Sponsored jointly by FAO and WHO


    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 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.



    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


    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

    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).


    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

    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,



    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

    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


    Additional mutagenicity assay.


    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,

    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)