First draft prepared by A.L. Black
    Department of Health, Housing and Community Services
    Canberra, Australia


         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.


    Short-term toxicity studies


         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,

         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


         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.


         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.


    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

         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.


    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,

    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.

    See Also:
       Toxicological Abbreviations
       Chlorothalonil (EHC 183, 1996)
       Chlorothalonil (HSG 98, 1995)
       Chlorothalonil (ICSC)
       Chlorothalonil (WHO Pesticide Residues Series 4)
       Chlorothalonil (Pesticide residues in food: 1977 evaluations)
       Chlorothalonil (Pesticide residues in food: 1981 evaluations)
       Chlorothalonil (Pesticide residues in food: 1983 evaluations)
       Chlorothalonil (Pesticide residues in food: 1985 evaluations Part II Toxicology)
       Chlorothalonil (Pesticide residues in food: 1987 evaluations Part II Toxicology)
       Chlorothalonil (Pesticide residues in food: 1990 evaluations Toxicology)
       Chlorothalonil  (IARC Summary & Evaluation, Volume 30, 1983)
       Chlorothalonil  (IARC Summary & Evaluation, Volume 73, 1999)