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    PESTICIDE RESIDUES IN FOOD - 1979


    Sponsored jointly by FAO and WHO






    EVALUATIONS 1979





    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, 3-12 December 1979



    CHLORDIMEFORM

    Explanation

    Chlordimeform was evaluated in 1971, when an ADI and Residue Limits
    were established on a temporary basis.  In 1975 the temporary ADI was
    maintained and the list of crops covered by limits was increased.  In
    1976 and again in 1977 the Meeting noted that, as an immediate
    response to preliminary findings of these ongoing long term studies on
    mice which showed a potential carcinogenic response following long
    term exposure, the manufacturers had voluntarily and temporarily
    suspended the distribution of chlordimeform.

    In 1978 when the Meeting reviewed the situation in the light of the
    results of some further toxicological and residue studies, the
    temporary ADI was reduced and the need for further toxicological
    information was confirmed.  At the same time some temporary MRLs were
    proposed for certain crops on a basis of evidence of no detectable
    residues at the point of consumption.

    New and additional toxicological data are reviewed in this monograph
    addendum.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    Adverse effects in mice fed diets containing chlordimeform and its two
    principle metabolites, N-formyl-4-chloro-o-toluidine and
    4-chloro-o-toluidine respectively, were reported in 1976 as
    implicating these chemicals as tumor producing, inducing dose-related
    malignant hemangioendotheliomas of vascular origin in various tissues,
    mainly connective tissue.  Two NCI studies with rats have since failed
    to demonstrate a carcinogenic potential of 4-chloro-o-toluidine in
    the species (see Ulland, et al., (1979) for the details of these
    studies).  Moreover, a previously reported long-term study with
    chlordimeform did not suggest a carcinogenic potential in rats.

    In 1978, as 4-chloro-o-toluidine, a major metabolite of
    chlordimeform was noted to be mutagenic in several short-term bioassay
    systems, and in view of the tumorigenic potential of chlordimeform in
    mice, the temporary ADI was reduced.  The 1978 Meeting was informed
    that there was another series of long-term carcinogenic studies in
    rats currently in progress and that the data would be available for
    review in 1979.  As there were no current uses for chlordimeform on
    crops other than cotton, from which food residues had not been
    detected at the limit of the analytical method, the 1978 Meeting
    requested that data be made available for review in 1979.

    Complete results of the NCI bioassay of 4-chloro-o-toluidine in mice
    and rats and two-year studies with chlordimeform in rats have been
    submitted in detail to the meeting.  Interim reports, through 105
    weeks of dietary administration, were submitted presenting data on the
    chronic toxicity in rats of 4-chloro-o-toluidine and
    N-formyl-4-chloro-o-toluidine, the two major chlordimeform

    metabolites.  Complete results of these studies were not available but
    preliminary summary data were made available.  Additionally,
    comparative metabolic and pharmacokinetic data in both rats and mice
    were presented as were additional short-term in vivo tests
    evaluating the potential for mutagenic hazard.  This monograph
    addendum includes only that new information received and does not
    constitute a full review of the toxicology of chlordimeform.

    BIOCHEMICAL ASPECTS

    Absorption, Distribution and Excretion

    Chlordimeform was orally administered to rats and mice at a dose of 25
    mg/kg (14C-ring-chlordimeform).  Both sexes were used in the study.
    The general excretion pattern found with rats and mice was similar. 
    Of the applied 14C-dose, more than 70% was excreted within 24 hours
    with approximately 80 to 95% of the excretion being via the renal
    route and 10-15% eliminated in the faeces.  At the end of 144 hours,
    from 95 to 113% of the administered dose was recovered.  Residues were
    found to exist in liver, kidney and blood.  The residual radioactivity
    in rats appeared to be slightly higher than that found in mice, and
    within each species there were higher residues noted in females than
    in males.  Urine was the major excretory route with levels of
    radioactivity ranging from 82 to 97% of the administered dose.  Tissue
    residues in both species never exceeded 1 ppm at the conclusion of the
    study (after 144 hours) (Ifflaender, 1977a).

    In a study to evaluate quantitative excretion patterns following the
    administration of chlordimeform to both species (25 mg/kg, 14C-ring
    labelled), chlordimeform was again rapidly excreted in the urine of
    rats and mice (74% and 85%, respectively, of the administered dose was
    excreted).  The quantitative differences in both rats and mice with
    respect to individual metabolites or classes of metabolites were very
    small.  The only significant difference with respect to individual
    metabolites was noted with the N-methyl formamidine (rat, 11.3%;
    mouse, 2.4%) and the phenylurea (rat, 6.3%; mouse,1.2%).  In addition,
    mice excreted more sulfuric acid conjugates than rats (20.8% versus
    14.0%).  Verification of the qualitative determination of the
    metabolite substantiated the standard thin-layer chromatographic
    identification.  The metabolic profile in both rats and mice followed
    a well-defined pattern of oxidative demethylation (Ifflaender, 1977b).

    A pharmacokinetic study following oral administration of chlordimeform
    to female mice was performed at two dose levels using either a single
    acute or multiple daily administrations (for up to 21 days).  The two
    dose levels applied to mice were 0.03 mg/mouse (corresponding to 1.2
    mg/kg and 120 mg/kg body weight).  The results of the experiment
    showed that chlordimeform, and/or its major metabolite(s), was rapidly
    eliminated and did not accumulate in the animal or within any specific
    tissue or organ.  Excretion was principally via the renal route with
    the major concentrations again being excreted in 24 hours.  Data
    obtained during the course of repeated daily administration showed no

    differences with respect to bio-accumulation at either dose.  At the
    high dose level, a slightly reduced 24-hour excretion pattern was
    observed with the single administration.  The delayed excretion
    pattern of the radioactivity following administration of a single high
    dose disappeared within two or three doses in the multiple dosing
    regime.  After a period of 21 days, the percentage excretion was the
    same irrespective of dose.  It was concluded that chlordimeform
    following oral administration was rapidly absorbed, distributed and
    excreted from the body.  Excretion was largely complete within 24
    hours of discontinuation of administration.  The unexcreted material
    did not cumulate in the body or any particular tissue (Kopp, et al.,
    1977).

    TOXICOLOGICAL STUDIES

    Special Studies on Mutagenicity

    Dominant Lethal Study

    A chlordimeform metabolite (N-formyl-4-chloro-o-toluidine) was
    administered to male mice at dosages of 0, 105 and 315 mg/kg in a
    single oral treatment.  The mice were mated weekly to untreated
    females for a period of six weeks in a standard dominant lethal study.
    The females mated to males which had been treated did not differ
    significantly from those females mated to controls with respect to
    mating, the number of implantations, and the incidence of embryonic
    deaths.  There was no indication of dominant and lethal effect over
    the mating period of six weeks.  In addition, no effect was observed
    on the progeny of the male mice treated with N-formyl-4-chloro-
    o-toluidine (Fritz, 1978).

    A similar study was reported with the chlordimeform metabolite,
    4-chloro-o-toluidine administered to male mice in dosages of 0, 110,
    and 330 mg/kg.  Again, there was no evidence of dominant lethal effect
    with this metabolite (Fritz, et al., 1978).

    Hamster

    Groups of Chinese hamsters (4 males and 4 females per group) were
    orally administered chlordimeform at dose levels of 0, 60, 120, 240
    mg/kg on two consecutive days.  A positive control using
    cyclophosphamide (64 mg/kg) and a negative control of
    carboxymethylcellulose was used in an in vivo mutagenesis assay
    evaluating chromatid-type and chromosome-type aberrations in bone
    marrow cells.  In the two highest dose groups, no aberrations were
    observed.  In the low dose group, two chromosome-type aberrations per
    400 cells were observed.  While some miscellaneous aberrations were
    observed in the negative control group, a high incidence of
    chromatid-type and chromosome-type aberrations were found in the
    positive control.  There was a slight incidence of chromosome-type
    aberrations at the low dose group, in excess of those found in the
    negative control.  However, this incidence was significantly lower

    than that observed in the positive control group and was not dose
    related (Hool and Mueller, 1978).

    Groups of Chinese hamsters (6 males and 6 females per group) were
    administered the N-formyl metabolite of chlordimeform at dosage levels
    of 0, 300, 600 and 1200 mg/kg on two consecutive days, each consisting
    of one oral application.  Positive and negative controls using
    cyclophosphamide (128 mg/kg) or carboxymethylcellulose were employed.
    The study was designed as an in vivo mutagenesis assay evaluating
    potential mutagenic development in bone marrow cells.  Results from
    all dosage groups, showing the percentage of cells displaying
    anomalies of the nuclei, did not differ significantly from negative
    controls although the positive control yielded a marked increase in
    observable anomalies (Langauer and Muller, 1978a).

    A similar study was performed with 4-chloro-o-toluidine administered
    to Chinese hamsters at dosage levels of 0, 100, 200 and 400 mg/kg body
    weight.  Again, there were no significant differences with respect to
    the treatment groups with the exception of the positive controls
    (Langauer and Muller, 1978b).

    It was concluded that under the conditions of these experiments there
    were no mutagenic events elicited as a result of the administration of
    chlordimeform metabolites.

    Long-Term Studies

    In three basically identical studies, chlordimeform and its two
    principal metabolites, N-formyl-4-chloro-o-toluidine and
    4-chloro-o-toluidine, were tested in rats using a standard protocol
    to define carcinogenic hazards from long-term dietary administration.

    Rats

    Groups of rats (90 males and 90 females/group)  housed under SPF
    conditions, were fed N-formyl-4-chloro-o-toluidine in the diet at
    dosage levels of 0, 2, 20, 100 and 500 ppm for two years.  These
    dosage levels corresponded to dietary intakes of 0, 0.1, 1.0, 5 and 30
    mg/kg/day for females and 0, 0.1, 1.0, 4.0 and 24 mg/kg/day for males.

    Groups of 10 males and 10 females were sacrificed at periodic
    intervals (26 and 53 weeks).  Complete hematological, clinical
    chemistry, urinalysis, and gross and microscopic pathological
    examinations were performed at these intervals.  At 24 months, 20
    males and 20 females were sacrificed and examined.

    Food intake and growth were affected over the course of the study in
    the high dose group where slight growth retardation was observed.
    Clinical signs of toxicity or adverse behaviour were not observed.
    There was no mortality attributable to the presence of the
    cholordimeform metabolite in the diet.

    The results of the hematological investigations, blood chemistry data,
    urinalysis, organ weights and ratios, and the gross and microscopic
    evaluations are not complete (Sachsse and Suter, 1979a).

    Groups of rats (90 males and 90 females/group) housed under SPF
    conditions, were fed 4-chloro-o-toluidine in the diet at dosage
    levels of 0, 2, 20, 100 and 500 ppm for two years.  These dosage
    levels corresponded to dietary intakes of 0, 0.1, 1.0, 5.0 and 28
    mg/kg/day for females and 0, 0.1, 1.0, 4.6 and 24.6 mg/kg/day for
    males.  Groups of 10 males and 10 females were sacrificed at periodic
    intervals (26 and 53 weeks).  Complete hematological, clinical
    chemistry, urinalysis, and gross and microscopic pathological
    examinations were performed at these intervals.  At 24 months, 20
    males and 20 females were sacrificed and examined.

    Food intake and growth were affected over the course of the study in
    the high dose group of females only, where slight growth retardation
    was observed.  There was no effect on male growth at any dose level.
    Clinical signs of toxicity or adverse behaviour were not observed.
    There was no mortality attributable to the presence of the
    chlordimeform metabolite.

    The results of the hematological investigations, blood chemistry data,
    urinalysis, organ weights and ratios, and the gross and microscopic
    evaluations are not complete (Sachsse and Suter, 1979b).

    Groups of rats (90 males and 90 females/group, Tif: RAIF strain) were
    maintained under SPF conditions and fed chlordimeform in the diet for
    24 months at dosage levels of 0, 2, 20, 100 and 500 ppm.  These
    dietary levels, based on food consumption data, were equivalent to a
    dosage level of 0, 0.1, 1.0, 5.0 and 24 mg/kg/day for males and 0,
    0.1, 1.2, 6.0 and 28 mg/kg/day for females.  Groups of 10 to 20 of
    each sex were sacrificed periodically (26 and 53 weeks) for clinical
    laboratory investigations including hematology, blood chemistry,
    urinalysis, and gross and microscopic examinations of tissues and
    organs.

    Excessive mortality was not observed over the course of the study.
    Growth and body weights were maintained in all groups with the
    exception of the 500 ppm level where growth in both sexes was slightly
    retarded.  There were no clinical signs of poisoning or abnormal
    behaviour.  Eye and ear examinations, performed at periodic intervals,
    revealed no adverse effects attributable to chlordimeform.
    Methemoglobinemia was observed at dosage levels of 20 ppm and above.
    Changes in several other blood chemistry parameters (Heinz body
    formation and reduced glucose levels) were observed only at 500 ppm.
    Ketonuria and proteinuria were observed at the highest dose level in
    young rats (noted only at the earliest examination).  Urinalysis
    performed at 13 weeks and thereafter for the remainder of the study
    did not reveal these occurrences.  Gross pathology and organ weight
    data did not show any significant dose-related responses.  While
    several organ weight and organ to body or brain weight ratios showed
    statistically significant differences from control animals, the

    findings were not dose-related and the biological significance of
    these random occurrences is doubtful.  Microscopic histophathologic
    analyses of tissues and organs indicated no significant changes
    attributable to chlordimeform in the diet.  Although numerous benign
    and malignant tumors were observed in both treated and control
    animals, the frequency and type of neoplasms were not dose-related nor
    could they be attributable to chlordimeform.  Several congenital
    degenerative or inflammatory changes were attributed to diseases
    common to older animals.  Based on the hematological occurrence of
    methemoglobinemia in young rats and several other clinical changes
    observed over the course of the study, the no-effect level of
    chlordimeform for both male and female rats is 2 ppm.  Over the course
    of the study there was, again, no indication of carcinogenic potential
    to rats as a result of the presence of chlordimeform in the diet
    (Sachsse, et al.,1979).

    COMMENTS

    Chlordimeform, an insecticide and acaracide, was reviewed in 1971,
    1975 and 1978 (FAO/WHO 1972; 1976; FAO, 1979).  In 1978, it was
    concluded that chlordimeform and its principle metabolites were
    carcinogenic in the mouse, producing a dose-related malignancy
    histologically characterized as hemangioendothelioma.  Carcinogenicity
    studies with rats produced conflicting data and were in the process of
    being repeated.  In addition, several bioassay systems had suggested a
    potential for mutagenicity based on short-term studies.

    The 1978 Joint Meeting reduced the temporary ADI.  A temporary ADI was
    estimated in 1978 to be 0-0.0001 mg/kg body weight with the assurance
    that current agricultural uses on cotton do not result in terminal
    residues within the limits of analytical detection and that exposure
    to workers has been minimized.

    Two studies have confirmed the carcinogenicity of a
    4-chloro-o-toluidine in mice and the lack of such carcinogenic
    events in chronic studies with rats.

    Further confirmation of the lack of carcinogenicity of chlordimeform
    in rats was presented to the meeting in new detailed long-term
    studies.  Chlordimeform is not carcinogenic to rats when administered
    in the diet over a 24-month period.  With consideration given to
    certain adverse clinical and hematological parameters, predominantly
    at the early stages of the trial, it was suggested that 2 ppm in the
    diet represented a no-effect level for rats.

    Preliminary results of chronic studies in rats with N-formyl-4-
    chloro-o-toluidine and 4-chloro-o-toluidine, were received.
    However, as the histopathology data were incomplete, these studies
    could not be utilized to assess the carcinogenic potential of the
    metabolites.

    Chlordimeform is rapidly absorbed, distributed, metabolized, and
    excreted in rats and mice in a similar manner with no evidence of
    bio-accumulation or unusual metabolite formation.

    Additional mutagenicity bioassays using an in vivo bone marrow assay
    in Chinese hamsters and a dominant lethal assay in mice again failed
    to demonstrate a mutagenic potential of chlordimeform.

    Several of the concerns expressed by previous meetings were resolved
    with data submitted to this Meeting.  Additional data on chronic
    studies with chlordimeform metabolites should be reviewed when
    completed.  A further long-term bioassay in an additional species
    would be of value in more fully evaluating the carcinogenic response
    noted in mice.  A no-effect level based upon somatic effects was
    ascertained with rats and dogs.  It was concluded that, based on all
    available data and considering the limited agricultural programme to
    which chlordimeform is being applied, a temporary ADI was reaffirmed. 
    The potential for carcinogenic response continues to be of concern to
    the Meeting and maintenance and reporting on an extensive occupational
    exposure surveillance programme is recommended for future evaluations.
    The Meeting was also concerned over the lack of response to the
    previous request for data on the potential for hemorrhagic cystitis.

    TOXICOLOGICAL EVALUATION

    Level Causing No Toxicological Effect

    Rat:  2 ppm in the diet equivalent to 0.1 mg/kg body weight.
    Dog:  250 ppm in the diet equivalent to 6.25 mg/kg body weight.

    Estimate of Temporary Acceptable Daily Intake for Man

    0-0.0001 mg/kg body weight.

    FURTHER WORK OR INFORMATION

    Required before July 1980:

    1.  Submission of data on completed long-term studies with
    chlordimeform metabolites.

    2.  Report on continued surveillance and epidemiological studies of
    occupationally exposed workers.

    3.  Report on the possible occurrence of hemorrhagic cystitis.

    Desired:

    1.  Confirmatory long-term animal bioassay using a third species for
    evaluating the potential carcinogenic hazard.

    REFERENCES

    Fritz, H.  Dominant Lethal Study, Mouse.  (1978) Unpublished report
    from Ciba-Geigy, Ltd., Basel, Switzerland, submitted by Ciba-Geigy,
    Ltd.

    Fritz, H., Becker, H. and Muller, D.  Dominant Lethal Study of
    (4-chloro-o-toluidine HCl), Mouse. (1978) Unpublished report from
    Ciba-Geigy, Ltd., Basel, Switzerland, submitted by Ciba-Geigy, Ltd.

    Hool, G. and Muller, D.  Chromosome Studies on Somatic Cells (C-8513),
    Chinese Hamster.  (1978) Unpublished report from Ciba-Geigy, Ltd.,
    Basel, Switzerland, submitted by Ciba-Geigy, Ltd.

    Ifflaender, U.  Distribution, Degradation and Excretion of
    Chlordimeform (CDF) in Mice and Rats.  (1977a) Unpublished report from
    Ciba-Geigy, Ltd., Basel, Switzerland, submitted by Ciba-Geigy, Ltd.

    Ifflaender, U.  Comparison of the Urinary Metabolite Pattern of Mice
    and Rats After Oral Application of 14C Chlordimeform.  (1977b)
    Unpublished report from Ciba-Geigy, Ltd., Basel, Switzerland,
    submitted by Ciba-Geigy, Ltd.

    Kopp, R., Hümpel, M., Kuhne, G., Fuchs, R. and Rzadkiewicz, M.
    Pharmacokinetics of Chlordimeform Following Single and Repeated Oral
    Administration of 0.03 and 3 mg in Female Mice (1.2 and 120 mg/kg).
    (1977) Unpublished report from Ciba-Geigy, Ltd., Basel, Switzerland,
    submitted by Ciba-Geigy, Ltd.

    Langauer, M. and Muller, D.  Nucleus Anomaly Test in Somatic
    Interphase Nuclei, Chinese Hamster.  (1978a) Unpublished report from
    Ciba-Geigy, Ltd., Basel, Switzerland, submitted by Ciba-Geigy, Ltd.

    Langauer, M. and Muller, D.  Nucleus Anomaly Test in Somatic
    Interphase Nuclei, Chinese Hamster.  (1978b) Unpublished report from
    Ciba-Geigy, Ltd., Basel, Switzerland submitted by Ciba-Geigy, Ltd.

    Sachsse, K., Suter, P., Luetkemeier, H., Zak, F., Hess, R. and Zakova,
    N.  Chlordimeform HCl Lifespan (Chronic Toxicity and Carcinogenicity)
    Feeding Study in Rats.  (1979) Unpublished report from Ciba-Geigy,
    Ltd., Basel, Switzerland, submitted by Ciba-Geigy, Ltd.

    Sachsse, K. and Suter, P.  Lifespan (Chronic Toxicity and
    Carcinogenicity) Feeding Study in Rats with
    N-formyl-N-chloro-o-toluidine. Status Report After 24 Months of
    Treatment.  (1979a) Unpublished report from Ciba-Geigy, Ltd., Basel,
    Switzerland, submitted by Ciba-Giegy, Ltd.

    Sachsse, K. and Suter, P.  4-chloro-o-toluidine HCl.  Lifespan
    (Chronic Toxicity and Carcinogenicity) Feeding Study in Rats. Status
    Report After 24 Months of Treatment.  (1979b) Unpublished report from
    Ciba-Geigy, Ltd.

    Ulland, B., Gordon, E., Cardy, R. and Creasia, D.  Bioassay of
    4-chloro-o-toluidine Hydrochloride for Possible Carcinogenicity.
    (1979) Unpublished Report of the NCI Frederick Cancer Research
    Center-NCI #C-02368.
    


    See Also:
       Toxicological Abbreviations
       Chlordimeform (EHC 199, 1998)
       Chlordimeform (ICSC)
       Chlordimeform (WHO Pesticide Residues Series 1)
       Chlordimeform (WHO Pesticide Residues Series 5)
       Chlordimeform (Pesticide residues in food: 1978 evaluations)
       Chlordimeform (Pesticide residues in food: 1980 evaluations)
       Chlordimeform (Pesticide residues in food: 1985 evaluations Part II Toxicology)
       Chlordimeform (Pesticide residues in food: 1987 evaluations Part II Toxicology)
       Chlordimeform (IARC Summary & Evaluation, Volume 30, 1983)