CHLOROTHALONIL
First draft prepared by A.L. Black
Department of Health, Housing and Community Services
Canberra, Australia
EXPLANATION
Chlorothalonil has been evaluated by the Joint Meetings in
1974, 1977, 1979, 1981, 1983, 1985, 1987, and 1990 (Annex I,
references 22, 28, 32, 36, 40, 44, 50, 59, and 65). In 1990 the
Joint Meeting allocated an ADI of 0.03 mg/kg bw for chlorothalonil,
based upon the results of a two-year feeding study in dogs. A WHO
Member State has since requested reconsideration of this ADI and a
clarification of the basis on which it was established. This request
and additional information submitted to the Meeting, including a
reproduction study in rats, were considered. The additional data are
summarized in this monograph addendum.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
Short-term toxicity studies
Dogs
In a 30-day feeding study, encapsulated chlorothalonil (97.9%
purity) was administered orally to groups of 2 male and 2 female
beagle dogs at 0, 50, 150, or 500 mg/kg bw/day. The following
tissues were examined macroscopically and microscopically at
necropsy: brain, liver, kidneys, testes with epididymis, ovaries,
adrenals, heart, thyroid and parathyroid. During treatment high-dose
dogs exhibited emesis and weight loss and reduced food consumption
(males only). Female dogs had slightly reduced bodyweight gains at
all doses. At necropsy, liver weights of high-dose females were
slightly increased. There were no microscopic changes in the tissues
examined. Due to the reduced body-weight gains of treated females, a
NOAEL was not established in this study (Fullmore & Laveglia, 1992).
In a two-year study, chlorothalonil (93.6% purity) was fed to
groups of four beagle dogs at dietary concentration of 0, 1500, 15
000 or 30 000 ppm equivalent to 0, 37.5, 375 or 750 mg/kg bw/day)
for two years. Biochemical and haematological parameters were
routinely monitored at days 21 and 45 and months 6, 9, 12, 18, and
24 months. Eight dogs, one of each dose/sex/group, were sacrificed
at 12 months and the remainder at 24 months. One dog of each
treatment group lost weight during the study. There was a tendency
for mild anaemia in four mid-dose dogs at two years and at earlier
intervals in two high-dose dogs. Biochemical and urine analyses were
unremarkable, apart from slightly decreased urinary specific gravity
at mid- and high-dose. At terminal necropsy, there were compound-
related changes in liver, thyroid and kidneys especially at mid- and
high-doses. Absolute and relative thyroid and kidney weights were
increased and liver/body-weight ratios were increased at mid- and
high-dose. Histopathological examination was performed only on
liver, thyroid, kidney, stomach, small and large intestine tissues
for mid- and low-dose dogs. Treatment-related changes occurred in
liver, thyroid, kidney and stomach of mid- and high-dose dogs.
Changes in low-dose dogs were equivocal. In the liver, the findings
were similar in nature but only slightly more pronounced at low-dose
than controls, but increased in severity at mid- and high-dose. They
included pericholangitis with associated portal fibrosis, bile duct
hyperplasia and pigmentation of hepatic cytoplasm and of macrophages
of sinusoids and portal triads. Generalized atrophy of hepatocytes
with cytoplasmic vacuolation and nuclei enlargement occurred at mid-
and high-doses. Renal glomerulosclerosis and degenerative renal
tubular changes (tubular hypertrophy and dilation) were found in the
kidneys of mid- and high-dose dogs. In the thyroid, markedly
increased pigmentation of follicular epithelia occurred in mid- and
high-dose dogs. Moderate to severe gastritis was found irregularly
in mid- and high-dose animals.
In summary, administration of chlorothalonil in the diet of
dogs at concentrations of 15 000 and 30 000 ppm caused irregular
body-weight reduction, borderline anaemia and histopathological
changes to liver, kidney, thyroid and stomach. At low-dose, 1500
ppm, the histopathological changes found in the liver were
qualitatively similar but minimally to slightly increased in
comparison to those found in control animals. Histopathological
changes to other tissues were otherwise unremarkable at the low-
dose. A NOAEL was not established in this study (Paynter & Busey,
1966).
In a 16-week dietary study, chlorothalonil (purity unspecified)
was fed at 0, 250, 500 or 750 ppm to groups of four beagle dogs.
There were no compound-related effects on appearance, behaviour,
appetite or body-weight. No changes in haematological parameters
were found at weeks 0, 4, 13 and 16. At termination, protein-bound
iodine was found to be increased in all treated dogs. Urinalysis at
weeks 6, 9, 13 and 16 was unremarkable. No compound-related
macroscopic or microscopic changes were found at necropsy. In
particular, only incidental changes were observed in liver and
kidneys. A NOAEL was not established in this study (Paynter &
Murphy, 1967).
Groups of beagle dogs (8 males and 8 females per group) were
fed chlorothalonil in the diet for 2 years at dosage levels of 0, 60
or 120 ppm. There were no effects noted on behaviour or growth over
the course of the study. Clinical chemistry values including
haematology, biochemistry and urine analysis, were comparable to the
controls at all levels of feeding. Gross and microscopic examination
of tissues and organs performed on animals sacrificed at 12 months
indicated a compound-related change in the kidney. Further
examination of tissues and organs at 24 months did not show
chlorothalonil-related abnormalities. A slight degree of renal
tubule vacuolation in two of four animals at 120 ppm after two years
in the absence of other changes (urinalyses values) was considered
questionable, especially as a slight degree of vacuolation was noted
in control as well as other treated animals (Holsing & Voelker, 1970
- cited in Annex 1, reference 23).
Reproduction study
Rats
In a two-generation, two litter per generation, reproduction
study in Charles River CD rats, groups of 35 animals of each sex
received technical chlorothalonil (98.1% purity) at dietary
concentrations of 0, 500, 1500 or 3000 ppm for 10 (F0) and 14
(F1) weeks prior to mating and thence continually. At the time of
mating, low-dose males consumed approximately 25 mg/kg bw/day and
females 32 mg/kg bw/day; mid-dose males consumed approximately 75
mg/kg bw/day and females 100 mg/kg bw/day; for the high-dose groups,
males consumed approximately 156 mg/kg bw/day and females 205 mg/kg
bw/day. There was no mortality or clinical signs of toxicity in the
parental animals. Body-weight depression occurred in the parents of
both generations with males being more sensitive than females. The
F0 rats had a dose-related decrease in body-weight of high-dose
and mid-dose males and high-dose females, while, in the F1
parents, the depression of body-weight occurred in the high-dose
groups of each sex only. Mating and fertility indices and duration
of gestation were unaffected by treatment. Litters were culled at
day 4 to 8 pups/litter and litter weights were determined at days 0,
4, 7, 14 and 21. At necropsy the parental animals exhibited similar
pathological findings of forestomach and kidneys to those found in
previous studies (Annex I: 46); mainly hyperkeratosis and squamous
epithelial hyperplasia of the forestomach and epithelia hyperplasia,
tubular hypertrophy and clear cell hyperplasia of the kidney. Males
were more sensitive to these renal effects than females. There was
no treatment-related effect on the incidence of malformations,
livebirths or stillbirths, lactation index or sex ratio of the pups.
At day 21 only pup weights of all high-dose groups were
significantly reduced; the mean body-weights of mid-dose F1b were
reduced at days 4, 7, 14 and 21 and there was a slight (ca. 10%)
depression of mean pup body-weight of the low-dose F2b at day 21
only. Necropsy findings for all groups were unremarkable. The NOAEL
for maternotoxicity in this study was 1500 ppm, equal to 75 mg/kg
bw/day (Lucas & Benz, 1990).
Although inadequate by contemporary standards, a previous
multigeneration reproduction study with chlorothalonil showed no
effect of chlorothalonil on reproduction at high doses that were
maternally toxic (suppression of body-weight gain, Annex 1,
reference 23). Other reproduction studies with the metabolite of
chlorothalonil, 4-hydroxy-2,5,6-trichloroisophthalonitrile, have
previously shown diverse effects (reduced fertility index, reduced
litter size and weight and increased pup mortality as well as
reduced maternal bodyweight gain (Annex 1, references 23 and 33). A
NOAEL for reduction of pup bodyweight by the metabolite of the order
of 10-30 ppm was indicated (Annex 1, references 37 and 41).
The present 2-generation reproduction study confirmed
depression of maternal body-weight, without other adverse effects on
reproduction per se, as the most sensitive endpoint. The pup body-
weight depression seen at 21 days could be attributed to direct
consumption of feed containing chlorothalonil. The NOAEL of 1500 ppm
for this study does not take into account the toxicity to
forestomach and kidneys for which NOAELs have been established in
previous studies undertaken at lower doses.
COMMENTS
The new reproduction study in rats showed a NOAEL of 1500 ppm,
for maternotoxicity without adverse effects on reproduction, equal
to 75 mg/kg bw/day.
The ADI allocated in 1990 was based on the NOAEL of 120 ppm
equivalent to 3.0 mg/kg bw/day, determined by the Joint Meeting in
1974 on review of a two-year feeding study in beagle dogs. This
NOAEL was revised by the 1987 Joint Meeting to 60 ppm, equivalent to
1.5 mg/kg bw/day, but it was subsequently restored to its original
value, 3.0 mg/kg bw/day, by the 1990 Meeting after consideration of
an independent review of the histopathology which indicated that the
renal tubular epithelial vacuolation found in the study was in all
probability an artifact of fixation.
Concern has been raised recently over the validity of the 1970
study in dogs. The study has again been reviewed by the present
Meeting and found to be adequate for evaluation.
Three additional studies with chlorothalonil at higher doses in
beagle dogs were considered. None of these showed a no-effect level.
In a 30-day study at 0, 50, 150 or 500 mg/kg bw/day, reduced body-
weight gain occurred. In a 16-week study at 0, 250, 500 or 750 ppm,
protein-bound iodine was increased at all doses. In a two-year study
at 0, 1500, 15 000 or 30 000 ppm, weight-loss occurred at all doses.
In addition, thyroid and kidney weight and liver/bodyweight ratios
were increased at mid- and high-doses. Treatment-related
histopathological changes occurred in the liver, kidneys, and
stomach of mid- and high-dose dogs.
A range of genotoxicity studies, in vivo and in vitro, were
considered by the Joint Meeting in 1985 and 1987. The present
Meeting confirmed that the data previously reviewed did not show a
genotoxic hazard of chlorothalonil for humans.
Feeding chlorothalonil to rats for two years produced gastric
and renal toxicity, hyperplasia and neoplasia. Renal epithelial
hyperplasia and forestomach hyperplasia/hyperkeratosis occurred with
a NOAEL of 1.5 mg/kg bw/day. Renal tumours, adenomas and carcinomas,
and non-glandular gastric papillomas and squamous cell carcinomas
occurred with a NOAEL for these effects of 3.3 mg/kg bw/day.
Similar findings in a two-year study in mice have increased
concern over the carcinogenic potential of chlorothalonil. Mice had
demonstrated similar sensitivity to gastric hyperplasia and
hyperkeratosis (NOAEL 15 ppm, equal to 1.6 mg/kg bw/day) and
papilloma formation (NOAEL 21 mg/kg bw/day) but they are somewhat
less susceptible than rats to chlorothalonil renal toxicity (NOAEL
for renal epithelial tubular hyperplasia in males, 4.5 mg/kg bw/day)
and renal neoplasia (NOAEL for males, 21 mg/kg bw/day).
The 1990 Joint Meeting concluded that the gastric lesions in
rats and mice were attributable to the irritancy of chlorothalonil
and so had little relevance for humans. The present Meeting
confirmed this interpretation and agreed that the gastric lesions
occurring in rodents were an inappropriate basis for the estimation
of an ADI.
Previous Joint Meetings considered the results of comparative
metabolic studies in rats, germ-free rats, monkeys and dogs.
Quantitative differences in the absorption, distribution,
metabolism, and excretion of chlorothalonil and its metabolites were
noted. The urinary metabolites of chlorothalonil differed in each
case. Orally-dosed normal rats excreted significantly more urinary
thiols than orally-exposed germ-free or dermally-exposed normal
rats. Monkeys excreted significantly lower levels of thiols than
rats. Thiols were not detected in the urine of treated dogs. This
suggested that the intestinal flora of the rat significantly
influences the metabolic fate of chlorothalonil in that species and,
indirectly, its renal toxicity. Accordingly, the 1990 Joint Meeting
considered that these results suggested "that the dog or the monkey
may be more suitable models than the rat for predicting the
metabolism of chlorothalonil by man." The present Meeting recalled
that the rat is well known to have significantly different
gastrointestinal flora than humans (WHO, 1987).
Studies reviewed by previous Joint Meetings have shown that
chlorothalonil reacts in vitro with glutathione (GSH) to produce
mono-, di-, tri- and possibly tetra-conjugates with chlorothalonil.
Dithiodichloroisophthalonitrile and trithiochloro-isophthalonitrile,
in both sulphhydryl free and methylated forms, are known to occur as
metabolites of chlorothalonil in rat urine. Orally administered
monoglutathione conjugates of chlorothalonil are further conjugated
with GSH in the gastrointestinal tract of rats prior to absorption.
In a 90-day gavage study in rats with the monoglutathione conjugate
of chlorothalonil, renal toxicity was induced at 150 mg/kg bw/day. A
similar study with equimolar concentrations of chlorothalonil showed
renal toxicity at 75 mg/kg bw/day with a similar pattern of urinary
metabolites. A mechanism for glutathione conjugation in oncogenesis,
and for the causation of nephrotoxicity and renal carcinogenicity by
certain chloroalkenes in rats, has been established (Neal et al.,
1990; Deleant et al., 1990). These findings suggest a role for
glutathione conjugation in the biotransformation and renal toxicity
of chlorothalonil in rats.
Overall, the Meeting considered that there was sufficient
concordance between the results of metabolism and toxicity studies
to establish that normal rats were sufficiently different from germ-
free rats, monkeys and dogs to be discounted as a model for ADI
estimation. Accordingly the Meeting used the most sensitive
toxicological endpoint that it considered to be appropriate, the
NOAEL established in the two-year study in dogs. A safety factor of
100 was applied.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Mouse: 15 ppm in the diet, equal to 1.6 mg/kg bw/day (two-
year study reviewed by the 1987 JMPR)
Rat: 1.5 mg/kg bw/day (two-year study reviewed by the 1990
JMPR)
Dog: 120 ppm in the diet, equivalent to 3.0 mg/kg bw/day
(two-year study)
Estimate of acceptable daily intake for humans
0-0.03 mg/kg bw
Studies which will provide information valuable in the continued
evaluation of the compound
1. Further clarification of the mechanism of nephrotoxicity
and renal carcinogenicity in rats and mice
2. Information on the relevance of findings in animal studies
to humans, including results of the metabolism study in
dogs known to be in progress
3. Observations in humans.
REFERENCES
W. Deleant et al. (1990) A mechanism of haloalkene-induced renal
carcinogenesis. Environmental Health Perspectives, 88: 107-110.
Fullmore, G.E. & Leveglia, J. (1992) A thirty day toxicity study in
dogs with T-117-2. Unpublished report 5092-91-0554-TX003 by Ricerca
Inc. Submitted to WHO by ISK Biotech. Corporation, Mentor, Ohio,
USA.
Holsing, G.C. & Voelker, R.W. (1970) (cited in Annex I: 23). 104-
week dietary administration - dogs, Daconil 2787 (technical).
Unpublished report by Hazleton Laboratories Inc. Submitted to WHO by
Biotech Corporation, Mentor, Ohio, USA.
Lucas, F. & Benz, G. (1990) A two generation reproduction study in
rats with technical chlorothalonil. Unpublished report
Chlorothalonil: No. 1722-87-0121-TX-003 from Ricerca, Inc.,
Painesville, Ohio, USA. Submitted to WHO by Biotech Corporation,
Mentor, Ohio, USA.
Neal, G.E., Moss, E.J. & Manson, M.M. (1990) Glutathione conjugation
in oncogenesis. In Glutathione Conjugation. H. Sies & B. Kitterer
(Eds.) Academic Press, London, pp 281-308.
Paynter, O.E. & Busey, W.M. (1966) Two year dietary administration -
dogs DAC 2787. Unpublished report by Hazleton Laboratories Inc.
Submitted to WHO by Biotech Corporation, Mentor, Ohio, USA.
Paynter, O.E. & Murphy, J.C. (1967) 16-Week dietary administration -
dogs DAC-2787. Unpublished report by Hazleton Laboratories Inc.
Submitted to WHO by Biotech Corporation, Mentor, Ohio, USA.
WHO (1987) Principles for the safety assessment of food additives
and contaminants in food. Environmental Health Criteria 70, Section
5.2, Geneva.