IPCS INCHEM Home

    CHLOROTHALONIL

    EXPLANATION

    First draft prepared by Dr. E. Arnold, 
    Health and Welfare Canada, Ottawa, Canada

         Chlorothalonil has been reviewed by seven previous Joint Meetings
    from 1974 to 1987 (Annex 1, 1975ab, 1978ab, 1980ab, 1982ab, 1984,
    1985a, 1986ac, 1987b, and 1988b).  Data considered on chlorothalonil
    itself include metabolism and biotransformation studies in mice, rats
    and dogs; pharmacological studies in mice and rats; acute toxicity in
    several species; short-term toxicity studies in mice, rats and dogs;
    in vivo and in vitro genotoxicity studies; teratology studies in
    rats and rabbits; and a number of long-term toxicity/oncogenicity
    studies in mice and rats.  Data on the metabolite 4-hydroxy-2,5,6-
    trichloroisophthalonitrile have also been considered including
    genotoxicity, reproduction and carcinogenicity studies.  In 1987, a
    temporary ADI of 0-0.003 mg/kg bw based on toxicity data for
    chlorothalonil, not the 4-hydroxy derivative, was extended.  However,
    because of concern for the demonstrated oncogenicity in rats and
    pending completion of an ongoing oncogenicity study in rats, a high
    safety factor was used.  The carcinogenicity study in rats together
    with further metabolism data have now been submitted.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Metabolism

    Oral administration

    Rats

         A single oral dose of 14C-chlorothalonil at 50 mg/kg bw in
    aqueous (0.75% methyl cellulose) suspension was given to nine
    germ-free Sprague-Dawley male rats in a volume of 10 ml/kg bw.  A
    tenth rat was given the vehicle alone as a control.  Urine and faeces
    were collected at 24 hour intervals to 96 hours.  At study termination
    at 96 hours, blood was collected and the kidneys and the rest of the
    carcass were saved.  All samples were frozen pending analysis.

         Urinary excretion of radioactivity accounted for 2.36-4.02% (mean
    3.12%) of the administered dose while faecal excretion accounted for
    61.3-95.6% (mean 84.4%).  Blood levels accounted for 0.0098-0.0229%
    (mean 0.0143%) of the dose in 8 of the 9 rats and 0.0517% in the
    remaining rat.  The kidney contained 0.037-0.066% of the dose in all
    rats.  Carcass radioactivity accounted for 0.177-0.285% of the dose in
    the 8 rats with similar blood levels and 1.78% in outlier rat. 
    Urinary excretion of test material occurred mainly in the first 24
    hours after dosing except in one rat which showed almost equal
    excretion at 0-24 and 24-48 hours.  Faecal excretion also was
    predominantly in the first 24 hours except in the one rat in which
    peak excretion was observed in the period 24-48 hours.

         Eight 0-24 hour and four 24-48 hour urine samples were analyzed
    for methylated thiols.  No methylated thiols were detected in the 0-24
    hour urine of 6/8 rats or in 24-48 hour urine from 2/4 rats.  No
    monothiol derivative was detected in any urine sample. The dithiol
    derivative was detected only in one 24-48 hour sample. The trithiol
    derivative was detected in two samples of 0-24 hour urine and two of
    24-48 hour urine representing urine from three rats.  The rat which
    excreted trithiols in both periods was also the rat which excreted the
    dithiol derivative.  The highest concentration of thiols detected
    accounted for 0.053% of the administered dose (Magee et al., 1990).

    Dogs

         Two male beagle dogs were given 14C-chlorothalonil by gelatin
    capsule at an average dose of 49.9 mg/kg bw.  Urine was collected for
    24 hour periods for 10 days and faeces for 24 hour periods for 12
    days.  Recovery of test material during the study was virtually

    complete (mean 100.1% of the administered dose).  Most of the material
    was excreted in the faeces (mean 99.6% of the administered dose) with
    only small amounts in urine and cage washings (0.2% and 0.3%,
    respectively).  However, white particles were observed in the 0-24
    hour faeces which may have been unabsorbed material.  Urine samples
    were analyzed for mono-, di-, and trithiols but none were detected at
    3 ng, the limit of sensitivity of the method (Savides et al.,
    1989a).

         In order to confirm the results of the above study, a second
    study in dogs was carried out.  Three male beagle dogs were given
    14C-chlorothalonil suspended in 0.75% aqueous methylcellulose by
    gavage at a nominal dose level of 50 mg/kg bw (estimate of actual dose
    level based on the recovery data: 28-63 mg/kg bw).  The dog which
    received the highest amount (63 mg/kg) regurgitated about 16% of the
    dose one hour and fifteen minutes after dosing.  Two of these dogs
    were the same ones dosed in the above study which had received control
    diet for approximately 5 months between the studies.  For collection
    of urine the dogs were catheterized for 4, 10 and 24 hours,
    respectively.  Subsequent urine samples were collected from the time
    of catheter removal to 24 hours and then at 24 hour intervals until 8
    days after dosing.  Faeces were collected every 24 hours.

         Faecal radioactivity in the first 24 hours after dosing accounted
    for 53.9-96.4% of the nominal administered dose or 76.2-98.1% of the
    estimated actual dose.  A further amount of 0.53-8.6% of the nominal
    dose (0.96-6.8% of estimated actual dose) was recovered in faeces on
    days 2-3.  Urinary excretion accounted for 0.66-1.17% of the nominal
    dose (0.73-1.86% of the estimated actual dose) and was predominantly
    recovered in the first 24 hours. Results from catheterization
    indicated that most of the excretion occurred during the first 10
    hours after dosing.

         Aliquots of urine from two of the dogs were analyzed to generate
    chromatographic profiles.  For both dogs the profiles of 4 to 10 hour
    samples were sufficiently similar to permit pooling.  The 12 and 14
    hour samples from one of the dogs were pooled and the sample collected
    from 10-24 hours in the other dog was also analyzed.  No methylated
    thiols were detected in any of the samples.  Less than half of the
    radiolabel in urine was extractable in acidified ethyl acetate
    suggesting that the metabolites were more polar than those observed in
    the rat (Savides et al., 1990a). 

    Monkeys

         Four male Chinese rhesus monkeys (Macaca mulatta) were given
    uniformly benzene ring labelled 14C-chlorothalonil, suspended in 0.75%
    aqueous methylcellulose, orally by gavage at a dose of 50 mg/kg bw. 
    An indwelling catheter was placed in the saphenous vein of the lower
    leg to permit periodic blood sample collection.  Urine was collected

    using an external catheter device for 48 hours after dosing and then
    the catheters were removed and the monkeys were housed in metabolism
    cages for 96 hours after dosing and urine and faeces were collected at
    72 and 96 hours.

         Blood concentration vs. time plots indicated considerable
    variability between animals.  Peak blood levels were seen 3-18 hours
    after dosing.  In two of the monkeys blood levels rose rapidly; in one
    of these the blood level declined rapidly while in the other blood
    levels declined rather slowly.  The other two animals took longer to
    attain peak blood levels and the blood levels declined at an
    intermediate rate.  In these latter two animals higher peak blood
    levels were reached than in the two which reached the peak rapidly. 
    Blood elimination half life was 6.9, 7.6, 19.8 and 35.0 hours in the
    four animals.  Area under the blood concentration curve was
    52,000-158,000 ng-eq hr/ml for the 0-30 sampling interval which was
    compared to 94,000 ng-eq hr/ml following the same dose in the rat. 
    Over 96 hours a total of 1.75-4.13% of the administered dose was
    excreted in urine and 52.3-91.6% in faeces.  In 3/4 monkeys, urine
    excreted in the period 24-48 hours contained the highest amount of
    test material.  The other animal excreted most of the radioactivity
    12-48 hours after dosing.  The same 3/4 monkeys had peak faecal
    excretion also in the 48 hour sample.  The other monkey had high
    excretion in the first 12 hours and virtually complete faecal
    excretion by 48 hours after dosing.

         Extraction of urine with acidified ethyl acetate recovered 32-
    65% (mean 49%) of the radiolabel in the urine.  Under similar
    conditions,  about 75% of the radiolabel was extractable in rat urine. 
    This suggests that in monkeys the radiolabel is excreted as more polar
    metabolites than in the rat.  Monothiols were not detected in any of
    the urine samples.  Dithiol was detected in urine of only one of the
    monkeys while trithiols were detected in all urine samples.  The total
    amount of thiols excreted by monkeys was 0.001 to 0.01% of the
    administered dose.  This was compared to the excretion in the rat in
    which 1.63% of the administered dose was excreted in the form of di-
    and trithiols (Savides et al., 1990b).

    Dermal administration

    Rats

         Four groups of 5 male CD Sprague-Dawley (Charles River) rats were
    treated dermally with 14C-chlorothalonil in acetone at a dose of about
    5 mg/kg bw.  Urinary excretion in the first 48 hours after dosing
    accounted for about 3% of the administered dose (range 2.52-4.0%) with
    approximately equal amounts excreted after 0-24 and 24-48 hours. 
    There was considerable variability between the four groups with
    respect to thiol excretion in urine.  Total thiols represented 0.07,
    0.01, 0.007 and 0.001% of the administered dose in the four groups. 

    Monothiol was detected in the 0-24 hour sample of one group only. 
    Dithiols were detected in urine from two of the four groups.  Trithiol
    was detected in all urine samples. These data were compared to the
    results of oral studies.  Urinary excretion was greater following oral
    than following dermal treatment (8%  3%) and there was at least a 20
    fold difference between the amount of the administered dose excreted
    as thiols (Savides et al., 1989b).

    Special in vitro study on metabolism

         Liver and kidney mitochondrial preparations were prepared from
    male CD Sprague-Dawley (Charles River) rats.  The preparations were
    incubated with one of the following sulfur analogs of chlorothalonil:
    the monothiol analog, the dithiol analog, the monoglutathione analog
    and the diglutathione analog. With the dithiol analog no increase in
    oxygen consumption was observed following the addition of adenosine
    diphosphate (ADP) to either liver or kidney mitochondrial preparations
    indicating complete inhibition of state 3 respiration.  The monothiol
    analog also affected oxygen uptake by liver mitochondria but had no
    effect on kidney mitochondria.  Neither of the glutathione analogs had
    an effect on oxygen uptake (Savides et al., 1988). 

    Toxicological studies

    Short-term studies

    Dogs

         Renal pathology data from a two year dog study, evaluated by the
    JMPR in 1974, were re-examined.  In this study 8 males and 8 females
    were given diets containing 0, 60 or 120 ppm of chlorothalonil.  Four
    dogs/sex/group were sacrificed at one year and the remaining four
    dogs/sex/group at two years.  Renal tubule vacuolation was observed in
    all of the females except one high dose dog at two years.  In males
    the lesion was observed in 0/4, 0/4 and 3/4 dogs at one year and 2/4,
    0/4 and 1/4 dogs at two years at 0, 60 and 120 ppm, respectively.  A
    second pathologist examining these tissues concluded that the observed
    lesion was probably an artifact of fixation.  The NOAEL of 120 ppm,
    originally established in 1974, was confirmed by this re-examination
    of the data.

    Long-term/Oncogenicity studies

    Rats

         Groups of 55 weanling Charles River Fischer 344 rats/sex/dose
    level were given diets containing chlorothalonil at dose levels to
    provide nominal intakes of 2, 4, 15 or 175 mg/kg bw/day for 99-125
    weeks.  Additionally, groups of 10 rats/sex/dose were given the same
    diets for one year to serve as interim sacrifice animals.  The target

    lower dose levels for the study were 1.5 and 3 mg/kg bw/day but the
    possibility of reduced bioavailability in the diet due to binding
    dictated the use of the higher (2.0 and 4.0 mg/day) levels.  The diet
    was presented twice a week and was frozen prior to use to minimize
    binding and increase bioavailability. Achieved concentrations provided
    dose levels of at least 1.5, 3.3, 14.7 and 173 mg/kg bw/day.

         At the 175 mg/kg bw/day dose level discolored urine was observed
    during the first year of the study, which occurred at a lower
    incidence during the second year.  Labored breathing was observed in
    a few animals in each group, which was increased at the end of the
    study in the high dose group.  Survival was reduced in males at 175
    mg/kg bw/day and this group was sacrificed at 99 weeks.  The other
    groups of males were sacrificed at 111 weeks at which time survival in
    the 15 mg/kg bw/day group was lower than in controls.  All groups of
    females were sacrificed at 125 weeks.  The females at 175 mg/kg bw/day
    had lower survival than controls at termination.  

         Body weights were lower than the controls in both sexes at 175
    mg/kg bw/day and in males at 15 mg/kg bw/day.  A slight but consistent
    depression in body weight was also seen at the nominal 4 mg/kg bw/day
    level but the difference was not considered to be biologically
    significant.  Food consumption was reduced in both sexes at 175 mg/kg
    bw/day during week 1 and increased on a "g/kg bw" basis for the
    remainder of the study.  There were no treatment-related effects on
    haematological parameters or ophthalmology.  At 175 mg/kg bw/day both
    sexes showed a number of changes in clinical chemistry: increased
    phosphorus and cholesterol levels and reduced alkaline phosphatase and
    alanine amino transferase levels.  Males at 175 and 15 mg/kg bw/day
    had increased BUN levels.  Males at 175 mg/kg bw/day showed increased
    urinary volume and reduced specific gravity at 18 and 23 months. 
    Absolute kidney weights were increased in males and females at 175
    mg/kg bw/day at 12 months and to a lesser extent in females given
    nominal levels of 4 or 15 mg/kg bw/day.  At terminal sacrifice only
    the females at 175 mg/kg bw/day were affected.  

         Chronic progressive nephropathy was observed in all groups but
    was more severe at 175 mg/kg bw/day and to a lesser extent at 15 mg/kg
    bw/day than in the low dose groups.  Focal epithelial hyperplasia in
    kidney was observed in all groups but was significantly increased in
    incidence at 175 mg/kg bw/day and slightly increased in incidence in
    females at 4 and 15 mg/kg bw/day.  Epithelial hyperplasia and
    hyperkeratosis of the non-glandular stomach were seen at doses of 4
    mg/kg bw/day and higher and were dose-related in incidence and
    severity.  In the kidney, tubular carcinomas were observed in both
    sexes at 175 mg/kg/ bw/day (7/55 males and 11/55 females) but in no
    other group.  Tubular adenomas were seen in 1/55, 1/54, 1/54, 3/54 and
    17/55 males at 0, 2, 4, 15 and 175 mg/kg bw/day, respectively, and in
    24/55 females at 175 mg/kg bw/day.  In the stomach, non-glandular
    papillomas were seen in 0, 0, 3, 2 and 5 males and 1, 1, 2, 4 and 7

    females at 0, 2, 4, 15 and 175 mg/kg bw/day, respectively.  No
    squamous cell carcinomas were observed in males  but one was seen in
    each the control and 15 mg/kg bw/day groups and 3 in the 175 mg/kg
    bw/day females.  The NOEL for non-neoplastic effects in this study was
    1.5 mg/kg bw/day. There was a possible slight increase in incidence of
    kidney and stomach neoplasms at 15 mg/kg bw/day but no tumorigenicity
    was observed at the nominal 2 and 4 mg/kg bw/day levels (actual 1.5
    and 3.3 mg/kg bw/day) (Wilson and Killeen, 1989).

    COMMENTS

         A study in germ-free rats was conducted to test the hypothesis
    that at least part of the metabolism of chlorothalonil in the rat is
    carried out by the microflora in the gastrointestinal tract.  In germ-
    free rats, urinary excretion of radioactivity accounted for about 3%
    of the administered dose, about half the amount observed in urine of
    non-germ-free rats dosed at the same level.  Thiols in urine accounted
    for about 0.05% of the administered dose in germ-free rats compared to
    1.6% of the administered dose in non-germ-free rats.  These data
    support the theory that the microflora of the gastrointestinal tract
    are involved in the metabolism of chlorothalonil in the rat. 

         Studies in dogs and monkeys also indicated lower urinary
    excretion of radioactivity than with non-germ-free rats:  about 1% of
    the administered dose in the dog and 1.8-4.1% of the administered dose
    in the monkey.  No thiols were detected in dog urine and only small
    amounts in monkey urine (0.001-0.01% of the administered dose).

         An in vitro study with rat liver and kidney mitochondria
    demonstrated a toxic effect on mitochondria from both tissues with the
    dithiol metabolites of chlorothalonil, but no effects with glutathione
    conjugates.

         In an oncogenicity study in rats, kidney and stomach tumours were
    observed at 175 mg/kg bw/day.  There were slight increases in kidney
    tubule adenomas and nonglandular stomach papillomas at 15 mg/kg bw/day
    but there was no evidence of tumorigenicity at lower dose levels. 
    Focal epithelial hyperplasia in kidney and epithelial hyperplasia and
    hyperkeratosis of the nonglandular stomach were observed in a dose-
    related incidence.  The NOAEL for non-neoplastic lesions in the kidney
    and stomach was 1.5 mg/kg bw/day.  The NOAEL for neoplastic lesions
    was 3.3 mg/kg bw/day.

         The metabolism of chlorothalonil in the rat differs from that in
    the monkey and dog.  This difference is related to a considerable
    degree to the gut flora in the rat.  This fact suggests that the dog
    or the monkey may be more suitable models than the rat for predicting
    the metabolism of chlorothalonil by man.

         The existing data on reproduction were considered to be
    unsatisfactory.  However, a new rat reproduction study is presently
    underway.

         A two-year study in dogs was evaluated in 1974.  No treatment-
    related effects on the kidney were observed at 24 months in four males
    and four females given 120 ppm chlorothalonil in the diet.  Since the
    present rat oncogenicity study demonstrates a NOAEL and the metabolism
    data assist in elucidating the mechanism, concern regarding
    carcinogenicity has been alleviated.  Since the dog is now considered

    to be a more relevant model than the rat, the dog study has been
    selected as the most suitable basis for estimating an ADI.  Therefore,
    a safety factor 100 was applied to the NOAEL in the study (3.0 mg/kg
    bw/day) to estimate of the ADI.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Mouse:    15 ppm in the diet, equal to 1.6 mg/kg bw/day
         Rat:      1.5 mg/kg bw/day
         Dog:      120 ppm in the diet, equivalent to 3.0 mg/kg bw/day

    Estimate of acceptable daily intake for humans

         0-0.03 mg/kg bw

    Studies which will provide information valuable to 
    the continued evaluation of the compound

         -    Reproduction study in rats known to be in progress
         -    Observations in humans

    REFERENCES

    Magee, T.A., Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr.,
    (1990).  Study to evaluate the metabolic pathway of chlorothalonil
    (14C-ASC-2787) in germ-free rats. Unpublished report no.
    3060-88-0219-AM-001 from Ricerca Inc., Painesville, Ohio, USA.
    Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA.

    Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1988). A
    study to evaluate the effects of sulfur-containing analogs of
    chlorothalonil on mitochondrial function. Unpublished report no.
    1479-87-0037-AM-001 from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA.

    Savides, M.C., Marciniszyn, J.P.,and Killeen, J.C.,Jr., (1989a). Study
    to compare the metabolism of chlorothalonil in dogs with its
    metabolism in rats following oral administration of
    14C-chlorothalonil. Unpublished report no. 1626-88-0008-AM-001 from
    Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta
    ASC, Mentor, Ohio, USA.

    Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1989b).
    Study to determine the metabolic pathway for chlorothalonil following
    dermal application to rats. Unpublished report no. 1625-87-0057-AM-001
    from Ricerca Inc., Painesville, Ohio, USA. Submitted to WHO by
    Fermenta ASC, Mentor, Ohio, USA.

    Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1990a).
    Study of the urinary excretion of radiolabel by catheterized dogs
    following oral administration of 14C-chlorothalonil by gavage.
    Unpublished report no. 3086-89-0041-AM-001 from Ricerca, Inc.,
    Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor,
    Ohio, USA.

    Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr. (1990b).
    Study to evaluate the urinary metabolites of chlorothalonil from male
    rhesus monkeys. Unpublished report no. 3349-89-0179-AM-001 from
    Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta
    ASC, Mentor, Ohio, USA.

    Wilson, N.H., and Killeen, J.C., Jr. (1989). A tumorigenicity study of
    technical chlorothalonil in rats. Unpublished report no.
    1102-84-0103-TX-007 from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA.


    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: 1992 evaluations Part II Toxicology)
       Chlorothalonil  (IARC Summary & Evaluation, Volume 30, 1983)
       Chlorothalonil  (IARC Summary & Evaluation, Volume 73, 1999)