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    CHLOROTHALONIL

    EXPLANATION

         Chlorothalonil was evaluated by the Joint Meetings of 1974, 1977,
    1979, 1981 and 1983 (Annex 1, FAO/WHO, 1975a, 1978a, 1980a, 1982a, and
    1984). A toxicological monograph was prepared by the Joint Meeting in
    1974 (Annex 1, FAO/WHO, 1975b) and monograph addenda were prepared in
    1977, 1979, 1981, and 1983 (Annex 1, FAO/WHO, 1978b, 1980b, 1982b, and
    1985a). A corrigendum to the 1983 monograph addendum was published in
    1985 (Annex 1, FAO/WHO, 1985c). The 1981 JMPR reduced the temporary
    acceptable daily intake (TADI) from 0.03 to 0.005 mg/kg b.w., which
    was endorsed by the 1983 Meeting because of insufficient metabolism
    data and inadequate data on the carcinogenic potential of
    chlorothalonil. These data, plus additional mutagenicity data, have
    been submitted and are reviewed in this monograph addendum.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution and excretion

         The absorption of 14C-chlorothalonil (purity 99.7%) through the
    skin was assessed in male Sprague-Dawley rats. A dose of 5 mg/kg was
    applied (46.7 /cm) to the clipped back (25 cm) of each rat. Twenty-
    seven animals were treated and groups of three rats were subsequently
    killed at 2, 4, 8, 12, 24, 48, 72, 96, and 120 hours after
    application. The treated skin, blood, kidneys, liver, intestinal
    contents, remaining carcass, urine, faeces and cage washes were
    analyzed for radioactivity.

         The rate of absorption from the skin was relatively constant
    (6.3% of the applied dose per day) from 24 to 120 hours after
    application. Animals exposed for 120 hours had absorbed 27.7% of the
    dose and excreted 18% of the dose in the faeces, 6% in the urine, with
    20% lost at the time of application due to evaporation. Approximately
    4% of the dose remained in the carcasses of animals exposed for 120
    hours. Mean concentration of radioactivity in blood, liver and kidney
    appeared to plateau after 72 hours. Excretion of radioactivity in
    faeces appeared to be related to the blood concentrations, but urinary
    excretion appeared to be independent of blood concentrations. The
    urinary excretion pattern, attaining constancy of 1.2% of the applied
    dose per day, suggested that the renal excretory mechanism for
    chlorothalonil and/or its metabolites becomes saturated and is an
    active, rather than passive, form of excretion. Residues that remain
    on the skin surface, nonetheless, constituted the bulk of activity.

    Data suggest that the rate of absorption of chlorothalonil was
    constant and that the amount of the dose absorbed was dependent upon
    the exposure time (Marciniszyn et al., 1984a).

         Biliary excretion of ring labeled 14C-chlorothalonil (purity
    99.7%) was examined in Sprague-Dawley rats orally gavaged with
    5 mg/kg. Animals (8 males, 4 females) were fasted, except for water,
    16 hours prior to bile duct cannulation. Fifty percent of the males
    and females had sodium taurocholate (a choleretic substance) infused
    at a rate of 25 mg/hour. Animals were restrained and bile samples
    collected at hourly intervals from 0 to 48 hours after dosing. Blood
    was sampled at 6 and 24 hours and at termination. Urine and faecal
    samples were also collected periodically. Levels of radioactivity were
    determined in each bile, blood, urine and faecal sample and in the
    G.I. tracts, carcasses and cage washings.

         Approximately 91% of the administered radioactivity was
    recovered. The presence of activity in the blood, urine and bile
    demonstrate that absorption via the gut occurs. The data indicate that
    approximately 33% of the administered dose was absorbed, with the non
    absorbed material (67%) found in the faeces and G.I. tract. Biliary
    excretion accounted for 17-21% of the administered dose, with maximum
    concentrations eliminated within 2 hours of dosing. Urinary excretion,
    of about 8-12% of the labeled dose, shows this to be a significant
    route of elimination, but not a major one. No appreciable tissue
    binding was demonstrated as evidenced by low residual carcass levels,
    approximately 2% of the administered dose. Absorption via blood was
    also minimal with the maximum concentration less than 0.4% of the
    labeled dose (Marciniszyn et al., 1985a).

         The fate of orally administered 14C-chlorothalonil (purity
    99.7%) at three dose levels (5, 50 and 200 mg/kg) was investigated in
    Sprague-Dawley rats to determine the effects of increasing doses of
    the test material. Four animals per sex per dose were killed 2, 9, 24,
    96 and 168 hours after dosing and urine, faeces, and selected tissues
    assayed for radioactivity. The average recovery of the radiolabel at
    each of the dose levels was approximately 89% for males and 96% for
    females. The major route of elimination was via the faeces (83-87%)
    and was essentially complete by 48 hours in low-dose females and
    low/mid-dose males, and by 72 hours in the mid/high-dose females and
    high-dose males. A delay in stomach emptying time was observed for
    mid- and high-dose males and females. Urinary excretion was 92-93%
    complete for low-dose rats within 24 hours, 48 hours at mid-dose and
    95% complete for high-dose rats within 72 hours. Urinary excretion of
    the radiolabel at the three dose levels was 5-7% of the administered
    dose in males, and 5-11.5% in females. Urinary excretion was
    essentially saturated as the dose level was increased. The highest
    concentrations of radiolabelled material in non-G.I. tissues were
    found in the kidney, being approximately 0.7% of the dose per gram of
    kidney for males and 0.4% in females at peak concentration (2 hrs) for

    the 5 mg/kg dose level. Kidney concentrations were greatest at 2, 9
    and 24 hours for low, mid and high dose, respectively (Marciniszyn
    et al., 1984b and 1985b).

         Male Sprague-Dawley rats were administered, via oral gavage,
    14C-chlorothalonil (purity 99.7%) at a dose level of 200 mg/kg in
    order to isolate and identify the urinary metabolites. Urine was
    collected at 17, 24 and 48 hours after dosing. Urinary metabolites
    accounted for 2.4% of the administered dose and, except for 30%
    of the radiolabel which was nonextractable from the urine, were
    determined to be trimethylthiomonochloroisophthalonitrile and
    dimethylthiodichloroisophthalonitrile. This suggests the formation of
    glutathione (GSH) conjugates. These thiols were excreted in urine both
    as free thiols and as their methylated derivatives. The authors
    suggest a metabolic pathway such that hepatic metabolism proceeds
    through conjugation with GSH followed by enzymatic degradation. The
    smaller conjugates are then transported via the bloodstream to the
    kidney where they are converted to thiol metabolites and excreted in
    the urine (Marciniszyn et al., 1985c).

         In order to determine if chlorothalonil would react in vitro
    with GSH, chlorothalonil was incubated with GSH prior to isolation of
    biliary metabolites. In vitro studies have indicated that GSH forms
    mono, di, tri, and possibly tetra conjugates with chlorothalonil. On
    the other hand, data available on the isolation and identification of
    metabolites in bile of rats dosed with 14C-chlorothalonil suggest
    that GSH conjugates of chlorothalonil may be formed in the liver and
    eventually excreted in urine. Data thus far suggest that the major
    metabolite in bile is the di-GSH conjugate of chlorothalonil, while
    mono-GSH was not detected (Savides et al., 1985a).

         Data from a multiple dose study at 1.5, 5, 50 or 160 mg/kg, each
    administered five times at 24 hour intervals to male Sprague-Dawley
    rats, indicate that there were shifts in the times to peak blood
    concentrations with increasing single and multiple doses of
    chlorothalonil for both sexes. Significant depletion (> 50%) of the
    radiolabel from blood occurred by 24 hours post-dose for both sexes at
    dose levels less than or equal to 50 mg/kg. At 160 mg/kg, an apparent
    plateau in radiolabel concentration in blood was reached after a
    single dose, suggesting saturation of blood between 50 and 160 mg/kg.
    The concentrations of radiolabel in kidneys after single dose
    administration showed no apparent sex-related differences, but the
    times to peak kidney concentrations did appear to increase with
    increased dose level for both sexes. With multiple doses, the maximum
    kidney concentration was found 2 hours after the fifth dose at all
    dose levels. As with blood levels, peak kidney concentrations may have
    reached a plateau by the final 160 mg/kg dose. The maximum kidney
    concentration after five doses was proportional to the total
    administered dose at 1.5 mg/kg (3.12 g equiv/g) and 5 mg/kg (8.03 g

    equiv/g); at 50 mg/kg (31.5 g equiv/g) and 160 mg/kg (105 g
    equiv/g), kidney concentrations were proportional with one another,
    but kidney concentrations were not proportional between the two
    lower and two higher doses. In this multiple dose study, kidney
    concentrations at 1.5, 5 and 160 mg/kg decreased 50% by 24 hours, but
    decreased only 20% by 27 hours at 50 mg/kg. By 7 days after the
    fifth dose, kidneys contain 14, 16, 23 and 25% of their maximum
    concentrations at 1.5, 5, 50 and 160 mg/kg, respectively. The authors
    suggest that these data demonstrate apparent saturation of blood,
    plateau of radiolabel in kidneys, and a trend toward slower depletion
    (or greater retention) of radiolabel from kidney caused by increased
    and/or repeated doses of chlorothalonil. The authors further suggest
    that shifts in metabolism occur between doses of 5 and 50 mg/kg/day
    for some parameters and between 50 and 160 mg/kg/day in other
    parameters. (Savides et al., 1985b).

         The effect of a single administration of chlorothalonil (purity
    97.8%) on liver and kidney GSH concentrations was assessed in male
    Sprague-Dawley rats, administered 5 mg/kg chlorothalonil i.p. or
    5000 mg/kg via oral gavage. Concentrations of GSH in liver and kidney
    determined 2 hours after i.p., or 24 hours after oral gavage,
    demonstrated no differences between control and i.p. groups regarding
    GSH levels. However, chlorothalonil administered orally caused lower
    hepatic GSH and higher renal GSH concentrations. The authors suggest
    that this supports the proposed metabolic pathway, which includes a
    GSH conjugate formed in the liver which is subsequently metabolized in
    the kidney to a sulfur-containing, potentially nephrotoxic, compound
    (Sadler et al., 1985a)

         Groups of Sprague-Dawley male rats (5 per dose) were administered
    5000 mg/kg chlorothalonil (purity 97.8%) via oral gavage to measure
    the time course of the acute effect of a single dose on body weight,
    liver and kidney weights and liver and kidney GSH concentrations. Rats
    were sacrificed at 1, 3, 9, 18, 24 or 48 hours post-dosing. The data
    demonstrated significantly-increased relative liver and kidney
    weights, reduced hepatic GSH concentration up to 24 hours post-dosing,
    and significantly-increased renal GSH concentration up to 48 hours
    after treatment. The authors suggest that the hepatic GSH changes were
    related to its conjugation with chlorothalonil, but that the results
    were inconclusive regarding the renal GSH changes (Sadler et al.,
    1985b).

    Toxicological studies

    Special Study on Carcinogenicity

    Rat

         Groups of Fischer 344 rats (60 males and 60 females per group)
    were administered chlorothalonil (98.1% pure) in the diet at dosage

    levels of 0, 800, 1600 or 3500 ppm (equal to 0, 40, 80 and 175 mg/kg
    b.w./day) for 116 weeks (males) or 129 weeks (females). Rats were
    examined daily for gross signs of toxicity, including mortality. Body
    weight and food consumption were recorded periodically throughout the
    study. Haematology, clinical chemistry and urinalysis parameters were
    examined routinely througout the study and at termination in 10 rats
    per sex per dose. All animals were necropsied, selected organs weighed
    and a complete list of tissues/organs examined microscopically (Wilson
    et al., 1985c).

         Survival was comparable in all groups, both sexes, for the first
    24 months. Continuation on study decreased survival in high dose males
    resulting in all males sacrificed at 27 months. Females were
    terminated on schedule at 30 months. The major cageside clinical
    observation included dark yellow urine in high-dose males and females
    from weeks 27-91. An increased brown/red staining around the
    anogenital region of mid- and high-dose females was also observed.
    There was a significant body-weight decrease (10-29%) in high-dose
    males and females throughout the study, as well as a 5-12% body-weight
    decrease in both sexes at the mid dose. There was no body-weight
    reduction in low-dose animals. Food consumption was unaffected, except
    for an increase in high-dose animals, generally towards the last half
    of the study.

         Monuclear cell leukemia is a common finding (approx. 20%) in
    Fischer 344 rats at an average age of 2 years (so-called "Fischer rat
    leukemia"). In this particular study there was an inverse relationship
    with dose in that this finding was most pronounced in controls. This
    was supported by numerous haematological, clinical chemistry and
    micropathological findings. These effects were most noticeable in
    control males. They included: decreased red blood cells, haemoglobin,
    haematocrit, and platelet counts, with increased mean corpuscular
    volume, mean corpuscular haemoglobin, reticulocytes, nucleated red
    blood cells and segmented neutrophils. These changes were accompanied
    by enlarged spleen at 0 and 40 mg/kg, and are suggestive of a
    macrocytic normochromic regenerative anaemia. Also, in control males,
    there were increases in total bilirubin, aspartate amino transferase,
    alanine amino transferase and alkaline phosphatase levels, findings
    which are common in Fischer rats in later stages of this disease.

         Parameters measured which were compound related and associated
    with the effects on the kidneys included increased blood urea nitrogen
    and serum creatinine in high-dose males and females, decreased serum
    albumin and serum glucose in high-dose males and females, increased
    urine volume and decreased specific gravity in all treated males
    throughout the study, and in all treated females initially (first
    year), but in high-dose females only, after the first year. The
    relative kidney weights were significantly increased in all treated
    males and in mid-dose and high-dose females only. Relative liver
    weight was affected in the same groups, being significantly increased

    in all dosed males, and mid- and high-dose females. Gross necropsy of
    all animals demonstrated a compound-related effect on the kidneys and
    stomach. In all dosed male groups and the high-dose female group there
    were kidney masses and/or nodules as well as increased granularity of
    the surface of the kidneys (the latter observed in all dose groups).
    There were increased incidences of erosions and ulcerations in the
    non-glandular stomach of all dosed rats as well as a significant
    incrase in discoloration of the mucosa in high-dose males.

         Histologically there was evidence of compound-related effects on
    the kidneys, oesophagus, stomach and duodenum. Non-neoplastic changes
    in the kidney included: chronic glomerulonephritis which increased in
    severity in a dose-related manner in all groups; dose-related increase
    in cortical tubular hyperlasia in dosed rats; increased incidence of
    tubular cysts in dosed rats; and increased incidence in dosed males
    only of hyperplasia of the papillary/pelvic epithelium. Other changes
    included increased hyperplasia/hyperkeratosis of the squamous mucosa
    of the oesophagus (all dose groups); increased mucosal hypertrophy of
    the duodenum (all dose groups); hyperplasia of the parathyroid (all
    dosed-male and high-dose female groups, considered a secondary lesion
    as a result of severe chronic renal disease); increased hyperplasia/
    hyperkeratosis of the squamous mucosa of the stomach in all dose
    groups; increased incidences of foci of necrosis or ulcers in the
    glandular stomach (all dose groups) increased incidence of suppurative
    prostatitis in all male dose-groups (considered associated with
    treatment-related renal lesions); complete involution of the thymus
    was increased in high-dose males and all female dose-groups.
    Interesting inverse dose-related changes included: chronic
    interstitial prostatitis (lower incidences in mid- and high-dose male
    groups); medullary tumours of the adrenal (lower incidence in mid-
    and high-dose female groups); osteoschlerosis of the femur and sternum
    (lower incidence in female dose-groups); and basophilic cell
    focus/foci of the liver (lower incidence in dosed females; this change
    is a common finding in aging Fischer 344 rats). There were also
    inverse dose-related changes for pituitary adenomas and fibromas of
    the skin.

         Neoplastic changes associated with treatment were observed in
    kidneys and stomach (forestomach). Tubular adenomas and carcinomas,
    anaplastic renal carcinomas, and transitional cell carcinomas were
    originally described in the kidney of treated rats only, being
    statistically significant in all dosed rats except low-dose females
    (Table 1). There was also a possible decrease in time to tumours in
    high-dose rats for renal adenomas and carcinomas. Re-examination of
    the renal tissues was performed by an independent pathologist, who did
    not identify transitional cell or anaplastic renal carcinomas (Busey,
    1985d; Table 2).

         In the original pathological examination there was poor
    correlation between the incidence of cortical tubular hyperplasia and

    the observation of a tubular adenoma or carcinoma (Table 3). However,
    in the re-evaluation by an independent pathologist there was very good
    correlation between the incidence of epithelial hyperlasia (in the
    proximal convoluted tubules) and the observation of tubular adenoma
    and carcinoma (Tables 4 and 5).

         The incidence of papillomas and carcinomas of the stomach were
    dose-related, but statistically significant only in high-dose females
    (0/60, 1/60, 1/60, 2/60 for males and 0/60, 1/60, 2/60, and 6/60 for
    females at 0, 40, 80 and 175 mg/kg dose levels, respectively) (Table
    6). Although there was no apparent correlation between forestomach
    tumours and the incidence of hyperplasia or hyperkeratosis, the non-
    neoplastic changes may have been obscured by the progression to
    tumour. Nonetheless, there was a dose-related increase in the severity
    of hyperplasia/hyperkeratosis in the forestomach.

         Results of this study demonstrate that chlorothalonil produced
    renal adenomas and carcinomas in Fischer 344 rats (both sexes) at
    > 40 mg/kg b.w. Secondary to this response was a dose-related
    increase in papillomas of the stomach (McGee & Brown, 1985).

        Table 1.  Incidencea of renal tumours of epithelial origin, original report
                                                                                                

    Tumour type             Control         40 mg/kg/day        80 mg/kg/day        175 mg/kg/day
                            M      F        M         F         M         F         M         F
                                                                                                 

    Tubular adenoma         0      0        2         2         4         4         11b**     9**

    Tubular carcinoma       0      0        5         1         2         2         7*        11**

    Transitional-cell       0      0        0         0         0         0         2b        0
     carcinoma

    Anaplastic renal        0      0        0         0         1         0         0         3
     carcinoma

    Total animals with      0      0        7*        3         7*        6*        19**     23**
     these tumours
                                                                                                 

    a    Kidneys from 60 animals of each sex were examined for all groups.
    b    Includes one male with tubular adenoma and transitional cell carcinoma.
    *    Statistically different from control - p< 0.05 (Fisher's exact test).
    **   Statistically different from control - p< 0.01 (Fisher's exact test).

    Table 2.  Incidence of renal tumours of epithelial origin, independent evaluation.
                                                                                           

    Tumour type           Control       40 mg/kg/day       8mg/kg/day        175 mg/kg/day
                          M      F      M         F        M        F        M          F
                                                                                           

    Tubular adenoma       0      0      2         3        5        10       7a         15b

    Tubular carcinoma     0      0      4         1        2        0        14a        12b

    Total animals with    0      0      7         4d       7        10e      19         24c
     these rumours
                                                                                           

    a    Includes 2 males with combined incidence of tubular adenoma and tubular
         carcinoma.
    b    Includes 3 females with combined incidence of tubular adenoma and tubular
         carcinoma.
    c    Includes 1 female with atubular carcinoma, originally diagnosed as
         invasive lipomatous tumour.
    d    Includes 1 female with a tubular adenoma, originally diagnosed as negative.
    e    Includes 4 females with a tubular adenoma, originally diagnosed as negative.

    Table 3.  Correlation of renal hyperplasia with tubular adenoma and carcinoma, original
              report (males).
                                                                                        

    Pathological             Control     40 mg/kg/day       80 mg/kg/day   175 mg/kg/day
    finding
                                                                                        

    Glomerulonephritis       39/60          56/60               56/60          60/60

    Cortical tubular         0/60           7/60                9/60           22/60
     hyperplasia

    Kidney adenoma           0/60           7/60                7/60           19/60
     or carcinoma

    Number of tumour-        0/0            0/7                 0/7            3/19
     bearing rats
     with renal
     hyperplasia
                                                                                        

    Table 4.  Correlation of renal hyperplasia with tubular adenoma and carcinoma, independent
              evaluation (males).
                                                                                               

    Pathological             Control        40 mg/kg/day        80 mg/kg/day      175 mg/kg/day
    finding
                                                                                               

    Chronic progressive      47/60              52/60               54/60             57/60
     nephropathy

    Focal epithelial         0/60               6/60                20/60             6/60
     hyperplasia
     (prox. conv. tub.)

    Epithelial hyperplasia   0/60               32/60               30/60             36/60
     (prox. conv. tub.)

    Kidney adenoma or        0/60               7/60                7/60              19/60
     carcinoma

    Number of tumour-        0/0                6/7                 7/7               19/19
     bearing rats
     with renal
     hyperplasia
                                                                                               
    
    Special Studies on Mutagenicity

         Three recent chromosomal aberration studies, carried out on
    technical chlorothalonil, are summarized in Table 7. The rat and mouse
    bone marrow cytogenetic assays were negative. The Chinese hamster bone
    marrow cytogenetic assay was positive, but no dose-response
    relationship was established.

         A series of in vitro gene mutation assays were conducted in
    Salmonella typhimurium with and without a metabolic system obtained
    from rat kidney (Table 8). The compounds tested included technical
    chlorothalonil, four manufacturing impurities and eight known or
    potential metabolites. The results did not provide any evidence of a
    mutagenic potential of any of the tested compounds.

        Table 5.  Correlation of renal hyperplasia with tubular adenoma and carcinomm,
              independent evaluation (females).
                                                                                    

    Pathological            Control    40 mg/kg/day   80 mg/kg/day     175 mg/kg/day
     finding
                                                                                    

    Chronic                 45/60      49/60          47/60              51/60
     progressive
     nephropathy

    Focal epithelial        6/60       22/60          34/60              42/60
     hyperplasia
     (prox. conv. tub.)

    Epithelial              5/60       35/60          39/60              48/60
     hyperplasia
     (prox. conv.
     tub.)

    Kidney adenoma          0/60       4/60           10/60              24/60
     or carcinoma

    Number of tumour-       0/0        4/4            10/10              21/24
     bearing rats
     with renal
     hyperplasia
                                                                                    
    
    Short-term Studies

         Additional electron and light microscopic evaluations wee
    conducted on kidneys from the short-term rat and mouse studies
    reviewed by the 1983 Joint Meeting in order to investigate and further
    clarify ultrastructural renal changes.

    Mouse

         Histopathological re-evaluation of the kidneys in the Shults
    (1983) study identified microscopic kidney changes in males at
    750 ppm. These changes, which consisted of hyperplasia of the
    epithelium of the proximal convoluted tubules, were minimal to slight
    (severity), involved only 4/15 males, and were not considered to be
    clearly treatment-related effects (Busey 1985b & c).

        Table 6.  Incidencea of tumours in the gastric mucosa
                                                                                          

    Site/                 Control      40 mg/kg/day        80 mg/kg/day      175 mg/kg/day
    Tumour type           M     F      M         F         M         F       M         F
                                                                                          

    Forestomach:
      Papilloma           0     0      1         1         1         2       2         6*

      Squamous
      carcinoma           0     0      0         0         0         0       1         1

      Total number
      of animals with
      fore-stomach
      rumours             0     0      1         1         1         2       3         7*

    Fundle stomach:
      Mucosal polyp       1     0      0         0         0         0       0         0

      Adenocarcinoma      0     0      0         0         0         1       0         0
                                                                                          

    a    Stomachs from 60 animals of each sex were examined.
    *    Statistically different from control - p < 0.05 (Fisher's exact test).
    
    Rat

         EM and light microscopy of renal tissue from the Wilson et al.
    (1984) study confirmed the absence of a demonstrated microscopic
    change in female kidneys. In males there was evidence of an increased
    incidence of hyperplasia of the proximal convoluted tubules at
    40 mg/kg b.w. Electron and light microscopy identified a compound-
    related increased number of irregular intracytoplasmic inclusion
    bodies in the proximal convoluted tubules of all males, including
    controls. The number of such inclusions increased with dose at
    > 1.5 mg/kg b.w. but showed a tendency to reversal at the low dose
    (105 mg/kg) only following a 13 week recovery period (Wilson et al.,
    1985a & 1985b).

         The exact toxicological significance of these inclusion bodies is
    unknown since there were no associated degenerative renal changes, a
    spontaneous occurence in controls was also observed, and there was a
    tendency to reversal after a 13 week recovery period. The NOEL is
    > 1.5 mg/kg b.w. The previous NOEL was 3 mg/kg b.w. (Busey, 1985a;
    Colley, 1983).

        Table 7. Results of mutagenicity assays of chlorothalonil
                                                                                                   

    Test system              Test substance      Dose level/         Results             Reference
                                                 concentration
                                                                                                   

    Mouse bone               Chlorothalonil      250, 1250, &        Negativea           Mizens et
     marrow cytogenetics                         2500 mg/kg,                             al., 1985a
     assay                                       orally
     - in vivo

    Rat bone marrow          Chlorothalonil      500, 2500, &        Negativeb           Mizens et
     cytogenetics                                5000 mg/kg,                             al., 1985b
     assay -                                     orally
     in vivo

    Chinese hamster          Chlorothalonil      500, 2500, &        Weak clastogen      Mizens et
     bone marrow                                 5000 mg/kg          positive            al., 1985c
     cytogenetics                                given as            in treated
     assay -                                     single treatment;   groups: 5 
     in vivo                                     50, 125, & 250      50 & 5  250
                                                 mg/kg given         mg/kg. No
                                                 as 5 daily          dose-response
                                                 treatments          relationship.
                                                 orally
                                                                                                   

    a    Positive control (MMS) gave expected positive response at 65 mg/kg.
    b    Positive control (MMS) gave expected positive response at 130 mg/kg.

    Table 8.  Results of mutagenicity assays of technical chlorothalonil, manufacturing impurities,
              and possible metabolites of technical chlorothalonil
                                                                                                           

    Test system              Test                          Dose level/
    (Ames test)              substance                     concentration       Results        Reference
                                                                                                           

    S. typhimurium           Chlorothalonil                Non-activation      Negative       Jones et al.,
     TA98, TA100,                                          0.16, 0.8, 4.0,                    1984
     TA1535, TA1537,                                       8.0, & 16.0
     & TA1538 W/S9                                         g/plate.
     and W/O S9*                                           Activation
                                                           0.5, 2.5, 12.5,
                                                           25, & 50 g/plate

    S. typhimurium           2,5,6-                        20, 100, 500,       Negative       Jones et al.,
     TA98, TA100,            Trichloro-3-                  1000, & 2000                       1985j
     TA1535, TA1537,         cyanobenzamide                g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)

    S. typhimurium           2,4,5,6-                      Non-activation      Negative       Jones et al.,
     TA98, TA100,            Tetrachloro-3-                6, 30, 150,                        1985k
     TA1535, TA1537,         cyanobenzamide                300, & 600
     & TA1538 W/S9                                         g/plate.
     and W/O S9*                                           Activation
                                                           10, 50, 250,
                                                           500, & 1000
                                                           g/plate
                                                                                                           

    Table 8.  (Con't)
                                                                                                           

    Test system              Test                          Dose level/
    (Ames test)              substance                     concentration       Results        Reference
                                                                                                           

    S. typhimurium           2,5,6-                        Non-activation      Negative       Jones et al.,
     TA98, TA100,            Trichloro-4-                  20, 100, 400,                      1985l
     TA1535, TA1537,         hydroxy-3-                    800, & 2000
     & TA1538 W/S9           cyanobenzamide                g/plate.
     and W/O S9*                                           Activation
                                                           40, 400, 1000,
                                                           3000, & 6000
                                                           g/plate

    S. typhimurium           2,3,5,6-                      20, 100, 500,       Negative       Jones et al.,
     TA98, TA100,            Tetrachlorobenzonitrile       1000, & 2000                       1985d
     TA1535, TA1537,                                       g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)

    S. typhimurium           2,4,5,6-                      100, 500,           Negative       Jones et al.,
     TA98, TA100,            Tetrachlorobenzamide          2500, 5000 &                       1985e
     TA1535, TA1537,                                       10,000 g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)

    S. typhimurium           2,4,5-Trichloro-              20, 100, 500,       Negative       Jones et al.,
     TA98, TA100,            3-cyanobenzamide              1000, & 2000                       1985f
     TA1535, TA1537,                                       g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation)
                                                           & non-activation)
                                                                                                           

    Table 8.  (Con't)
                                                                                                           

    Test system              Test                          Dose level/
    (Ames test)              substance                     concentration       Results        Reference
                                                                                                           

    S. typhimurium           2,5,6-Trichloro               Non-activation      Negative       Jones et al.,
     TA98, TA100,            4-thioisophthalonitrile       250, 400, 630,                     1985g
     TA1535, TA1537,                                       1000, 1600,
     & TA1538 W/S9                                         & 2500 g/plate.
     and W/O S9*                                           Activation
                                                           400, 630, 1000,
                                                           1600, 2000, 2500,
                                                           3000, 4000, & 5000
                                                           g/plate

    S. typhimurium           2,5,6-Trichloro-              100, 500, 2500,     Negative       Jones et al.,
     TA98, TA100,            3-carboxybenzamide            5000, & 10,000                     1985h
     TA1535, TA1537,                                       g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)

    S. typhimurium           2,4,5-                        0.5, 2.5, 10,       Negative       Jones et al.,
     TA98, TA100,            Trichloroisophthalonitrile    35, & 70                           1985i
     TA1535, TA1537,                                       g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)

    S. typhimurium           2,3,5,6-                      4, 20, 100,         Negative       Jones et al.,
     TA98, TA100,            Tetrachloroterphthalonitrile  200, & 400                         1985a
     TA1535, TA1537,                                       g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)
                                                                                                           

    Table 8.  (Con't)
                                                                                                           

    Test system              Test                          Dose level/
    (Ames test)              substance                     concentration       Results        Reference
                                                                                                           

    S. typhimurium           Isophthalonitrile             40, 200, 1000,      Negative       Jones et al.,
     TA98, TA100,                                          2000, & 4000                       1985b
     TA1535, TA1537,                                       g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)

    S. typhimurium           Pentachlorobenzonitrile       10, 50, 250,        Negative       Jones et al.,
     TA98, TA100,                                          500, & 1000                        1985c
     TA1535, TA1537,                                       g/plate
     & TA1538 W/S9                                         (for both
     and W/O S9*                                           activation
                                                           & non-activation)
                                                                                                           

    *    The S9 fraction was prepared from rat kidney homogenate
    
         Histopathological re-evaluation of the Wilson et al., (1981)
    rat study by two separate pathologists confirmed the finding of
    epithelial hyperplasia of the proximal convoluted tubule in males at
    all levels (e.g > 40 mg/kg) and in females at > 175 mg/kg b.w.
    Cytoplasmic inclusion bodies or "hyaline droplets" were also
    identified using neutral red stain in both sexes at all doses. The
    angular material (cytoplasmic inclusions), which represent an
    analogous finding to the E.M. evaluation, was seen only in males
    (Trump et al., 1985; Busey 1985a).

    COMMENTS

         Chlorothalonil was evaluated by the Joint Meetings in 1974, 1977,
    1979, 1981 and 1983 and additional metabolism data and a rat
    carcinogenicity study were requested. Chlorothalonil has also been
    evaluated by IARC (1983) and classified as a compound with limited
    evidence of oncogenic potential to humans.

         The additional data provided to the 1985 Joint Meeting
    demonstrated preferential excretion via the bile and faeces, with
    secondary excretion in the urine. These metabolism data suggest
    metabolic pathways involving initial hepatic biotransformastions,
    conjugation with reduced glutathione (GSH) and enzymatic degradation.
    There is additional metabolism in the kidney, formation of the thiol
    metabolites, and excretion in the urine. There was a suggestion of the
    formation of a sulfur-containing, potentially nephrotoxic, compound in
    the G.I. tract or kidney. At higher doses there was a plateau of
    radioactivity in kidneys with subsequent slower removal. It was
    apparent that there is a shift in metabolism which occurs between
    doses of 50 and 160 mg/kg b.w. suggesting saturation of an active,
    rather than passive, uptake mechanism in the kidney.

         Rat and mouse bone-marrow cytogenetic assays were negative. A
    hamster bone-marrow cytogenetic assay was positive.

         In 1983 the Meeting expressed concern for the demonstrated
    nephrotoxicity and potential tumourigenicity which was evident from
    earlier studies in rats and mice. The mouse oncogenicity study
    reviewed in 1983 demonstrated compound-related effects on the kidney
    at > 750 ppm, with a compound-related increased incidence of renal
    cortical tubular adenomas and carcinomas in males.

         The rat oncogenicity study reviewed by this Meeting demonstrated
    compound-related neoplastic changes in kidneys of treated males
    (> 800 ppm) and females (> 1600 ppm). Renal tubular adenomas and
    carcinomas were significantly increased in all dosed rats except
    low-dose females. There was also a decrease in time to tumour
    formation which was evident at the high-dose level. Exposure of these
    rats to chlorothalonil in the diet produced a pronounced increase with
    dose in the occurrence and severity of renal tubular epithelial
    hyperplasia and chronic glomerulonephritis.

         In 1974 the Meeting evaluated five long-term studies in which
    rats were administered chlorothalonil at doses of 4 to 15,000 ppm with
    no evidence of a tumourigenic effect on the kidney, although there was
    clear evidence of epithelial degeneration and hyperplasia at doses
    > 60 ppm.

         The Meeting was informed of additional ongoing oncogenicity
    studies in mice and rats, exposed via the diet to chlorothalonil. In
    consideration of these new studies, which are being conducted at lower
    doses than previous rat and mouse oncogenicity studies, the recognized
    change in metabolism at doses approximating 50 mg/kg b.w., the absence
    of demonstrated mutagenic potential in a complete battery of such
    assays, the absence of tumourigenic response in several long-term
    studies reviewed in 1974, and the conflicting evidence from
    carcinogenicity studies conducted by the National Cancer Institute
    (USA), the Meeting extended the TADI and required the submission of
    these ongoing tests when completed. However, because of concern for
    the demonstrated oncogenicity in rodents the safety factor used in
    estimating the TADI was increased.

    TOXICOLOGICAL EVALUATION

    LEVEL CAUSING NO TOXICOLOGICAL EFFECT

         Rat: 10 ppm in the diet, equivalent to 0.5 mg/kg b.w.

         Dog; 120 ppm in the diet, equivalent to 3 mg/kg b.w.

    ESTIMATE OF TEMPORARY ACCEPTABLE DAILY INTAKE FOR MAN

         0-0.0005 mg/kg b.w

    FURTHER WORK OR INFORMATION REQUIRED (by 1987)

    1.   Carcinogenicity studies in rats and mice are understood to
         be in progress. Although the Meeting recognized that these
         studies are not scheduled for completion until September of 1988
         and June of 1987, the Meeting recommended any available data be
         submitted for evaluation when available.

    2.   Further metabolism data to identify the change in metabolic
         pattern with increasing dose as, well as further characterization
         of the GSH conjugation occurring in the G.I. tract and kidney.

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    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: 1987 evaluations Part II Toxicology)
       Chlorothalonil (Pesticide residues in food: 1990 evaluations 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)