weeks. All animals were
approximately 6 weeks old at the initiation of the test. The
initial concentration of chlorobenzilate to male rats was 0, 1,600,
or 3,200
mg/kg and for females was 0, 1,175 or 2,350 mg/kg. (A time-weighted
average dietary concentration was 0, 1,600 or 2,995 mg/kg for males
and O, 1,175 or 2,229 mg/kg for females respectively). The basal
diet contained 2% corn oil. In week 58 of the study,
administration of chlorobenzilate to the high dose male group was
interrupted for one week because of signs of toxic poisoning.
After this one week, administration was reinitiated and continued
for four weeks at the original concentration of 3,200 mg/kg. A
pattern of cyclic administration was instituted for the remainder
of the 78 weeks dosing interval. The same intake reduction was
employed for the high dose female group beginning with week 63 and
continuing until the conclusion of the treatment interval at 78
weeks. All animals were maintained on control diets for an
additional period of 32-33 weeks until termination of the study.
Growth, food consumption, toxicity, and behaviourial data were
recorded periodically throughout the study. Gross, and where
possible, microscopic examination of tissues and organs were
performed on animals that died during the course of the study. At
the conclusion of the study, gross and microscopic examination of
tissues and organs were performed and the data were analysed
statistically.
A dose-related depression of growth was observed in both male and
female rats beginning with day 10 and continuing throughout the
course of the study. The appearance and behaviour patterns
observed among the treated animals was similar to that of controls
during the first year. As the study progressed, more and more
signs of toxicity (hunched appearance and urine stains) were
observed. These were relieved somewhat in the high dose groups by
the intermittent treatment after one year of dietary exposure.
There appeared to be no substantial effect on survival as a result
of chlorobenzilate in the diet. Gross examination during the last
six months of the study revealed undersized gonads in several dosed
male rats, an observation subsequently confirmed at necropsy with
the finding of an increased incidence of compound-related
testicular atrophy (an incidence of 31/49, 26/49 and 9/44 in the
high dose, low dose, and control respectively).
Gross and microscopic examination at the conclusion of the study
revealed a wide variety of nonproliferative lesions of spontaneous
disease in both control and treated animals. A variety of
neoplasms were also observed among both treated and control
animals. A statistical analysis was performed with respect to
individual and total tumour incidence. It was reported that there
was an increased incidence of cortical adenoma of the adrenal gland
in both male and female rats. In males, there was a significantly
higher incidence of these tumours in the low dose group than in the
high dose or in the control groups. For female rats, there was a
significantly higher proportion of these tumours in the high dose
group than in the low dose or in control groups.
The incidence of adrenal cortical adenomas in the control rats of
this study was lower than the incidence in historical controls in
the same laboratory. The incidence of cortical adenomas of the
adrenal gland in rats administered chlorobenzilate in the diet was
elevated relative to both the controls utilised with the study and
to the historical controls reported for the laboratory performing
the study. The statistically significant increase observed with
respect to the concurrent controls was not noted with respect to
the historical controls.
Statistically, there was the possibility of a negative correlation
between chlorobenzilate administration and an incidence of
hemangiosarcoma in both male and female rats.
Under the conditions of this bioassay, it was concluded that
chlorobenzilate was not carcinogenic in Osborne-Mendel rats (NCI,
1978).
EVALUATION
COMMENTS
Chlorobenzilate was evaluated by the 1965 and 1968 Joint Meetings
(FAO/WHO 1966; 1969) and an Acceptable Daily Intake for man was
estimated to be 0-0.02 mg/kg body weight/day.
Data from carcinogenicity studies and a re-evaluation of previously
reported testicular damage served as a basis for re-evaluation of
this chemical.
Data have been presented that suggest a correlation between the
administration of chlorobenzilate at high dietary dosages and the
development of hepatomas in mice. In two older studies, as well as
a recent study, a carcinogenic potential with rats was not
observed. The finding of a carcinogenic response in the mouse
alone and the lack of such a response in the rat gave the Meeting
assurance that food residues would not represent a hazard to
humans.
The Meeting re-evaluated the potential for chlorobenzilate to
induce testicular damage as noted previously in chronic and
reproduction studies, and concluded that insufficient evidence
exists to suggest that testicular damage would result from
chlorbenzilate exposure.
Further evidence and supportive claims for safety with respect to
male reproduction have been made with new in vivo cytogenetic
studies in the mice which revealed no mutagenic effects on the
germinal epithelium and on spermatocytes in animals exposed to
chlorobenzilate. Additional mutagenicity studies are desirable.
The residues of chlorobenzilate in food and the human exposures
from such areas as occupational exposure do not appear to reflect
a high level of risk in light of the experimental data. An
evaluation of the
risk associated with the continued agricultural use of
chlorobenzilate and the resultant exposure of humans was made,
based on the potential carcinogenic and reproductive hazards
suggested by animal bioassays.
The Meeting concluded that a no-effect level could be established
from studies with rats and dogs and an ADI for man was reaffirmed.
Level causing no significant toxicological effect
Rat: 40 mg/kg in the diet, equivalent to 2 mg/kg bw/day.
Dog: 500 mg/kg in the diet, equivalent to 12.5 mg/kg bw/day.
Estimate of acceptable daily intake for man
0-0.02 mg/kg bw/day
FURTHER WORK OR INFORMATION
Additional mutagenicity assay.
REFERENCES
Hool, G. and Müller, D. Chromosome studies in male germinal
epithelium - mouse. (1978) Unpublished report from Ciba-Geigy,
Ltd., submitted to the World Health Organization by Ciba- Geigy,
Ltd.
Fahrig, R. Comparative Mutagenicity Studies with Pesticides in
Chemical Carcinogenesis Essays, I.A.R.C. Scientific Publication No.
10 p. 161-81.
Innes, J.R.M., Ulland, B.M., Valerio, M.G., Pettrucelli, L.,
Fishbein, L., Hart, E.R., Pallotta, A.J., Bates, R.R., Falk, H.L.,
Gart, J.J., Klein, M., Mitchell, I. and Peters, J. Bioassay of
Pesticides and Industrial Chemicals for Tumorigenicity in Mice: A
preliminary Note. J. Natl. Cancer Inst. 42: 1101-14
National Cancer Institute. Bioassay of Chlorobenzilate for Possible
Carcinogenicity. NCI Carcinogenesis Technical Report Series No. 75.
DHEW Publication No. (NIH) 78-1325.
See Also:
Toxicological Abbreviations
Chlorobenzilate (ICSC)
Chlorobenzilate (FAO Meeting Report PL/1965/10/1)
Chlorobenzilate (FAO/PL:1968/M/9/1)
Chlorobenzilate (WHO Pesticide Residues Series 2)
Chlorobenzilate (WHO Pesticide Residues Series 5)
Chlorobenzilate (Pesticide residues in food: 1977 evaluations)
Chlorobenzilate (IARC Summary & Evaluation, Volume 5, 1974)
Chlorobenzilate (IARC Summary & Evaluation, Volume 30, 1983)