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 µ/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 µg equiv/g) and 5 mg/kg (8.03 µg
equiv/g); at 50 mg/kg (31.5 µg equiv/g) and 160 mg/kg (105 µg
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 µg/plate.
and W/O S9* Activation
0.5, 2.5, 12.5,
25, & 50 µg/plate
S. typhimurium 2,5,6- 20, 100, 500, Negative Jones et al.,
TA98, TA100, Trichloro-3- 1000, & 2000 1985j
TA1535, TA1537, cyanobenzamide µg/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 µg/plate.
and W/O S9* Activation
10, 50, 250,
500, & 1000
µg/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 µg/plate.
and W/O S9* Activation
40, 400, 1000,
3000, & 6000
µg/plate
S. typhimurium 2,3,5,6- 20, 100, 500, Negative Jones et al.,
TA98, TA100, Tetrachlorobenzonitrile 1000, & 2000 1985d
TA1535, TA1537, µg/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 µg/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, µg/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 µg/plate.
and W/O S9* Activation
400, 630, 1000,
1600, 2000, 2500,
3000, 4000, & 5000
µg/plate
S. typhimurium 2,5,6-Trichloro- 100, 500, 2500, Negative Jones et al.,
TA98, TA100, 3-carboxybenzamide 5000, & 10,000 1985h
TA1535, TA1537, µg/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, µg/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, µg/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, µg/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, µg/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.
REFERENCES
Busey, W.M. A subchronic toxicity study of T-117-11 in Rats (SDS
(1985a) Biotech Study No. 5TX-81-0213), Revised Pathology Report,
Experimental Pathology Laboratories, Inc., SDS Document No.
753-5TX-85-0056-002. Submitted to WHO by SDS Biotech.
Busey, W.M. A 90-day feeding study in mice with T-117-11, Revised
(1985b) Pathology Report, Experimental Pathology Laboratories, Inc.,
SDS Document No. 753-5TX-85-0053-002. Submitted to WHO by
SDS Biotech.
Busey, W.M. A chronic dietary study in mice with T-117-6 (SDS Biotech
(1985c) Study No. 5TX-79-0102); Pathology report. Experimental
Pathology Laboratories, Inc. Submitted to WHO by SDS
Biotech.
Busey, W.M. A two-year tumorigenicity study of T-117-11 in rats (SDS
(1985d) Biotech Study No. 5TX-80-0234); Pathology report.
Experimental Pathology Laboratories, Inc. Submitted to WHO
by SDS Biotech.
Colley, J., et al. A 13-week subchronic toxicity study of T-117-11
(1983) in rats (followed by a 13-week withdrawal period). Electron
and Light Microscopy of Kidneys, Huntingdon Research Centre,
SDS Document No. 5TX-81-0213. Submitted to WHO by SDS
Biotech.
IARC. Monographs on the evaluation of the carcinogenic risk of
(1983) chemicals to humans. Vol 30.
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1984) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with technical
chlorothalonil, report No. 694-5TX-84-0064-002. Submitted to
WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985a) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,3,5,6-
tetrachloroterphthalonitrile, report No. 694-5TX-84-
0090-002. Submitted to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985b) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with isophthalonitrile,
report No. 694-5TX-84-0094-002. Submitted to WHO by SDS
Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985c) mammalian-microsome plate incorporation assay (Ames
Test) with and without renal activation with
pentachlorobenzonitrile (PCBN), report No. 694-5TX-84-
0095-002. Submitted to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985d) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,3,5,6-
tetrachlorobenzonitrile, report No. 694-5TX-84-0091-002.
Submitted to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985e) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,4,5,6-
tetrachlorodibenzamide, report No. 694-5TX-84-0092-002.
Submitted to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985f) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,4,5-trichloro-3-
cyano-benzamide, report No. 694-5TX-84-0093-002. Submitted
to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985g) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,5,6-trichloro-4-
thio-isophthalonitrile, report No. 694-5TX-84-0124-002.
Submitted to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985h) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,5,6-trichloro-3-
carboxybenzamide, report No. 694-5TX-84-0139-002. Submitted
to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985i) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,4,5-
trichloroisophthalonitrile, report No. 694-5TX-84-0086-002.
Submitted to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr; & Ignatoski, J.A. Salmonella/
(1985j) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,5,6-trichloro-3-
cyanobenzamide, report No. 694-5TX-84-0088-002. Submitted to
WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(1985k) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,4,5,6-tetrachloro-
3-cyanobenzamide, report No. 694-5TX-84-0087-002. Submitted
to WHO by SDS Biotech (unpublished).
Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/
(19851) mammalian-microsome plate incorporation assay (Ames Test)
with and without renal activation with 2,5,6-trichloro-4-
hydroxy-3-cyanobenzamide, report No. 694-5TX-84-0089-002.
Submitted to WHO by SDS Biotech (unpublished).
McGee, D.H., & Brown. A tumorigenicity study of T-117-11 in rats
(1985) (5TX-80-0234), International Research and Developmental
Corporation, SDS Document No. 099-5TX-80-0234-006. Submitted
to WHO by SDS Biotech.
Marciniszyn, J.P., Savides, M.C., Killeen, J.C., Jr., &
(1984a) Ignatoski, J.A. Study of the dermal absorption of
14C-chlorothalonil by male rats, report No. 649-4AM-84-
0010-001. Submitted to WHO by SDS Biotech (unpublished).
Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Study of the
(1984b) distribution of radioactivity following oral administration
of 14C-chlorothalonil to male Sprague-Dawley rats, report
No. 631-4AM-83-0011-002. Submitted to WHO by SDS Biotech
(unpublished).
Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Pilot study of the
(1985a) biliary excretion of radioactivity following oral
administration of 14C-chlorothalonil to Sprague-Dawley
rats, report No. 633-4AM-83-0062-002. Submitted to WHO by
SDS Biotech (unpublished).
Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Study of the
(1985b) distribution of radioactivity following oral administration
of 14C-chlorothalonil to female Sprague-Dawley rats, report
No. 631-4AM-84-0078-002. Submitted to WHO by SDS Biotech
(unpublished).
Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Identification of
(1985c) metabolites in urine and blood following oral administration
of 14C-chlorothalonil to male rats: the thiolmetabolites in
urine, report No. 621-4AM-83-0061-001. Submitted to WHO by
SDS Biotech (unpublished).
Mizens, M., Killen, J.C., Jr., Claudio, G., & Ignatoski, J.A. In Vivo
(1985a) bone marrow chromosomal aberration assay in mice with a
single dose of technical chlorothalonil, report No. 625-5TX-
83-0029-002-001. Submitted to WHO by SDS Biotech
(unpublished).
Mizens, M., Killen, J.C., Jr., Claudio, G., & Ignatoski, J.A. In Vivo
(1985b) bone marrow chromosomal aberration assay in rats with a
single dose of technical chlorothalonil, report No. 625-5TX-
83-0028-002-001. Submitted to WHO by SDS Biotech
(unpublished).
Mizens, M., Killen, J.C., Jr., & Ignatoski, J.A. Acute and subchronic
(1985c) in vivo bone marrow chromosomal aberration assay in
Chinese hamsters with technical chlorothalonil, report No.
625-5TX-83-0014-003. Submitted to WHO by SDS Biotech
(unpublished).
Sadler, E.M., Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A.
(1985a) Acute effect of technical chlorothalonil on hepatic and
renal glutathione content in rats, report No. 732-5TX-84-
0006-001. Submitted to WHO by SDS Biotech (unpublished).
Sadler, E.M., Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. Time
(1985b) course of the acute effect of technical chlorothalonil on
hepatic and renal glutathione content in male rats, report
No. 751-5TX-85-0032-001. Submitted to WHO by SDS Biotech
(unpublished).
Savides, M.C., Marcinsizyn, J.P., Killeen, J.C., Jr., &
(1985a) Ignatoski, J.A. Isolation and identification of metabolites
in the bile of rats orally administered 14C-chlorothalonil,
I. Synthesis and characterization of glutathione conjugates
of chlorothalonil, report No. 633-4AM-84-0104-001. Submitted
to WHO by SDS Biotech (unpublished).
Savides, M.C., Marcinsizyn, J.P., Killeen, J.C., Jr., &
(1985b) Ignatoski, J.A. Study of the distribution of radioactivity
following repeated oral administration of 14C-
chlorothalonil to male Sprague-Dawley rats, interim report
No. 631-4AM-84-0079-001. Submitted to WHO by SDS Biotech
(unpublished).
Shults, S.K. A 90-day feeding study in mice with technical
(1983) chlorothalonil. Unpublished report from SDS Biotech Corp.
Submitted to WHO by Diamond Shamrock Corp.
Trump, B.F., et al. Re-evaluation of the renal tissue from the "90-day
(1985) toxicity study of T-117-11 in rats", 5TX-80-0200, Warwick
Institute for Science and the Environment, SDS Document No.
753-5TX-85-0055-002. Submitted to WHO by SDS Biotech.
Wilson, N.H., et al. A 90-day toxicity study of technical
(1981) chlorothalonil in rats. Report submitted to WHO by Diamond
Shamrock Corp. (unpublished).
Wilson, N.H., Killeen, J.C., Jr., Haley, B.L., & Ignatoski, J.A.
(1984) A subchronic toxicity study of technical chlorothalonil in
rats, report amendment No. 562-5TX-81-0213-004-001.
Submitted to WHO by SDS Biotech (unpublished).
Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. Histopathologic
(1985a) re-evaluation of renal tissue from a subchronic toxicity
study of technical chlorothalonil in rats (5TX-81-0213).
Document No. 753-5TX-85-0056-002. Submitted to WHO by SDS
Biotech (unpublished).
Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. Histopathologic
(1985b) re-evaluation of renal tissue from a 90-day toxicity study
of technical chlorothalonil in rats (5TX-80-0200). Document
No. 753-5TX-85-0055-002. Submitted to WHO by SDS Biotech
(unpublished).
Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. A tumorigenicity
(1985c) study of technical chlorothalonil in rats, document No.
099-5TX-80-0234-008. Submitted to WHO by SDS Biotech
(unpublished).