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.