PESTICIDE RESIDUES IN FOOD - 1983
Sponsored jointly by FAO and WHO
EVALUATIONS 1983
Data and recommendations of the joint meeting
of the FAO Panel of Experts on Pesticide Residues
in Food and the Environment and the
WHO Expert Group on Pesticide Residues
Geneva, 5 - 14 December 1983
Food and Agriculture Organization of the United Nations
Rome 1985
CHLOROTHALONIL
TOXICOLOGY
Explanation
Chlorothalonil was evaluated by the Joint Meetings of 1974, 1977,
1979 and 1981 (FAO/WHO, 1975, 1978, 1980 and 1982). The 1981 Meeting
reduced the temporary acceptable daily intake (ADI) from 0.03 to
0.005 mg/kg b.w. inadequate metabolism data and a low no-effect level
(NOEL) in a rat reproduction study with the 4-hydroxy-2, 5,
6-trichloro-isophthalonitrile metabolite. Additional data were
required by 1983 in order to clarify the metabolism, nephrotoxicity
and mutagenicity of the parent compound and the 4-hydroxy metabolite.
These data are reviewed in this monograph addendum.
Temporary maximum residue limits (MRLs) were estimated during
previous meetings for a variety of commodities. Certain studies on
residues were required by 1983 and others considered desirable.
Desirable data and information have been provided in response to
several questions raised at the 1981 Meeting. One such question
concerned the efficiency of tumble extraction for field-incurred
residues of chlorothalonil, hexachlorobenzene (HCB) and
pentachlorobenzonitrile (PCBN). Another concerned apparent
discrepancies between two studies on residues resulting from the
cooking of chlorothalonil-treated foods. A revised report has been
provided, based on the original data.
In response to the suggestion of the 13th Session of the Codex
Committee on Pesticide Residues (CCPR) that the recommended limit for
clorothalonil on grapes did not represent good agricultural practice
(GAP), information on GAP and additional residue data were provided to
the 1981 Meeting, but were received too late for evaluation. These
data are also reviewed in this addendum, together with new or
additional residue data on other commodities, information on national
tolerances, and monitoring data on selected commodities from one
country.
The Meeting re-examined the definition of the residue in response
to a request of the 14th Session of the Codex Committee on Pesticide
Residues (CCPR).
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOCHEMICAL ASPECTS
Absorption, Distribution and Excretion
Chlorothalonil (2, 4, 5, 6-tetrachloroisophthalonitrile,
DS-2787), labelled in the benzene ring, was orally administered as a
water suspension to male Sprague-Dawley rats (five per group) housed
in metabolism cages. Urine and faeces from rats administered a single
dose of 0, 5 or 200 mg/kg were analysed by high performance liquid
chromatography (HPLC) to determine the extent to which chlorothalonil
was metabolized to 4-hydroxy- 2 3, 5-trichloroisophthalonitrile
(DS-3701) and/or other metabolites, at 2, 9, 24, 36, 48, 60, 72, 84
and 96 hours after dosing. Blood and other tissue samples were
collected at necropsy, but not analysed.
HPLC analyses of high dose faecal extracts showed that 28 percent
of the administered dose was eliminated as DS-2787 and 5 percent as
DS-3701. A third peak of radioactivity eluted from the HPLC in the
same time frame as the trichloroacid amide (DS-46851) and accounted
for 1.4 percent of the administered dose. Analyses of faecal extracts
from the low dose level showed that chlorothalonil, DS-3701 and
DS-46851 (tentative) accounted for 1.6, 6.2 and 2.6 percent of the
administered dose, respectively.
HPLC analyses of urine samples showed that, at the 200 mg/kg dose
level, chlorothalonil, DS-3701 and DS-46851 represented 0.05, 0.29 and
3.53 percent, respectively, of the administered dose. At the 5 mg/kg
dose level, DS-2787, DS-3701 and DS-46851 accounted for 0.08, 0.58 and
4.48 percent, respectively, of the administered dose (Ignatoski et
al 1983a).
Chlorothalonil, labelled in the benzene ring, was orally
administered to male Sprague-Dawley rats at dose levels of 0, 5, 50
and 200 mg/kg b.w. to determine the distribution of radioactivity in
rats at 2, 9 and 24 hours after dosing. Approximately 80 percent of
the administered dose was found in the contents of the small intestine
at 2 hours, 90 percent in the large intestine at 9 hours and 95
percent in the large intestine at 24 hours after dosing. Accumulation
of radioactivity in all tissues except the gastro-intestinal tract was
less than 1 percent of the dose. The total µg equivalents (expressed
as chlorothalonil) found in the gastro-intestinal tract contents were
directly proportional to the dose administered.
Profiles of the concentration of labelled material in blood and
other tissues as a function of time showed that only at the 5 mg/kg
dose was the concentration essentially zero 24 hours after dosing. At
the 50 and 200 mg/kg doses, blood and tissue concentrations of the
labelled material (s) were persistent in significant quantities 24
hours after dosing when compared to the 5 mg/kg dose (Ignatoski et
al 1982).
14C-Chlorothalonil was administered orally as a microparticulate
suspension in 0.5 percent methylcellulose to male Charles River CD-1
mice (16 per group) at dose levels of 0, 1.5, 15 or 105 mg/kg b.w.
Urine, faeces, blood and other selected tissues were assayed for
radioactivity. The total recoveries of radioactivity ranged from 93 to
99 percent for the low dose, 78 to 85 percent for the mid dose and 73
to 77 percent for the high dose.
The major route of elimination of radiolabelled material was via
the faeces. Faecal elimination was complete by 24 hours for the low
and mid doses and by 96 hours for the high dose. Delayed faecal
elimination at the high dose resulted from large amounts of
radioactivity remaining in the stomach contents at 9 and 24 hours.
Approximately 20 percent and 9 percent of the high dose was found in
the stomach contents at 9 and 24 hours, respectively.
There was also a high amount of radio activity in the large
intestine (13 percent) at 9 hours. The high dose of chlorothalonil may
have had an effect on stomach emptying time, which delayed faecal
elimination of radioactivity. Furthermore, the soft consistency of the
faeces suggested that there was increased motility or water retention
in the lower gastro-intestinal tract but no increased faecal
elimination at the high dose.
Urinary excretion at all doses generally varied between 5 and 10
percent of the administered dose. The total amount of radioactivity
found in nine tissues and blood was less than 3 percent of the
administered dose (Ignatoski et al 1983b).
14C-Chlorothalonil was administered intraduodenally to male rats
(donor animals) at a dose level of 5 mg/kg b.w. and bile was collected
for 24 hours after dosing. The bile collected during the first 6 hours
post-dosing was pooled from individual animals and administered
intraduodenally to recipient rats. The recipient rats received 0.2
percent to 2.4 percent of the total radioactivity administered to the
donor rats. Bile from the recipient animals was also collected for 24
hours after dosing.
Data from the donor animals indicated that 1 to 6 percent of the
administered radioactivity was excreted in bile within 24 hours
after dosing. Approximately 19 percent of the radioactivity
administered to recipient rats was excreted within 24 hours after
dosing. Characterization of the radioactivity with respect to the
parent compound or metabolites was not performed (Ignatoski et al
1983c).
14C-chlorothalonil was administered intraduodenally in maize oil
to Sprague-Dawley rats (six per group) at dose levels of 0.5, 5 or
50 mg/kg b.w. or in methyl-cellulose at a dose level of 5 mg/kg b.w.
The percent of the administered dose excreted in bile from animals
receiving 5 mg/kg b.w. in maize oil (31.2 percent) was essentially
identical to those animals receiving 0.5 mg/kg b.w. in maize oil (35.8
percent). At 50 mg/kg b.w. in maize oil, only about 11 percent of the
administered dose was excreted in bile.
When methylcellulose was the vehicle at 5 mg/kg b.w., about
7 percent of the administered dose was absorbed and excreted in bile
within 24 hours, suggesting that absorption was facilitated by maize
oil as compared with methylcellulose (Ignatoski et al 1983d).
14C-Chlorothalonil was administered intraduodenally in maize oil
to male Sprague-Dawley rats (five per dose) at six dose levels (0.5,
5, 10, 50, 100 and 200 mg per kg). Data suggested that when
chlorothalonil is administered intraduodenally to rats, the excretion
profile in bile is dependent upon the dose and further that there is
possibly a pharmacokinetic overload in response to increasing doses of
chlorothalonil (Ignatoski et al 1981).
Two male Sprague-Dawley rats were administered orally 5 mg
14C-chlorothalonil/kg b.w. as a microparticulate suspension in 0.5
percent methylcellulose. Blood samples, obtained at periodic intervals
by orbital sinus puncture, showed peak blood levels between 4 and 7
hours post-dosing, which rapidly declined thereafter. The total amount
of radioactivity in the blood never exceeded one percent (1 percent)
of the administered dose, with the majority of the activity in plasma
(70 percent) and the remainder in the red blood cells (packed cell
volume) (Ignatoski et al 1983e).
In a separate study there were dose related changes in the mean
blood concentrations (ng equivalents/ml), versus time. The kinetics
were non-linear at 200 mg/kg with a shift in the time of peak
concentration at 50 and 200 mg/kg caused possibly by affecting stomach
emptying and a resultant lag in absorption (Ignatoski et al
1983f).
TOXICOLOGICAL STUDIES
Short-Term Studies
Mouse
Groups of CD-1 Charles River mice (15 males and 15 females/group)
were administered chlorothalonil (98.9 percent pure, containing 0.03
percent HCB or less) in the diet at dosage levels of 0, 7.5, 15, 50,
275 and 750 ppm for 13 weeks. Five males and five females/group were
subjected to interim necropsies after six weeks of compound
administration. Routine examinations were performed for gross signs of
toxicity, physical examinations, body weight changes, food consumption
and selected clinical chemistry parameters. At terminal sacrifice all
surviving animals were subjected to complete gross necropsy, brain and
kidneys were weighed and a histological evaluation made of stomach and
kidneys.
There were no compound-related deaths during the study or any
other untoward signs of toxicity, mood consumption and body weight
gains were comparable among groups. Mean compound consumption ranged
between 1.2 and 141.2 mg/kg/day for the respective doses. There were
no compound related effects on the clinical chemistry parameters,
including blood urea nitrogen (BUN) and creatinine. Alkaline
phosphatase levels were increased in high-dose females. There were no
significant differences in brain weight; however, absolute and
relative kidney weights were increased in females, but not in males,
at 275 and 750 ppm. Gross necropsy was unremarkable except for
proteinaceous plugs in the lumen of the bladder in males, generally
distributed among all groups, with slight increased incidence in high-
dose males. Microscopic examination of the stomach revealed an
increased finding of hyperplasia and hyperkeratosis of the squamous
epithelial cells in both sexes at 50, 275 and 750 ppm. Histologic
evaluation of the kidney did not reveal any compound-related changes.
Generalized vacuolation of the cortical tubular epithelial cells and
hyperplasia of the epithelial cells of the proximal convoluted tubules
in high-dose males at the interim sacrifice were not evident at
terminal sacrifice. Other changes were sporadic and not considered
compound-related.
Chlorothalonil was demonstrated to be without adverse effects on
mice at levels up to and including 15 ppm (2.5-3.0 mg/kg/day) when
incorporated in the diet for 90 days (Shults 1983).
Rat
Groups of Charles River CD rats (20 sex/dose) were administered
chlorothalonil (98 percent pure, with 0.03 percent HCB) in the diet at
dosage levels of 0, 40, 80, 175, 375, 750 and 1 500 mg/kg b.w./day for
90 days. Animals were examined routinely for mortality and gross
signs of toxicity; body weights and food consumption were measured
periodically and haematological, clinical chemistry and urinalysis
parameters examined pre-test, at 30 days and at the termination of the
study. All surviving animals were necropsied, selected organs weighed
and a complete list of tissues/organs examined microscopically.
Survival was comparable among all groups. Indication of cathartic
action related to compound ingestion was evident in both sexes at 750
and 1 500 mg/kg/day. Evidence of soft stools, reduced faecal output,
mucus in stools, swelling and irritation of the anus occurred with
greater frequency and severity in the two highest dose groups. There
were significant dose-related body weight reductions in both sexes at
dose levels of 375 mg/kg/day end greater. Food consumption comparisons
indicated compound-related increases throughout the study. There were
significant but spurious increases in haemoglobin, haematocrit and
erythrocyte counts in males, which are not considered to be compound-
related. Mean corpuscular volume, mean corpuscular haemoglobin and
mean corpuscular haemoglobin concentration were unaffected by
treatment. Red blood cell morphology was normal in all groups. There
were compound-related decreases in glucose levels at > 375 and
> 750 mg/kg in males and females, respectively. There were similar
reductions in BUN at > 80 and > 375 for males and females,
respectively. All other clinical chemistry determinations were normal
except for depressed SGPT activity, present in both sexes in all
treatment groups. This effect was generally dose-related. A special
evaluation of serum thyroxine (T-4) and triiodothyronine (T-3)
demonstrated depressed T-4 levels at > 175 and 1 500 mg/kg in males
and females, respectively. This depression correlates with the
decreased rate of body weight gain at those levels.
There were dose-related increases in specific gravity and
decreased urine volume for males given > 375 mg/kg of
chlorothalonil. There was also an increased incidence of dark urine as
the dose was increased from 375 to 1 500 mg/kg. There were no similar
findings in females.
Although several absolute and relative organ weight changes were
determined, only the kidney weight changes are considered to be
compound-related. Relative kidney weights were increased in both sexes
at all treatment levels, but gross and histopathological evaluations
revealed no correlative compound-related effects. Gross necropsy
findings were unremarkable among all groups. The only dose-related
histologic effect of treatment, which was inversely related to dose,
was a finding of acute gastritis in the non-glandular portion of the
stomach in all treatment groups.
Based on the relative kidney weight changes at all levels, with
compound-related effects on specific gravity and urine volume at
> 375 mg/kg, a clear no-adverse-effect level has not been
demonstrated. The depressed SGPT activity at all treatment levels in
both sexes, considered to be compound-related, is difficult to
interpret, particularly since relative liver weights were increased at
> 750 mg/kg for both sexes (Wilson et al 1981).
Technical grade chlorothalonil (98.2 percent pure) was
administered in the diet to groups of Sprague-Dawley rats
(25/sex/dose) at dose levels of 0, 1.5, 3, 10 and 40 mg/kg for 13
weeks. Selected animals from each group were continued on a control
diet for an additional 13-week recovery period. Serial sacrifices were
conducted at 6 weeks (5/sex/group), 13 weeks (10/sex/group) and 26
weeks (all remaining animals). Mortality was monitored daily and
physical examinations conducted periodically. Body weight gain and
food/water consumption were recorded weekly. Haematological, clinical
chemistry and urinalysis parameters were determined throughout the
study. Complete necropsies were performed and selected organs weighed;
microscopic examination was done on all animals. In addition, EM
examination of kidneys was also performed.
There was no mortality associated with treatment. Physical
examinations were unremarkable as were body weight, food consumption,
urinalysis and haematological determinations. The only differences
between control and treatment groups regarding clinical chemistry
evaluations were reduced alkaline phosphatase and glutamic pyruvic
transaminase activities in both sexes at 10 and 40 mg/kg. The
differences were more pronounced in males and remained depressed thru
week 13, while females tended to recover by week 13.
Gross necropsies did not reflect treatment-related effects,
except for increased kidney weights in males and females at 3, 10 and
40 mg/kg/day and increased liver weights in males at 40 mg/kg/day.
Microscopic examination revealed an increased incidence of
epithelial hyperplasia and hyperkeratosis in the non-glandular portion
of the stomach in both sexes given 10 and 40 mg chlorothalonil/kg
b.w./day. Examination of the kidney revealed marginal increases in the
incidence of dilated medullary tubules but no effects on cortical
tubules at 10 and 40 mg/kg/day. There were no other adverse effects
noted in other organs/tissues. After the 13-week recovery period,
these apparent compound-related effects had reversed. Microscopic
examinations did not provide evidence for the cause of organ weight
changes observed in the liver and kidneys.
Chlorothalonil was without adverse effects on the Sprague-Dawley
rat at doses up to and including 3 mg/kg/day (Wilson et al 1983a,
b).
Groups of Sprague-Dawley CD rats (10 males and 10 females/group)
were fed 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (99.6 percent
pure) in the diet at dosage levels of 0, 10, 20, 40, 75, 125, 150,
500 and 750 mg/kg b.w./day for 61 to 69 days. Complete physical
examinations were conducted routinely and body weight and food
consumption measured throughout the study. Haematology, clinical
chemistry and urinalysis parameters were routinely determined. Gross
necropsy, organ weights and histologic examinations were performed at
the termination of the study.
Mortality was significantly increased in males at > 125 mg/kg
and in females at > 75 mg/kg. There were no mortalities in dose
groups of 0 to 40 mg/kg. Gross observation included dose-related
occurrence of piloerection in all dose groups and pale skin and eyes
in males and females at > 20 and > 75 mg/kg respectively. Body
weights were depressed by treatment at levels > 40 mg/kg in both
sexes. Food consumption was similarly decreased at the same dietary
levels.
Haematological evaluations presented evidence of anaemia in
treated groups. There were significant decreases in the red blood cell
count, haematocrit, haemoglobin, mean corpuscular volume and mean
corpuscular haemoglobin at > 75 and > 40 mg/kg in males and
females, respectively. Reticulocytes were similarly increased in males
and females at > 75 and > 40 mg/kg, respectively. Nucleated red
blood cells were increased in both males and females at > 40 mg/kg.
Clinical chemistry parameters demonstrated similar compound-
related effects with increases in alkaline phosphatase, BUN, SGPT and
SGOT at > 125 and > 75 mg/kg in males and females, respectively.
Both males and females at > 75 mg/kg had reduced total protein,
albumin and globulin levels, with an increased albumin globulin (A/G)
ratio at the same dosage level.
Urinalyses were unremarkable at doses up to and including
75 mg/kg. There were no evaluations conducted at higher doses.
Gross necropsies presented occurrences of pale liver,
pale/discoloured kidney, gastric ulceration, distended urinary bladder
and flaccid heart at 75 mg/kg in both sexes. Since body weight
decreased at 40 mg/kg, brain weight was used for comparison of
relative organ weights. Accordingly, males and females at 40 mg/kg had
reduced heart and kidney-to-brain weights. Females at that level also
had reduced ovarian weights. At 75 mg/kg males, had reduced liver,
spleen and testes weights, while females had reduced liver weights.
Histopathological examination demonstrated compound-related
effects in males and females at > 40 mg/kg in the form of erythroid
hyperplasia and depressed granulopoiesis in bone marrow and spleen.
There were also increased incidences of ovarian follicular
degeneration and necrosis and renal cortical atrophy in females (the
latter evident in males at > 75 mg/kg). At > 75 mg/kg, males and
females had increases of hepatic haemosiderosis, toxic centrilobular
hepatitis and myocardial degeneration. In females, there was also
evidence of increased focal adrenal cortical necrosis, vacuolative
degeneration and renal cortical tubular degeneration. These became
evident in males at > 125 mg/kg, along with an increased incidence
of testicular degeneration. Females were more sensitive to the toxic
effects of the metabolite than were males. There were no adverse
effects demonstrated at dose levels up to and including 20 mg/kg
(Murchison et al 1979).
Special Studies on Reproduction
Rat
A one-generation reproduction study was performed using Charles
River CD rats, wherein 4-hydroxy-2, 5, 6-trichloroisophthalonitrile
was administered in the diet. Five groups containing 12 males and 24
females each were given dosage levels of 0, 10, 20, 30, 60 and 120 ppm
for 18 weeks before mating and continuously through two successive
reproduction cycles. F1a and F1b offspring were necropsied at weaning
with tissues preserved from 5 males and 5 females in each group from
the Flb pups. These tissues were not examined microscopically.
Litters were culled at day 4 to 10 per litter (sexes equal) and the
litter weights were determined at days 0, 4, 7, 10, 14 and 21. Indices
of reproductive performance were determined.
Mortality, body weight and food consumption of F0 parents were
unaffected by treatment, with the exception of increased food
consumption in high-dose males. The overall mating index in F1a and
F1b litters was poor, being 62.5 percent and 54.2 percent,
respectively. Gestation index for both control litters was similarly
reduced, being 73.3 percent and 69.2 percent, respectively. Overall
mating and gestation indices in the dose groups were greater than
control values and, therefore, unaffected by treatment. There were no
compound-related effects on maternal body weights during gestation and
lactation. There were no compound-related effects observed on the mean
number of live pups in either F1a or F1b litters from birth through
lactation. Live born and stillborn indices were comparable among all
groups in both litters. Pup viability in the high dose group at day
4 was significantly lower than controls in the F1a and F1b litters.
There were no compound-related effects on the lactation or litter
viability indices, although slightly reduced litter viability indices
were reported for both litters (86.7 and 84.6 percent, respectively)
at the high dose.
No statistically significant differences in mean live pup weights
between control and treatment groups were reported at birth or day 4,
although there was a negative trend in the F1a high dose group at day
4. This was not evident in the F1b litter. There were, however,
statistically significant differences for the high dose group in both
litters at days 7, 10, 14 and 21 and at 60 ppm on days 10, 14 and 21
in the F1b litter. There were no compound-related gross findings in
any of the animals necropsied. The no-effect level in this study was
demonstrated at 30 ppm (Ford 1982).
Special Studies on Teratogenicity
Rat
Groups of Sprague-Dawley rats (25 females/groups) were
administered chlorothalonil orally, via gavage, doses of 0, 25, 100
and 400 mg/kg/day from day 6 through 15 of gestation. Surviving
females were necropsied on day 20 and foetuses delivered by
hysterotomy. The number and position of viable/non-viable foetuses,
early/later resorptions, mean number of corpora lutea and total
number of implantations were recorded. External, internal and skeletal
examinations of foetuses were performed for evidence of abnormalities
and anomalies. Half of the foetuses were evaluated for soft tissue
anomalies and the other half for skeletal effects.
There was no dose related mortality in the 25 and 100 mg/kg/day
groups. However, the deaths during treatment of three dams in the
400 mg/kg group were considered related to compound ingestion. There
were no abortions in any group. General appearance and behaviour were
unremarkable, except for evidence of cathartic action at 400 mg/kg
(e.g. loose faeces, matting of urogenital fur). Mean maternal body
weights were significantly different (less) than control at the high
dose level. Food consumption was significantly reduced in all
treatment groups initially (days 6 to 9) and in the high-dose group
throughout the dosing period (days 6 to 15). There were no differences
compared to controls for mean number of viable foetuses, implantation
sites, corpora lutea or foetal weights. There was a significant
increase in the number of early resorptions in the high-dose group, as
well as post-implantation losses, when compared to controls. There
were no reported effects on number or percentage of foetuses/litters
with external, internal or skeletal malformations or developmental
variations at any dose level administered.
Chlorothalonil was considered maternally toxic to rats at
400 mg/kg but there was no evidence of teratogenicity at any level
tested (Rodwell et al 1983).
Rabbit
Groups of pregnant Japanese White rabbits (9/group) were
administered orally, via gastric intubation, doses of 0, 5 and
50 mg/kg of chlorothalonil from day 6 through 18 of gestation. All
does were sacrificed on day 29, pups delivered by caesarean section,
and the number of implants, live and dead foetuses recorded. Pups were
examined for gross malformation, visceral organs removed and examined,
and carcases stained with Alizarin Red S for skeletal evaluation.
Body weights were reduced in high-dose females and 4/9 does
aborted. Foetal resorptions were increased at 5 and 50 mg/kg compared
to controls. The number of implants and number of live foetuses were
reduced in the high-dose group when compared to controls. External
examination revealed 1/74 foetuses at 5 mg/kg with hydrocephaly and
1/36 foetuses at 50 mg/kg with cleft palate. There were no visceral or
skeletal anomalies reported. Chlorothalonil is considered maternally
toxic at 5 and 50 mg/kg, but there was no evidence presented of
teratogenic effects (Shirasu & Teramoto 1975).
In a pilot teratology study, groups of rabbits were orally dosed
with 4-hydroxy-2, 5, 6-trichloroisophthalonitrile at 1, 5, 10, 25 and
50 mg/kg/day from day 6 through 18 of gestation. Maternal death and
toxicity occurred at doses of 10, 25 and 50 mg/kg with no adverse
effects observed at 1 mg/kg. In the 5 mg/kg dose group, one female
aborted, one was not pregnant and one female presented a litter of
runts. The other females in this group had normal pregnancies and pups
(Wazeter & Goldenthal 1976).
Groups of Dutch Belted rabbits (10 per/group) were orally
intubated with 4-hydroxy-2, 5, 6-trichloroisophthalonitrile at dose
levels of 0, 1, 2.5 and 5 mg/kg/day from day 6 through 18 of
gestation. Pups were delivered by Caesarean section on day 28 and the
number, location and distribution of live/dead foetuses, corpora
lutea, implantations and early/late resorptions recorded. All
foetuses were examined grossly, sectioned for visceral anomalies and
stained for skeletal anomalies.
There were three deaths, one at 2.5 and two at 5 mg/kg. There
were no other gross signs of toxicity reported and body weight gains
were uniform among all groups. There were no compound-related effects
on the number of implantation sites, foetal weights, sex ratios or
foetal anomalies. The number of females with early resorbed foetuses
and number of females aborting was increased compared to controls at
the high dose. However, most of these early resorptions in the high-
dose group occurred in one female which had 10 implantation sites and
10 resorptions. Otherwise, resorptions were comparable among treatment
and control groups. There were no visceral or skeletal anomalies or
abnormalities directly attributable to compound ingestion at doses up
to and including 5 mg/kg/day. However, this dose did present marginal
effects of maternal toxicity, which were not apparent at 2.5 mg/kg
(Wazeter & Goldenthal 1976).
Special Studies on Carcinogenicity
Mouse
Groups of CD-1 mice (60 males and 60 females/group) were
administered technical chlorothalonil in the diet at dosage levels of
0, 750, 1 500 and 3 000 ppm for 24 months. Mice were six weeks old at
the initiation of the study. Animals were observed daily for mortality
along, with other cage-side observations for signs of toxicity.
Individual body weights, food consumption, physical examinations and
haematological parameters were evaluated regularly throughout the
study. All animals were necropsied, organ weights determined and
selected tissues/organs examined histologically.
There were no apparent compound-related effects on body weight
gain, food consumption or general toxic response. Mortality was
increased in high-dose males (40/60) compared to control males
(29/60). Mortality in control females was 42/60. There was abdominal
swelling in mid-and high-dose males, which was evident for the first
six months, but no differences between control and treatment groups
after six months. Distended abdomens were noted generally among all
groups. Compound consumption resulted in 0, 119, 251 and 517 mg
chlorothalonil/kg b.w./day for males; and 0, 134, 278, and 585 mg
chlorothalonil/kg b.w./day for females in the 0, 750, 1 500 and
3 000 ppm dose groups, respectively.
Haematology measurements revealed an increased incidence of
abnormal erythrocyte morphology, which was unrelated to other normal
findings and not considered significant. Decreased haemoglobin,
haematocrit and red blood cell values were evident in high-dose males
at 24 months and in high-dose females at 18 and 24 months. There was
an increase of hyperplastic bone marrow (sternum) for males and
females of all treatment groups, in relation to corresponding
controls, as well as hyperplasia of the red pulp in the spleen of
treated males. The spleens of all female groups were comparable with
control females, which had the most significant increase in
hyperplastic red pulp. Haemosiderosis was unremarkable or not evident.
Examination of organ weights demonstrated an increase in absolute
and relative spleen weights for high-dose females but no significant
pathological changes. Gross necropsy indicated the spleen was enlarged
for mid- and high-dose males. Absolute and relative ovary weights in
all females were decreased, with no significant dose-related or
histopathologic changes. Absolute and relative testes weights for
high-dose males were decreased but no compound related histomorphic
changes were observed. Absolute and relative liver weights were
increased in mid- and high-dose males and all females but were not
statistically significant and were not accompanied by histopathologic
changes. Absolute and relative kidney weights were increased in all
treatment groups (p < 0.01) and were considered dose-related.
Gross necropsy of all organs revealed compound-related effects in
the kidney, described as renal enlargement, discolouration, surface
irregularities, pelvic dilation, cysts, nodules and masses in treated
groups only. There were no other significant compound-related effects
on other organs or tissues reported.
Histopathological data demonstrated compound-related effects on
the stomach, esophagus and kidneys. The incidence and severity of
hyperplasia and hyperkeratosis of the squamous mucosa in the
oesophagus of treated males and females was significantly increased in
a dose- and compound-related manner. Microscopic examination of the
stomach demonstrated a more significant dose-related increase in the
incidences of hyperplasia and hyperkeratosis of the squamous mucosa in
male and female mice in the treated groups. This finding was virtually
non-existent in control groups. There was a significant increase in
squamous cell tumours in the 1 500 ppm females but the incidence was
not dose-related. The incidence of squamous cell tumours in males was
0/60, 1/60, 5/60 and 2/60 in control, 750, 1 500 and 3 000 ppm groups,
respectively. In females the incidence was 0/60, 2/60, 6/60 and 5/59
in control, 750, 1 500 and 3 000 ppm groups, respectively. Glandular
epithelial tumours were increased in the treated groups but were not
significant or dose-related. The incidence of glandular epithelial
tumours in males was 0/60, 1/60, 2/60 and 0/60 in control, 750, 1 500
and 3 000 ppm groups, respectively. In females the incidence was 0/60,
1/60, 1/60 and 2/59 in control, 750, 1 500 and 3 000 ppm groups,
respectively.
Microscopic examination of the kidney revealed a significant
finding of chronic glomerulonephritis present in all control and
treatment groups, which was increased predominantly in males at
3 000 ppm. Tubular degeneration was increased in males at 750 and
1 500 ppm and in females at 1 500 ppm. The incidence of cortical cysts
was increased in all treated males and in high-dose females. The
neoplastic changes, reported as adenomas and carcinomas of the renal
cortical tubules, were increased in the males in all treated groups
but not in females. The incidence was 0/60, 6/60, 4/60 and 4/60 for
control, 750, 1 500 and 3 000 ppm male groups, respectively. There
were no neoplastic changes in the female groups, except for one
low-dose female with a kidney haemangiosarcoma.
Chlorothalonil has presented evidence of causing nephrotoxicity
in earlier studies in rats, mice and dogs, predominantly in males. In
an NCI rat study there was presumptive evidence of adenomas and
carcinomas of the renal tubular epithelium. The NCI mouse study was
negative for evidence of kidney tumours. Although primary renal
tumours in rodents are rare, there was no positive trend for adenomas
and carcinomas in the renal cortical tubules of male mice in this
study, and therefore the evidence for tumourigenicity of
chlorothalonil in the kidney remains elusive. The effects on the
kidney, in this study, are nonetheless considered compound-related
(Wilson et al 1983a; Tierney 1983).
Groups of six-week old CD-1 mice (60 males and 60 females per
group) were administered the chlorothalonil metabolite 4-hydroxy-2, 5,
6-trichloroisophthalonitrile in the diet at dosage levels of 0, 375,
750 and 1 500 ppm for 105 consecutive weeks. Animals were observed
daily for mortality and other gross signs of toxicity. Individual body
weights, food consumption, physical examinations and haematological
parameters were evaluated regularly throughout the study. All animals
dying or sacrificed during and at the termination of the study were
necropsied; organ weights were determined, and selected tissues/organs
were examined histologically.
The mortality in the low- and mid-dose females reached 75 percent
during month 20 and, therefore, all surviving low- and mid-dose
females, as well as 10 control females (to serve as concurrent
controls) were terminated at month 20. Mortality in the high-dose
females reached 75 percent at month 22 at which time all females in
that group and in the control group were sacrificed. Survival of the
male mice was comparable between control and treated groups.
There were no significant differences between control and
treatment group animals on the basis of cage-side observations for
signs of toxicity. Body weights were significantly reduced in both
males and females given 1 500 ppm throughout the study. There were
also sporadic decreases in males and females at 750 ppm that were
evident during the first year of study and were comparable to controls
during the second year. There were significant increases in food
consumption for high-dose females compared to controls throughout the
study and for high-dose males during the first year.
Average compound intake throughout the study, on a mg/kg/day
basis, for males was 50-88 (at 375 ppm), 90-180 (at 750 ppm) and
190-425 (at 1 500 ppm); for females it was 58-103 (at 375 ppm),
100-275 (at 750 ppm) and 232-624 (at 1 500 ppm).
Haematological examinations conducted at 12, 18 and 20-24 months
revealed compound-related effects on red blood cells with decreases in
all treatment groups at 12 months, decreases in males at 750 ppm and
females at 750 and 1 500 ppm at 18 months, and decreases in males
given 1 500 ppm at 24 months. Reticulocyte counts were increased in
males given 1 500 ppm at 24 months and in females given 750 and
1 500 ppm at 20 months.
Evaluation of differential leukocyte counts demonstrated moderate
to marked increases in morphological changes in erythrocytes and
leukocytes at 18 months in all treated females and 750 ppm males. At
terminal sacrifice, all treatment and control groups presented
evidence of changes, but they were more noticeable in the high-dose
groups. Changes included anisocytosis, poikilocytosis, polychromic red
cells, target cells, large platelets, crenated red cells and nucleated
red cells.
Bone marrow differential counts, including M/E ratios,
demonstrated a decreasing trend of 2:1 for control females to
approximately 1:1 for treatment groups. Similarly, a ratio of 1:2 for
control males was approximately 0.9:1 for treated males.
Organ weights and organ-to-body weight measurements demonstrated
significant increases in spleen and liver weights and respective
organ-to-body weight increases at 20 months in 750 ppm females.
Absolute liver weights and liver-to-body weight ratios were also
increased in 1 500 ppm females at 22 months. Absolute kidney weights
and heart weights were also decreased at 22 months in the 1 500 ppm
females. Male mice given 375-1 500 ppm revealed significant increases
in liver weight and liver-to-body weight ratios and a positive trend
in the spleen-to-body weight ratio. Absolute kidney weights were
reduced in males at 1 500 ppm but were not considered significant,
based on decreased body weight.
Histopathological evaluations revealed an increase in
bronchioalveolar adenomas in treated males. However, the differences
between the control incidences were not dose-related or significant
and, therefore, were not considered compound-related. The reported
incidence in males was 2/59 (0 ppm), 6/57 (375 ppm), 6/57 (750 ppm)
and 4/59 (1 500 ppm). The incidence of benign hepatocellular neoplasms
and hepatocellular carcinomas was higher in control mice, with a
negative trend in the treatment groups. There was one tubular adenoma
and one tubular carcinoma of the kidney in the low-dose males but no
incidences in any other groups.
Non-neoplastic changes consisted of a dose-related increase of
amyloidosis in the thyroid of both males and females. There was an
overall marginal dose-related increase in the incidences of
amyloidosis in treated males and females reported for the stomach,
duodenum, ilex, liver, adrenal and urinary bladder. There was also an
overall increase in amyloidosis reported for all female groups with
respect to the spleen, kidney, jejunum and ovary. There was an
increase in pigmentation (haemosiderin) of the spleen, which was
compound-related and evident at all doses in both sexes. Chronic
interstitial nephritis of the kidney was also considered compound-
related and was increased in all treated females compared to controls.
The test material was not oncogenic in CD-1 mice under the
conditions of this study at doses up to and including 1 500 ppm.
However, marginal and significant non-neoplastic changes, including
amyloidosis, haemosiderin in the spleen, morphological changes in
leukocytes and erythrocytes, increase in reticulocyte count as well as
evidence of red cell haemolysis, failed to demonstrate a clear no-
effect level for these effects (Hozan & Auletta 1981).
Rat
Groups of Sprague-Dawley CD rats (75 males and 75 females/group)
were administered 4-hydroxy-2, 5, 6-trichloroisophthalonitrile in the
diet at dosage levels of 0, 0.5 and 3 mg/kg/day for 104 weeks.
Original dosage levels of 15 and 30 mg/kg/day were reduced at week 30
to 10 and 20 mg/kg/day, respectively, because of poor survival and
anaemia. Animals were observed daily for mortality, gross signs of
toxicity and general appearance. Individual body weights and food
consumption were measured regularly during the study. Clinical
laboratory studies were performed periodically throughout the study on
10 rats/sex/group at six-month intervals. Ophthalmological
examinations and urinalyses were performed routinely, and faeces were
collected and examined to evaluate the observed anaemia. Interim
sacrifices were performed after one year on 10 rats/sex in all groups,
except for the high-dose animals, which were all necropsied. Terminal
necropsies were performed on all surviving animals after two years,
selected organs weighed and complete histopathological examinations
conducted.
Pale skin and eyes were evident for the first 30 weeks in high-
dose males and females, with similar but less marked findings observed
in the 15 mg/kg group. Mortality was significantly increased in the
30 mg/kg males and females and in the 15 mg/kg females. The high-dose
group was sacrificed at 12 months after the dose level had been
reduced to 20 mg/kg at week 30. Decreasing the 15 mg/kg/day dose level
at week 30 to 10 mg/kg similarly improved the survival, which was
comparable to controls for the remainder of the study. Body weight was
reduced in the 10/15 and 20/30 mg/kg males and females throughout the
study, even after reduction of doses. Food consumption was
unremarkable except for decreases in 10/15 and 20/30 mg/kg females and
20/30 mg/kg males, which was consistent with decreased body weights
and increased mortality during the first 30 weeks. There were similar
decreases in total serum protein, albumin, globulin and cholesterol in
20/30 mg/kg males and females and 10/15 mg/kg females after six
months. These returned to control levels for the remainder of the
study after doses were reduced to 20 and 10 mg/kg, respectively.
There were significant haemopoietic effects in the 10/15 and
20/30 mg/kg animals, particularly females, during the first six
months. Evidence of microcytic anaemia was provided by reduced red
cell counts, haematocrit, haemoglobin, MCV and MCH, with accompanying
increases in MCHC, reticulocytes and metarubricytes. Segmented
neutrophiles were increased with a corresponding decrease in the
percentage of lymphocytes. Specially stained bone marrow presented
evidence of hypocellularity. Mallory's stain of liver tissue revealed
an increased iron content (haemosiderin). After 18 and 24 months
exposure, the 10/15 mg/kg group females continued to present evidence
of anaemia (decreased Hct, Hgb, MCV, MCH and increased MCHC) with a
positive bone marrow response (increased cellularity with a shift to
increasing number of immature erythyroid cell types and increased
number of animals with 1:1 M/E ratio). Prussian Blue staining
demonstrated the presence of haemosiderin in the 10 mg/kg males and
females which was not considered significant at 3 mg/kg. After 24
months exposure, there were decreased serum potassium levels in all
treated females. Urinalyses, and examination for faecal occult blood
were unremarkable, except for increased urine volume at six months in
the high-dose animals.
Ophthalmological examination at six months revealed increased
pale ocular structures and spontaneous hemorrhage in high-dose males
and females. At 24 months there were increased numbers of dilated
pupils (not responding to light) and increased bilateral cataract
disease in high-dose males.
Comparison of selected organ weights demonstrated decreased
absolute organ weights for kidney, heart and brain in high-dose males
with no significant relative organ-to-body weight changes. High-dose
females had decreased absolute kidney and heart weights with no
relative weight changes, except for spleen and brain. Microscopic
examination failed to confirm any compound-related effects on these
organs. There were no significant compound-related non-neoplastic
organ changes, except for haemosiderin in the liver of high-dose
females and haemorrhage in CNS tissues, hypocellular bone marrow and
post-mortem congestion of lymph in high-dose males and females.
Examination of tissues/organs for neoplastic changes did not
indicate any compound-related effects at any level tested. Data
presented in this study demonstrate that the metabolite 4-hydroxy-2,
5, 6-trichloroisophthalonitrile is without adverse effects on male and
female rats at levels up to and including 3 mg/kg/day for two years
(McGee et al 1983).
Special Studies on Mutagenicity
Bacteria
Chlorothalonil, dissolved in DMSO, was tested for mutagenic
activity in Salmonella typhimurium strains TA98, TA100, TA1535,
TA1537 and TA1538 according to the plate incorporation procedure of
Ames, at concentrations between 0.33 and 6.6 µg/plate with and without
metabolic activation. Under the test conditions reported
chlorothalonil (97.8 percent pure) did net induce point mutations in
the five histidine-requiring strains of S. typhimurium at the
concentrations tested (Banzer 1977a).
Chlorothalonil was examined for mutagenic activity in
S. typhimurium strains G-46, TA1530, C207, TA1531, C3076, TA1700,
D3056 and TA1724 implanted in male albino mice. Host mediated assay at
the dose level of 4 mg/kg (single IP administration) was carried out.
No increase in mutation frequency of chlorothalonil treated group over
the control was observed and the test compound (99 percent pure) was
not mutagenic in the host mediated assay at the dose level tested
(Legator 1974a).
Chlorothalonil was tested for its mutagenic activity in
S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538,
according to the plate incorporation procedure of Ames et al.
(1975), at concentration between 1 and 10 µg/plate without metabolic
activation and between 2 and 20 µg/plate with metabolic activation. In
addition, Escherichia coli B/r WP2 hcr+4 and its uv-sensitive
derivative WP2 hcr -5 were also employed in the plate incorporation
procedure with specific selective medium at concentrations between 10
and 500-µg/plate without metabolic activation and between 10 and
100 µg/plate with metabolic activation. Under the given experimental
test conditions, chlorothalonil (99.3 percent pure) failed to induce
any increase in the number of revertant colonies above that of the
control and, thus, was not mutagenic at the concentrations tested
(Shirasu et al 1977).
Cultured mammalian cells
Chinese hamster cells (V-79) and mouse fibroblast cells
(Balb/3T3) in culture, with and without metabolic activation, were
exposed to chlorothalonil at a concentration of 0.3 µg/ml and
0.03 µg/ml, respectively, to detect mutations at the Ouabain resistant
gene locus, according to the method of Schechtman. Acetone was the
vehicle control without activation. S-9 alone was the negative control
with activation. No evidence of mutagenic effect of chlorothalonil was
observed in these two in vitro mammalian cell mutagenesis assay
systems. The test compound (97.8 percent pure) was not mutagenic under
the experimental conditions tested (Banzer 1977b).
Cytogenic assay
Chlorothalonil, suspended in 0.5% (w/v) Methocel E 15 premium
(hydroxy-propyl methyl cellulose), was tested in rats, mice and
Chinese hamsters for its ability to induce chromosomal aberrations in
bone marrow cells. Prior to the chromosomal aberration test, a
preliminary study was conducted in animals to determine appropriate
dosage levels. In the cytogenetic assay, animals were orally dosed
with the test compound with a 24-hour interval between doses. Six
hours after the second dose, animals were killed, bone marrow removed
from the femurs and slides prepared for the cells. One hundred
metaphase cells per animal were evaluated for chromosomal aberrations.
Ten male rats (Wistar) per group were orally dosed with 0, 8, 40,
200, 1 000 and 5 000 mg/kg chlorothalonil twice, with a 24-hour
interval between doses.
Ten male mice (Swiss C.F.L.P>) per group were orally dosed with
0, 4, 20, 100, 500 and 2 500 mg/kg chlorothalonil twice, with a
24-hour interval between doses.
Ten male Chinese hamsters, per group were orally dosed with 0, 8,
40, 200, 1 000 and 5 000 mg/kg chlorothalonil twice, with a 24-hour
interval between doses.
No significant differences in the induced chromosomal aberrations
were observed for the test animals between the treated and control
groups at the dose levels tested. Under the given experimental test
conditions, chlorothalonil (98.2 percent pure) was not considered to
be a clastogenic agent (Siou et al 1981a).
Micronucleus test
Chlorothalonil, suspended in 0.5 percent (w/v) Methocel E 15
premium (hydroxy-propyl methyl cellulose), was tested in rats, mice
and Chinese hamsters for its ability to induce the formation of
micronuclei in polychromatic erythrocytes. Prior to the micronucleus
test, a preliminary study was conducted in animals to determine
appropriate dosage levels. In the micronucleus test, animals were
orally dosed with the test compound, with a 24-hour interval between
doses. Six hours after the second dose, animals were killed, bone
marrow removed from the femurs and slides prepared from the cells. Two
thousand polychromatic cells per treatment were examined for the
presence of micronuclei.
The number of rats and mice per group and the dosage levels of
test compound used in this study were identical with that used in the
previous cytogenetic assay (Siou et al 1981b).
Ten male Chinese hamsters per group were orally dosed with 0, 4,
20, 100, 500 and 2 500 mg/kg chlorothalonil twice, with a 24-hour
interval between doses.
No significant differences in the mean value of polychromatic
erythrocytes with micronuclei were observed between the treated and
control groups at the dose levels tested. Under the test conditions
reported, chlorothalonil (98.2 percentage pure) exhibited no
clastogenic activity in the polychromatic stem cells of treated
animals (Siou et al 1981b).
Chlorothalonil was tested for its ability to induce clastogenic
activity in the polychromatic erythrocytes of treated animals. Swiss
albino male mice were given an oral dose of 6.5 mg/kg/day for five
consecutive days. Three hours after the final administration, animals
were killed, bone marrow removed from the femurs and slides prepared
from cells. Two thousand polychromatic cells were examined for the
presence of micronuclei. No significant differences in the frequency
of micronuclei from the polychromatic erythrocytes were noted between
the treated and control groups at the dose level tested. Under the
test conditions of this experiment, chlorothalonil (98 percent pure)
was not a clastogenic agent in the micronucleus test (Legator 1974a).
Dominant lethal test
Ten male mice were orally gavaged with 6.5 mg/kg/day
chlorothalonil for 5 days. Following the treatment, the males were
sequentially mated to two females per week for 8 weeks. The number of
corpora lutea, total implantations and resorptions were counted for
each pregnant female. The same number of untreated mice served as
controls. No increase in the resorptions in the treated group was
observed when compared to the control group. Chlorothalonil was not
considered to induce dominant lethals in treated male mice at the dose
level tested (Legator 1974a).
DNA damage and repair
The DNA damaging capacity of chlorothalonil was investigated by
the bacterial system (PolA-/PolA+) using S. typhimurium strains
TA1538 (repair deficient) and TA1978 (repair competent) in a spot
test. The test compound was tested with and without metabolic
activation at concentrations of 2, 10, and 20 µl of a 1.0 mg/ml stock
solution. Chlorothalonil (97.8 percent pure) exhibited significantly
different preferential cell killing between the two strains of
S. typhimurium, with and without metabolic activation, at the
concentrations tested. The test compound appears to have reacted with
the DNA molecules in a manner causing preferential cell killing of
strains, which lack an excision repair enzyme system. It is considered
a DNA-modifying agent in this test (Banzer 1977c).
Chlorothalonil was tested in Rec Assay using Bacillus subtilis
H17 (recombination wild) and M44 (repair deficient) in a spot test.
The compound was tested at concentrations of 2, 5, 10, 20, 100 and
200 µg/disc without metabolic activation. Chlorothalonil (99.3 percent
pure) exhibited no marked difference in the growth inhibition zones of
the strain H17 and M44. It is not considered a DNA-modifying agent at
the concentrations tested (Shirasu et al 1977).
Cell transformation
Chlorothalonil, diluted in acetone and saline, was tested to
determine its ability to induce malignant transformation of Fischer
rat embryo (F1706 and H4536) cells in culture. Prior to the
transformation assay, a preliminary cytotoxicity test was conducted to
determine the maximum non-toxic dose level of the test compound. In
the transformation assay, three concentrations of the test compound
(0.001, 0.0001 and 0.00001 µg/ml) were incubated with each of the
described cell lines for seven days. At the end of incubation, each
culture was washed, refed with a complete medium and held for two
additional weeks to look for foci of transformed cells. The cell
cultures treated with the test compound were inoculated subcutaneously
in the back of a newborn Fischer rat and examined for the development
of tumours within three months. There was no increased number of
transformed foci over the control in either of the treated cell
cultures. The cultures that had been exposed to the test compound were
not tumourigenic when injected into newborn Fischer rats.
Chlorothalonil (96 percent pure) is not considered a transforming
agent in the Fischer rat embryo cell lines at the concentrations
tested (Price 1978a).
The results of mutagenicity assays carried out with
chlorothalonil are summarized in Table 1.
Special Studies on Mutagenicity of Metabolites
Bacteria
4-hydroxy-2, 5, 6-trichloroisophthalonitrils (DS-3701), dissolved
in DMSO, was tested for mutagenic activity in S. typhimurium strains
TA98, TA100, TA1535, TA1537 and TA1538 according to the plate
incorporation procedure of Ames et al. (1975) at concentrations
between 1 and 100 µg/plate, with and without metabolic activation.
Under the conditions of this test, DS-3701 (99 percent pure) did not
induce point mutations in the five histidine-requiring strains of
S. typhimurium at the concentrations tested (Banzer 1977d).
Table 1. Results of Mutagenicity Assays of Chlorothalonil
Test Organism Test Substance Results Reference
(% purity)
GENE MUTATION STUDIES
Bacteria
S. typhimurium 97.8 No mutagenic activity was Banzer 1977a
reported in TA98, TA100, TA1535,
TA1537 and TA1538 with or without
metabolic activation.
99.0 Negative response in G46, C207, Legator 1974a
TA1530, TA1531, C 3076, TA1700,
D3056 and TA1724 according to
host mediated assay.
99.3 No mutagenic activity was Shirasu et al
reported in TA98,TA100, TA1535, 1977
TA1537 and TA1538 without
metabolic activation.
E. coli 99.3 No mutagenic activity was Shirasu et al
reported in WP2 hcr+4 and WP2 1977
hcr-5 with or without metabolic
activation.
Cultured Mammalian
Cells
Table 1. (con't)
Test Organism Test Substance Results Reference
(% purity)
Chinese hamster cells 97.8 No mutagenic effect was reported Banzer 1977b
(V79) and Mouse Fibroblast in these two mammalian cell lines
Cells (Balb/3T3) with or without metabolic activation.
CHROMOSOMAL EFFECTS
Cytogenetics - In Vivo 98.2 No induced chromosomal aberrations Siou et al
Rat, Mouse, Chinese were reported in bone 1981a
hamster marrow cells of rat, mouse and
Chinese hamster.
Micronucleus - In Vivo 98.0 Negative responses were reported Legator 1974a
Rat, Mouse, Chinese in the polychromatic erythrocytes & Siou et al
hamster of treated animals. 1981b
Dominant Lethal - Mice No mutagenic activity to induce Legator 1974a
dominant lethals in male mice
were reported.
DNA DAMAGE AND REPAIR -
Bacteria
S. typhimurium 97.8 Positive response in demonstrating Banzer 1977c
significantly preferential
cell killing between the
TA1538 and TA1978 with or
without metabolic activation.
Table 1. (con't)
Test Organism Test Substance Results Reference
(% purity)
S. subtilis 99.3 Negative response. No marked Shirasu et al
difference in the inhibition zones 1977
of the strain H17 and M44 was
reported.
Cell Transformation - 96.0 Negative responses were reported Price 1978a
In Vitro in both treated cell lines.
Fischer Rat Embryo Cell
Lines (F1706 and H4536)
4-hydroxy-2, 5, 6-trichloroisophthalonitrile was examined for
mutagenic activity in S. typhimurium strains G46, TA1530, C207,
TA1531, C3706, TA1700, D3056 and TA1724 which was implanted in male
Swiss albino mice according to the method of host mediated assay at
the dose level of 6.5 mg/kg (single IP administration). No increase in
mutation frequency of the treated group over the control was observed
and the metabolite (99 percent pure) was not mutagenic in the host
mediated assay at the dose level tested (Legator 1974b).
Mammalian cells
Chinese hamster cells (V-79) and mouse fibroblast cells
(Balb/3T3) in culture were exposed to 4-hydroxy-2, 5,
6-trichloroisophthalonitrile (DS-3701) at a concentration of 30 µg/ml,
with and without metabolic activation, to detect mutations at the
Ouabain-resistant gene locus, according to the method of Schechtman
et al. Acetone was the vehicle control. No evidence of mutagenic
effect of DS-3701 was observed in these two in vitro mammalian
cell mutagenesis assay systems. The test compound (99 percent pure)
did not induce Ouabain-resistant mutations at the concentration tested
(Banzer 1977e).
Micronucleus test
4-hydroxy-2, 5, 6-trichloroisophthalonitrile (DS-3701) was tested
in Swiss albino male mice at 6.5 mg/kg/day for five days for its
ability to induce the formation of micronuclei in polychromatic
erythrocytes. At the concentration tested, this compound (99 percent
pure) did not increase the number of polychromatic cells with
micronuclei (Legator 1974b).
Dominant lethal test
Ten male albino rats per group received a single oral
dose or five daily doses of 0, 2, 4 or 8 mg/kg 4-hydroxy-2, 5,
6-trichloroisophthalonitrile (DS-3701). Following treatment, the male
rats were sequentially mated with two virgin female rats per week for
eight weeks. After 12 days, the females were sacrificed and their
uteri were removed, implantation sites examined, resorption sites and
total foetuses counted and foetal deaths noted. No significant
differences in body weights of males, percent fertility, foetal deaths
or resorptions were observed between the treated and the respective
untreated groups either on a weekly or total basis. The test compound,
DS-3701 (99 percent pure), did not induce dominant lethals at the dose
levels tested (Hastings & Clifford 1975).
4-hydroxy-2, 5, 6-trichloroisophthalonitrile was tested for its
ability to induce dominant lethals in treated male mice at 1, 3 and
6.5 mg/kg/day for five days. The compound (99 percent pure) was not
considered as a mutagenic agent and did not induce dominant lethals at
the dose tested (Legator 1974b, 1975).
DNA damage and repair
DNA damaging capacity of 4-hydroxy-2, 5,
6-trichloroisophthalonitrile (DS-3701) was evaluated by the bacterial
system (PolA-/PolA+) using S. typhimurium strains TA1538 (repair
deficient) and TA1978 (repair competent) in a spot test. The compound
was tested with and without metabolic activation at concentrations of
2, 10 and 20 µl of a 1.0 mg/ml stock solution. Under these test
conditions, DS-3701 (99 percent pure) did not interfere with the DNA
mechanisms of S. typhimurium and, thus, was not considered a
DNA-modifying agent (Banzer 1977f).
Cell transformation
4-hydroxy-2, 5, 6-trichloroisophthalonitrile (DS-3701), diluted
in acetone and saline, was tested to determine its ability to induce
malignant transformation of Fischer rat embryo (F1706 and H4536) cells
in culture. Prior to the transformation assay, a preliminary
cytotoxicity test was conducted to determine the maximum nontoxic dose
level. The test compound (0.1, 1 and 10 µg/ml) was incubated with each
of the described cell lines for seven days. There was no increased
number of transformed foci over control in either of the treated cell
cultures. The cultures exposed to the test compound were not
tumourigenic when injected into newborn Fischer rats. However, the
F1706 cells exposed to DS-3701 did produce late tumours in the rats.
The production of these late tumours was considered to be due to
spontaneous transformation and not due to transformation by the test
compound. DS-3701 (99 percent pure) is not considered a transforming
agent in the Fischer rat embryo cell lines at the concentrations
tested (Price 1978b).
The results of mutagenicity assays of 4-hydroxy-2, 5,
6-trichloroisophthalonitrile are summarized in Table 2.
COMMENTS
Excretion and elimination studies in rats demonstrated that
chlorothalonil is preferentially excreted in the faeces, with minor
elimination of the parent compound or its metabolites in the urine.
4-hydoxy-2, 3,5-trichloroisophthalonitrile was identified as a major
metabolite. Faecal elimination was complete (95 percent) within 24
hours on low doses, but was noticeably delayed (96 hours) at high
doses. The total amount of radioactivity in the blood following the
oral administration of labelled chlorothalonil was less than one
percent of the administered dose, with most of the activity being
found in the plasma. This information failed to clarify the general
metabolism of chlorothalonil, particularly regarding the 4-hydroxy
metabolite.
Table 2. Results of Mutagenicity Assays of 4-hydroxy-2,5,6-trichloroisophthalonitrile
Test Organism Test Substance Results Reference
(% purity)
GENE MUTATION STUDIES
Bacteria
Salmonella 99.0 No mutagenic activity was reported Banzer 1977d
typhimurium in TA98, TA100, TA1535,TA1536 and
TA1538 with or without metabolic
activation.
S. typhimurium 99.0 Negative response in G46, TA1530, Legator 1974b
C270, TA1531, C3076, TA1700, D3056
and TA1724 according to host
mediated assay.
Cultured Mammalian
Cells
Chinese hamster Cells 99.0 No mutagenic activity was reported Banzer 1977e
(V79) and Mouse Fibroblast in TA98,TA100, TA1535, TA1537 and
Cells (Balb/3T3) TA1538 without metabolic activation.
CHROMOSOME EFFECTS
Micronucleus - In Vivo 99.0 Negative response was reported in Legator 1974b
Mouse the polychromatic erythrocytes of
treated mice.
Table 2 (con't)
Test Organism Test Substance Results Reference
(% purity)
Dominant Lethal - Rodent
Rat 99.0 No induced dominant lethals were Hastings et
reported in treated rats. al 1975
Mouse 99.0 No induced dominant lethals were Legator 1974b,
reported in treated mice. 1975
DNA DAMAGE AND REPAIR -
Bacteria
S. typhimurium 99.0 Negative response. No marked Banzer 1977f
difference in the inhibition
zones of the strains TA1538 and
TA1978 were reported.
CELL TRANSFORMATION -
IN VITRO
Fischer Rat Embryo Cell 99.0 Negative responses were reported Price 1978b
Lines (P1706 and H4536) in both treated cell lines.
Short-term dietary studies in rats and mice exposed to
chlorothalonil demonstrated increased incidences of hyperplasia and
hyperkeratosis in the non-glandular portion of the stomach and
hyperplasia of the epithelial cells of the proximal convoluted tubules
in the kidney. Kidney and liver weights were also increased, but there
was no microscopic evidence of morphological change. The no-observed-
effect level (NOEL) was 3 mg/kg b.w. in both rats and mice.
A short-term dietary study in rats using the 4-hydroxy metabolite
at levels of 10-750 mg/kg presented evidence of anaemia and compound-
related liver effects at dietary levels greater than 20 mg/kg b.w.
A one-generation study in rats using the 4-hydroxy metabolite
demonstrated adverse reproductive effects at doses greater than
30 ppm. These included poor pup viability and reduced mean live pup
weights.
Teratology studies with rabbits and rats gavaged with
chlorothalonil produced no teratogenic responses; however, maternal
toxicity was evident at >5 mg/kg and 400 mg/kg in rabbits and rats,
respectively. Teratogenicity studies by gavage in the rabbit at
maternally toxic doses of 5 mg 4-hydroxy metabolite/kg failed to
induce terata. A wide range of mutagenicity studies, using both
chlorothalonil and the 4-hydroxy metabolite, did not indicate
mutagenic activity for either compound.
A carcinogenicity study in mice exposed to chlorothalonil at
dietary levels of 150-3 000 ppm produced glandular epithelial tumours
in treated groups, which were compound-related but not dose-related.
The neoplastic changes in the kidney, including adenomas and
carcinomas of the renal cortical tubules were increased in males in
all treated groups, but not in females. There was no positive trend
for these kidney adenomas and carcinomas and, therefore, the evidence
for the tumourigenicity of chlorothalonil in the kidney remains
elusive. A NOEL was not determined.
Carcinogenicity studies in rats and mice using the 4-hydroxy
metabolite did not demonstrate oncogenic potential at doses up to and
including 1 500 ppm in the diet (22.5 mg/kg b.w.) and 20 mg/kg b.w. in
mice and rats, respectively. There was, however, clear evidence of
anaemia at doses greater than 3 mg/kg b.w. in rats. A NOEL for non-
oncogenic effects was not demonstrated in the mouse study.
The Meeting expressed concern about the lack of adequate
metabolism data for chlorothalonil and the 4-hydroxy metabolite and
the need to clarify the incidence of kidney adenomas and carcinomas in
rodents. The Meeting was informed that a long-term oncogenicity
feeding study with chlorothalonil in the rat is currently being
conducted, but the results will not be available until 1985. The
Meeting, therefore, agreed that the temporary ADI should remain at the
present level until further data are made available to clarify
remaining questions on metabolism and carcinogenicity of
chlorothalonil.
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
0-0.005 mg/kg b.w.
FURTHER WORK OR INFORMATION
Required (by 1985)
1. Further work to elucidate the metabolism of chlorothalonil and
the 4-hydroxy metabolite.
2. Submission of the on-going long-term oncogenicity feeding study
in the rat.
Desirable
Information on data which may clarify the type of anemia which
has been demonstrated in mammals exposed to the 4-hydroxy
metabolite.
REFERENCES - TOXICOLOGY
Ames, B.N., McCann, J. & Yamasake, E. Methods for detecting
1975 carcinogens and mutagens with the Salmonella mammalian-
microsome mutagenicity test. Mutat. Res., 31: 347-364.
Banzer, C.B. Activity of DTX-77-0035 in the Salmonella/microsomal
1977a assay for bacterial mutagenicity. Report from
Microbiological Associates submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Banzer, C.B. Activity of DTX-77-0034 in an in vitro mammalian cell
1977b point mutation assay. Report from Microbiological Associates
submitted to WHO by Diamond Shamrock Corp. (Unpublished)
Banzer, C.B. Activity of DTX-77-0033 in a test for differential
1977c inhibition of repair deficient and repair competent strains
of Salmonella typhimurium: repair test. Report from
Microbiological Associates submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Banzer, C.B. Activity of DTX-0038 in the Salmonella/microsomal assay
1977d for bacterial mutagenicity. Report from Microbiological
Associates submitted to WHO by Diamond Shamrock Corp.
(Unpublished)
Banzer, C.B. Activity of DTX-77-0040 in an in vitro mammalian cell
1977e point mututation assay. Report from Microbiological
Associates submitted to WHO by Diamond Shamrock Corp.
(Unpublished)
Banzer, C.B. Activity of DTX-77-0039 in a test for differential
1977f inhibition of repair deficient and repair competent strains
of Salmonella typhimurium: repair test. Report from
Microbiological Associates submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Ford, W.H. A one-generation reproduction study in rats with DS-3701.
1982 Report submitted to WHO by Diamond Shamrock Corp.
(Unpublished)
Hastings, T.F, & Clifford, D. 8-week dominant lethal study of DAC-3701
1975 in rats. Report from Bio/tox Research Lab. Inc. submitted to
WHO by Diamond Shamrock Corp. (Unpublished)
Hozan, G.K. & Auletta, C.S. A chronic dietary study in mice with T-114
1981 (DS-3701). Report from Bio/dynamics, Inc. submitted to WHO
by Diamond Shamrock Corp. (unpublished)
Ignatoski et al. Dose-response determination of the excretion of
1981 radioactivity in rat bile following intraduodenal
administration of 14C-chlorothalonil (14C-DS-2787). Report
submitted to WHO by Diamond Shamrock Corp. (Unpublished)
Ignatoski et al. Balance study of the distribution of radioactivity
1982 following oral administration of 14C-chlorothalonil
(14C-DS-2787) to rats. Report submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Ignatoski et al. Distribution of radioactivity following oral
1983a administration of 14C-chlorothalonil to rats: extraction
and analysis of 14C-materials in excreta. Report submitted
to WHO by Diamond Shamrock Corp. (Unpublished)
Ignatoski et al. Balance study of the distribution of radioactivity
1983b following oral administration of 14C-chlorothalonil to male
mice. Report submitted to WHO by Diamond Shamrock Corp.
(Unpublished)
Ignatoski et al. Recirculation of radioactivity in rat bile following
1983c intraduodenal administration of bile containing
14C-chlorothalonil label. Report submitted to WHO by
Diamond Shamrock Corp. (Unpublished)
Ignatoski et al. Dose-response determination of the excretion of
1983d radioactivity in rat bile following intraduodenal
administration of 14C-chlorothalonil. Report submitted to
WHO by Diamond Shamrock Corp. (Unpublished)
Ignatoski et al. Method development for the determination of 14C-DS-
1983e 2787 in blood. Report submitted to WHO by Diamond Shamrock
Corp. (Unpublished)
Ignatoski et al. Levels of radioactivity in blood following oral
1983f administration of 14C-chlorothalonil to male rats. Report
submitted to WHO by Diamond Shamrock Corp. (Unpublished)
Legator, M.S. Mutagenic testing with DAC 2787. Report submitted to
1974a WHO by Diamond Shamrock Corp. (Unpublished)
Legator, M.S. Mutagenic testing with DAC 3701. Report submitted to WHO
1974b from Diamond Shamrock Corp. (Unpublished)
Legator, M.S. Dominant lethal evaluation of DAC 3701 at 1.0 and 3.0
1975 mg/kg for five days. Report submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
McGee, D.H. et al. A two-year toxicity and tumourigenicity study of
1983 T-114 in rats. Report from International Research and
Development Corp. submitted to WHO by Diamond Shamrock Corp.
(Unpublished)
Murchison, T.E. et al. A short-term dietary study in rats with
1979 T-114-2 (DS-3701). Report from Dawson Research Corp.
submitted to WHO by Diamond Shamrock Corp. (Unpublished)
Price, P. The activity of compound DTX-77-0037 in the Fischer rat
1978a embryo transformation assay system. Report from
Microbiological Associates submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Price, P. The activity of compound DTX-77-0041 in the Fischer rat
1978b embryo transformation assay system. Report from
Microbiological Associates submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Rodwell, D.E. et al. A teratology study in rats with T-117-11. Report
1983 from Wil Research Labs. Inc. submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Shirasu, Y. & Teramoto, S. Teratogenicity study of Daconil in rabbits.
1975 Report from Institute of Environmental Toxicology submitted
to WHO by Diamond Shamrock Corp. (Unpublished)
Shirasu, Y. et al. Mutagenicity testing on Daconil in microbial
1977 systems. Report from the Institute of Environmental
Toxicology submitted to WHO by Diamond Shamrock Corp.
(Unpublished)
Shults, K. A 90-day feeding study in mice with technical
1983 chlorothalonil. Report from SDS Biotech Corp. submitted to
WHO by Diamond Shamrock Corp. (Unpublished)
Siou, et al. The chromosomal aberration test in the rat, mouse and
1981 hamster using chlorothalonil. Report from C.E.R.T.I.
submitted to WHO by Diamond Shamrock Corp. (Unpublished)
Siou, G. et al. The micronucleus test in the rat, mouse and hamster
1981b using chlorothalonil. Report from C.E.R.T.I. submitted to
WHO by Diamond Shamrock Corp. (Unpublished)
Tierney, W.J. A chronic dietary study in mice with technical
1983 chlorothalonil. Report from Bio/dynamics, Inc. submitted to
WHO by Diamond Shamrock Corp. (Unpublished)
Wazeter, F.X. & Goldenthal, E.I. Teratology study in rabbits. Report
1976 from International Research and Development Corp. submitted
to WHO by Diamond Shamrock Corp. (Unpublished)
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. et al. A subchronic toxicity study of technical
1983a chlorothalonil in rats. Report submitted to WHO by Diamond
Shamrock Corp. (Unpublished)
Wilson, N.H. et al. Progress report for the electron microscopic
1983b evaluation of renal tissue from a subchronic toxicity study
of technical chlorothalonil in rats. Report from Huntingdon
Research Centre submitted to WHO by Diamond Shamrock Corp.
(Unpublished)
RESIDUES
USE PATTERN
Use patterns have been reviewed previously. Additional summary
information indicated that uses on grapes are registered or
recommended in Argentina, Australia (confirmed at the 13th Session of
the CCPR), Austria, Brazil, Greece, Italy, Spain, The Philippines, Sri
Lanka and Yugoslavia. Studies on the potential for chlorothalonil use
on grapes are or have been in progress in Canada, Chile, Colombia,
South Africa and the United States.
The most common good agricultural practice is said to be three to
six applications of either a 75 percent WP or a 500 g a.i./1 flowable
(water dispersible) formulation at 1.5-2.0 kg a.i./ha in a spray
volume up to 4 000 l/ha. The most common last-treatment-to-harvest
interval is said to be 7 to 14 days, but occasionally it is 21 to
28 days.
Diseases for which chlorothalonil is used are Plasmopara (downy
mildew), Uncinula necator (powdery mildew) and Botrytis cinerea
(grey mould). Good agricultural practice information from individual
countries was not provided except for Spain, in which the following
formulations and doses are recommended for fruit tree seeds, potatoes,
vines, strawberries and winter cereals:
Type of formulation Application rate
LC 75% 0.15 - 0.2%
LC 50% 0.25 - 0.3%
P 5% 20 kg/ha
RESIDUES RESULTING FROM SUPERVISED TRIALS
Residue trials have been conducted in The Netherlands on onions
and strawberries (Netherlands 1983) and on grapes in Australia,
Canada, The Federal Republic of Germany and South Africa (Tables 1-5;
Diamond Shamrock 1981).
Grapes
The current temporary maximum residue limit (MRL) of 5 mg/kg is
based on a 46-day last-treatment-to-harvest interval and on residue
trials in Canada, where the Meeting is informed that there are no
recognized uses reflecting good agricultural practices (GAP). The only
country in which the use of chlorothalonil on grapes is regarded as
good agricultural practice and for which residue trials data are
Table 1. Residues of chlorothalonil in grapes treated with
Chlorothalonil-Australia 1973-741
Application
Location Rate Number Pre-harvest Residue
(a,i.) interval (days) (mg/kg)
Hunter Valley 113 g/100 l2 7 -1 last spray 3.9
0-post-spray 6.1, 7.1
10 5.6 (8.6)4
225g/100 l3 7 -1 last spray 6.8
0-post-spray 10.7
10 8.7 (13.4)4
South Australia 0.5 kg/378 l2 6 1 1.4
7 0.6
18 1.6 (2.9)4
26 0.6, 0.3
1 kg/378 13 6 1 2.3
7 3.1
18 2.7 (4.9)4
26 0.8
1 BRAVO 75W wettable powder containing 75 percent active
ingredient was used for treatment. Information on spray volume
was not available.
2 Recommended rate.
3 2X recommended rate.
4 Figures in parentheses are corrected for recovery (64 percent
Hunter Valley; 55 percent South Australia).
Table 2. Residues of Chlorothalonil and D-3701 (4-hydroxy-2, 5,6-trichloroisophthalonitrile)
in Grapes Treated With Chlorothalonil - Canada 1979
Application1 Residues (mg/kg)2
Pre-Harvest
Location Rate Number Interval Chlorothalonil DS-3701
(kg a.i./ha) (days)
Vineland, Ont. 1.65 5 40 0.28 0.02
0.26 0.01
<0.01, <0.01 0.01, 01
- - - ND 0.02
St. Catherines, 1.65 4 30 1.94 0.13
Ont. 1.62 0.03
- - - <0.01 0.01
Jordan, Ont. 1.65 3 30 3.80 0.04
4.07 0.06
- - - ND(<0.005) 0.01
1.65 2 30 0.54 0.01
1.65 1 30 0.63 0.01
1.04, 1.21 0.03, 0.1
- - - ND 0.01
1 BRAVO 75W wettable powder containing 75 percent active ingredient was used for treatment.
Information spray volume was not available.
2 Uncorrected for 76 and 80 percent recoveries of chlorothalonil and DS-3701, respectively.
Table 3. Residues of Chlorothalonil in Grapes Treated With Chlorothalonil - Federal Republic of Germany
Application
Pre- Residues (mg/kg)
Spray Harvest
Location Year Rate Volume Number Interval Treated Untreated
(kg a.i.ha) (l/ha) (days)
Bad Münster 1972 4.5 3 000 6 1 12.0 0.5
(Trial 129) (0.2% DaconilR 15 6.2
2787, 75% WP) 22 6.0
29 7.0
71 3 4
Welhausen 1972 4.5 3 000 6 0 13.0 0.05
(Trial 128) (0.2% DaconilR 14 8.0
2787, 75% WP) 21 6.8
28 8.0
70 5.8 0.06
Rhodt 1972 1.5-3.75 1 000- 7 1 26.2 0.05
(Trial 47) (0.2% DaconilR 2 5001 14 12.4 0.03
2787) 21 5.0 0.02
28 5.8
68 2.0
Volkach/Main 1973 1.46/spray 1 000 8 2 2.71 0.03
51 0.38 0.04
Grossbottwar 1974 2.19 × 6; 1 000 8 0 26.5 0.06
2.92 × 2 21 17.13 0.22
(6×0.3% soln 28 8.0 0.06
2×0.4% soln) 35 7.0 0.06
42 4.25 0.03
Table 3. (con't)
Application
Pre- Residues (mg/kg)
Spray Harvest
Location Year Rate Volume Number Interval Treated Untreated
(kg a.i.ha) (l/ha) (days)
2.92/spray 1 000 8 0 28.0
(0.4% soln) 21 13.5
28 8.0
35 4.75
42 6.7
Mettenheim 1974 1.75/spray 800 6 0 1.45 0.05
(0.3% soln) 21 0.23 0.01
28 0.64 0.01
35 0.16 <0.01
42 0.13 <0.01
2.33/spray 800 6 0 1.9
(0.4% soln) 21 0.45
28 0.82
35 0.59
42 0.39
Deidesheim 1974 1.75/spray 800 6 0 3.7 <0.01
(0.3% soln) 21 1.07 0.02
28 0.55 <0.01
35 0.4 <0.01
42 0.23 <0.01
Table 3. (con't)
Application
Pre- Residues (mg/kg)
Spray Harvest
Location Year Rate Volume Number Interval Treated Untreated
(kg a.i.ha) (l/ha) (days)
2.33/spray 800 6 0 5.15
(0.4% soln) 21 2.15
28 1.07
35 0.72
42 0.21
Würzburg 1975 24.4 total 1/800 10 0 3.83 0.01
(8 × 0.15% 1/1 000 21 0.27 0.02
2 × 0.2%) 1/1 300 28 0.62 <0.01
1/2 000 35 0.63 0.01
2/1 600 42 0.59 0.01
4/2 500
Ebernburg 1975 28 total 2/1 500 10 0 1.87 <0.01
(8×0.15% soln) 8/2 500 21 0.49 <0.01
2×0.18% sola) 27 0.76 0.26
35 0.52 0.01
42 0.76 <0.01
Deidesheim 1975 31.9 total 2 500 10 0 2.38 <0.01
(6×0.15% soln 21 1.14 0.04
4×0.2% soln) 35 1.85 0.04
43 2.0 0.09
1 Spray volume varied from 1 000 to 2 500 l/ha, apparently increasing during the season from early to mature growth stages.
Table 4. Residues of Chlorothalonil in Wine From Grapes Treated With Chlorothalonil Federal Republic of Germany
Application
Pre- Residues (mg/kg)
Spray Harvest
Location Year Rate Volume Number Interval Treated Untreated
(kg a.i./ha) (1/ha) (days)
Hattenheim 1973 2.92 1 000 4 98 <0.01 <0.01
(0.4% DaconilR <0.01 <0.01
75% WP)
Hattenheim 1972 NA1 NA NA NA 0.003 0.00
0.004 0.003
1 NA = Information not available.
Table 5. Residues of Chlorothalonil and DS-3701
(4-hydroxy-2,5,6-trichloroisophthalonitrile) in Grapes
Treated With Chlorothalonil - South Africa 1979-80
Application1
Pre- Residues (mg/kg)2
Rate Number Harvest
(kg a.i./ha) Interval Chlorothalonil DS-3701
(days)
1.5/spray 8 1 18.4; 20.9 0.14; 0.14
2 12.4; 13.4 0.10; 0.11
4 3.6; 3.5 <0.05; <0.05
16 2.7; 2.5 0.06; 0.06
32 2.6; 2.3 0.07; 0.07
1 BRAVO 500, a water dispersible flowable formulation containing
500 g chlorothalonil/1, was used for treatment. Information on
spray volume was not available.
2 Limit of determination for both chlorothalonil and DS-3701 was
0.05 mg/kg. Uncorrected for recoveries of 89 percent and 94
percent respectively.
available is Australia (see Table 1), although specific information on
use patterns was not available. Data from residue trials were however
provided from The Federal Republic of Germany and South Africa
together with additional data from Canada. The trials conducted in
Australia therefore, warrant special consideration. Data were
available from recommended and twice the recommended application
rates, although the approved Australian pre-harvest interval (PHI) was
not known. At the recommended rate and 7-18 days, the most common PHI,
maximum residues were 5.6 mg/kg (or 8.6 mg/kg when corrected for 65
percent recoveries). These data represent chlorothalonil only, whereas
previously recommended limits also include the 4-hydroxy metabolite,
which can contribute approximately 5 percent of the total residue in
grapes.
Previously reviewed Canadian data at a 46-day PHI support the
current 5 mg/kg limit based on that PHI. So do Canadian data at 30-40
day PHI submitted to the Meeting (Table 2), although neither PHI is
commonly used.
In Table 3, data from The Federal Republic of Germany include
residues of 5-12-4 mg/kg at recommended pre-harvest intervals which
significantly exceed the current 5 mg/kg limit, but result from
exaggerated (twice the common recommended) application rates. Residues
at commonly recommended application rates and pre-harvest intervals
range from 4 to 17 mg/kg. Although these residues result from eight
applications as opposed to the six recommended, the data appear to be
atypically high when compared to other West German trials with six to
ten applications in which residues do not exceed the current limit.
Table 4 shows residues of 0.01 mg/kg in wine from grapes harvested 98
days after treatment. South African data (Table 5) are consistent with
the current limit.
Onions
Additional residue data provided to the Meeting (Netherlands
1983) reflect good agricultural practices of the Netherlands. In eight
trials conducted in 1981 represented by over 60 samples, residues in
mature silver onions and set onions ranged from <0.05 to 0.34 mg/kg
(0.57 mg/kg unwashed) at the 7-day last-application-to-harvest
interval that is the basis of the current 5 mg/kg limit, and were
<0.05 mg/kg (0.28 ppm unwashed) at the 28-day interval, which is
regarded as good agricultural practice in The Netherlands.
Strawberries
In a 1981 trial (Netherlands 1983) understood to represent good
agricultural practice in The Netherlands. residues in four samples of
mature berries ranged from 1.5 to 3 mg/kg (2.1 mg/kg mean) 14 days
after the last of five applications of a 75 percent WP formulation at
1.5 kg a.i./ha.
Residues of 4-hydroxy-2,5,6-trichloroisophthalonitrile (DAC 3701)
At the request of the 14th session of the Codex Committee on
Pesticide Residues (CCPR), the Meeting re-examined the definition of
the residue for chlorothalonil. Past evaluations of chlorothalonil by
the Meeting as well as new data were considered in order to estimate
the proportion of the major metabolite DAC 3701, in the total residue.
The results of supervised trials carried out at various locations in
the United States between 1965 and 1973 are summarized in FAO/WHO
1975, p.126-128. The concentration of DAC 3701 was lower than the
0.1 mg/kg limit of determination in all crops for which it was
analysed except lima beans (in pod) where it accounted for 10 percent
of the total residue, cantaloupe and grapes (5 percent) peaches and
onions (2 percent), celery (1 percent) and peanuts (0.1 percent). No
DAC 3701 was detected in crops grown in glasshouses. Similarly low
residues were reported in peaches and no residues of DAC 3701 in whole
banana or banana pulp, regardless of the rate of application (FAO/WHO
1978). DAC 3701 residues of less than 2 percent and 5 percent,
respectively, in onions and grapes were reported in later supervised
trials (FAO/WHO 1980). Data submitted to the Meeting more or less
confirm the estimate for grapes and indicate that DAC 3701 does not
exceed 5 percent of the parent compound residue in oranges.
Lima beans
In conducting the above review, the Meeting observed that the
current 0.5 mg/kg limit for lima beans was based on data for beans
without pod. The temporary MRL should be amended accordingly.
FATE OF RESIDUES
In Storage and Processing
The 1981 Meeting listed as desirable an explanation of apparent
inconsistencies between studies carried out in 1979 and 1980 on the
nature of residues resulting from the cooking of chlorothalonil-
treated foods. The main point of concern was the statement in the 1979
study that 14C-chlorothalonil was largely lost by volatilization
during cooking under open conditions while of the 14C in the organic
phase approximately 57 percent was characterized as the 4-hydroxy
metabolite whereas 43 percent was unidentified and was near the thin-
layer chromatography (TLC) origin. This was confirmed by a TLC
autoradiogram in the 1979 study.
In apparent conflict was the statement in the 1980 study that
chlorothalonil was the major residue in organic extracts of
chlorothalonil-fortified tomatoes after cooking with and without a pot
cover and that there were negligible quantities of the metabolite.
Again this was confirmed by an autoradiogram. It was further stated
that the effect of cooking chlorothalonil with green beans was similar
and this was again confirmed by a TLC autoradiogram.
In response to the questions raised by the 1981 Meeting, the
manufacturer submitted a redrafted report to present the data from the
1979 and 1980 studies in a more consistent manner (SDS 1983a). No
additional studies were provided. The new report confirms previous
conclusions that 85-98 percent of added chlorothalonil is lost through
cooking under open conditions. It emphasizes that the residue of the
hydroxy metabolite formed is only 2.4 percent of the chlorothalonil
originally present and that the percentages of degradation products
identified after cooking refer to the composition of the small residue
remaining.
EVIDENCE OF RESIDUES IN FOOD IN COMMERCE OR AT CONSUMPTION
Data were provided from Sweden on chlorothalonil residues found
in domestic and imported commodities for the period 1981 - 30 April
1983 (Sweden 1983). Of 1 085 samples analysed, residues in 1 070 were
below 0.21 mg/kg. Maximum residues with the number of samples having
residues over 0.21 mg/kg/total analysed in brackets were: cauliflower
0.41 mg/kg (1/165), celery 1.9 mg/kg (7/50), cucumbers 0.23 mg/kg
(1/580), gherkins 0.66 mg/kg (2/49, melons 0.71 mg/kg (2/98) and
strawberries 2.9 mg/kg (2/143). The highest levels found are well
below current Codex temporary MRLs except gherkins and strawberries
for which there are no Codex limits.
METHODS OF RESIDUE ANALYSIS
To compare the relative extraction efficiencies of tumble
extraction and maceration techniques, Bravo 500R was field-applied
to oranges at 12.9 l/ha and sampled at 0, 14 and 28 days after the
last of three applications (SDS 1983b). The formulation contained
40.6 percent chlorothalonil, 0.33 percent pentachlorobenzonitrile
(PCBN) and 0.21 percent hexachlorobenzene (HCB).
For surface extraction, residues were extracted with methylene
chloride by rotating the sample at 22 rpm for 2 h. One portion was
analysed for chlorothalonil by electron capture gas chromatography
after elution from a florisil column with a 50 percent methylene
chloride:48.5 percent hexane: 1.5 percent acetonitrile eluant after a
pre-wash with 20 percent methylene chloride:80 percent hexane. A
separate portion was analysed for HCB and PCBN after separation
from a florisil column by eluting successively with 20 percent
methylene chloride:80 percent hexane and 0.05 percent acetonitrile
in 1:1 hexane:methylene chloride.
For maceration extraction, samples were blended with 385 ml
acetone and 15 ml 1:1 (w/w) H2SO4:water, filtered, concentrated and
after pH adjustment in aqueous solvent, partitioned with hexane.
Clean-up and analysis for chlorothalonil, HCB and PCBN was as
described above. The DS-3701 metabolite was extracted from the aqueous
phase with 1:1 petroleum ether:diethyl ether after pH adjustment and
methylated for gas chromatographic analysis.
Analyses of samples extracted by both procedures were validated
by fortifying oranges in the extraction vessel with organic solutions
of chlorothalonil, HCB, PCBN and DS-3701 before analysis.
Fortification levels and mean recoveries are given in Table 6.
Determinations of field-incurred residues by surface extraction
and maceration techniques are summarized in Table 7.
Table 7 demonstrates that surface and maceration extraction
techniques give comparable results for chlorothalonil, HCB.
Table 6. Validation of Analytical Procedures for Determining Residues of Chlorothalonil and its Metabolites in Oranges.
Chlorothalonil HCB PCBN DS-3701
Added Mean Added Mean Added Mean Added Mean
mg/kg % mg/kg % mg/kg % mg/kg %
Recovery Recovery Recovery Recovery
Surface
extraction 0.5-2.1 103 0.01 88 0.01-0.02 95 - -
Maceration 0.5-2 85 0.01 77 0.01-0.02 82 0.03-0.05 69
Table 7. Comparison of Maceration and Surface Extraction Techniques for Measuring Field-lncurred Residues of Chlorothalonil
and Metabolites in Oranges.
Residue (mg/kg)1
Chlorothalonil D S-3701 HCB PCBN
Days after
Last Treatment Surface Macerated Macerated Surface Macerated Surface Macerated
0 11.2 10.1 0.04 0.006 0.004 0.1 0.07
(0.03) (0.05) (ND) (ND) (ND) (ND) (ND)
14 4.7 6.1 0.3 0.006 0.003 0.06 0.05
(0.03) (0.06) (ND) (ND) (ND) (ND) (ND)
28 5.3 4.1 0.014 0.003 ND 0.04 0.04
(ND) (0.13) (ND) (ND) (ND) (ND) (ND)
1 Each value is the mean of three replicates, each of which consists of four samples. Numbers in parentheses are
the means of two untreated controls where ND = <0.01 mg/kg chlorothalonil and DS-3701, <0.003 mg/kg HCB and
(0.005 mg/kg PCBN.
NATIONAL MAXIMUM RESIDUE LIMITS REPORTED TO THE MEETING
Country Commodity MRL (mg/kg)
The Netherlands banana 0.2
tomato 1
Sweden fruit and vegetables 1
potatoes 0.1
banana (without peel) 0.05
APPRAISAL
Limited information on good agricultural practice in the use of
chlorothalonil on grapes was made available to the Meeting. Data on
residue trials from several countries were also provided but from only
one country in which uses on grapes are known to be approved or
registered. The limited data reflecting approved uses, and some of the
other data suggest that the 5 mg/kg limit may be too low. In the
absence of data from residue trials and information on registered or
approved uses from other countries, there is no firm basis on which to
change the current limit. However, it should remain temporary
irrespective of the ADI status until this information is provided.
Additional residue data for onions do not warrant a revision in
the current limit, and data provided for strawberries were not
adequate to estimate a maximum residue level. Monitoring data from
Sweden indicated that previous estimates for cauliflower, celery,
cucumbers and melons are adequate.
The meeting reviewed a revised report on the effects of cooking
on chlorothalonil residues in food. In one of two previously reviewed
reports, the major residue remaining in beans after cooking under open
conditions was said to be unchanged chlorothalonil with little
degradation. In the other report, the major residue under similar
conditions was said to be the 4-hydroxy metabolite and unidentified
material (57 percent and 43 percent of the remaining residue,
respectively) with no chlorothalonil remaining. The redrafted report,
based on the original two reports, still does not fully clarify the
apparent inconsistencies noted by the 1981 Meeting, although the
results are more clearly presented. Whether the residue is
chlorothalonil, its hydroxy metabolite, a combination of those, or
57:43 hydroxy metabolite: unknown, the total residues remaining after
cooking chlorothalonil-fortified green beans under open conditions in
both studies are <7 percent of fortification levels. The 57:43
percent hydroxy metabolite: unknown is shown in the 1983 submission to
represent 2.4 and 1.8 percent, respectively of the originally added
chlorothalonil. For this reason, the Meeting concluded that the matter
had been adequately considered.
The Meeting also reviewed a study comparing surface and
maceration extraction techniques for determining field-incurred
residues of chlorothalonil, HCB and PCBN in oranges and data on levels
of the hydroxy metabolite (DS-3701) by the maceration technique. The
study showed the two techniques to give comparable results for field-
incurred residues. A similar conclusion had been reached for bananas
by the 1979 Meeting. Both procedures were validated by fortifying just
prior to extraction, but the study does not provide information on the
efficiency of extraction of field-incurred residues by either
technique. However, since the maceration extraction conditions are
similar to those used in earlier evaluations (FAO/WHO 1975), the
efficiency for the surface extraction technique is as high as that
considered adequate previously. Residues of chlorothalonil, HCB, PCBN
and DS-3701 are shown to decrease with time from application.
The Meeting reviewed the definition of the residue for
chlorothalonil. The results of supervised trials conducted in
different parts of the world between 1965 and 1983, which had been
reviewed by the Meeting, showed that levels of the major metabolite,
DAC 3701, were lower than the 0.1 mg/kg limit of determination in most
of the crops for which it was analysed. DAC 3701 accounted for
approximately 10 percent of the total residue in lima beans (in pod);
5 percent in cantaloupes, oranges and grapes; 2 percent in peaches and
onions; 1 percent in celery and 0.1 percent in peanuts.
The Meeting noted that the current 0.5 mg/kg limit for lime beans
is based on data for beans without pods and concluded that this should
be reflected in the commodity description.
RECOMMENDATIONS
Additional data reviewed by the Meeting do not warrant a revision
of current limits or support the estimation of new limits. The Meeting
concludes that the current 5 mg/kg temporary MRL for grapes should
remain temporary, irrespective of the status of the ADI, pending the
receipt of additional information on good agricultural practice and
data on residue trials, preferably from countries providing
information on good agricultural practices.
The Meeting recommends that the definition of the residue be
revised to include chlorothalonil only and concludes that no changes
in the numerical values of the maximum residue limits are necessary as
a result. The current 0.5 mg/kg limit for lima beans should be
expressed as applying to lima beans (without pod) to reflect the data
on which it was based.
FURTHER WORK OR INFORMATION
Required (by 1985)
1. Additional and more specific information on good agricultural
practice in the use of chlorothalonil on grapes, as well as
residue data from field trials in countries where uses on grapes
are known to be good agricultural practice.
2. Information, if it exists, on good agricultural practice for
those countries in which residue trials for data provided to the
1983 Meeting were conducted.
Desirable
1. Analyses of chlorothalonil-treated animal feed items (for
example, bean and peanut vines), processed and unprocessed, for
residues of PCBN (from the 1981 Meeting).
2. Information on possible PCBN residues in tissues and milk of
dairy cattle fed a diet containing chlorothalonil (from the 1981
Meeting).
REFERENCES-RESIDUES
Diamond Shamrock. (2, 4, 5, 6-tetrachloroisophthalonitrile),
1981 Compilation of Residue Data and Registration/Recommendation
Information on Use of Chlorothalonil on Grapes. Submitted to
FAO by Diamond Shamrock Corporation.
Netherlands. Information submitted to FAO by the government of The
1983 Netherlands, September 1983, MVD, Ref.: CL1983/22-Fr.
SDS. The effects of cooking 2,4,5,6-tetrachloroisophthalonitrile
1983a (chlorothalonil, DS-2787) with vegetables. Document No.
372-3EF-83-0004-001 submitted to FAO by SDS Biotech
Corporation.
SDS. Validation of analytical procedures for determining residues of
1983b 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil,
DS-2787), 4-hydroxy 2,5,6-trichloroisophthalonitrile
(DS-3701), hexachlorobenzene (HCB) and
pentachlorobenzonitrile (PCBN) on oranges. Document
No. 506-3CR-81-0182-001 submitted to FAO by SDS Biotech
Corporation.
Sweden. Chlorothalonil residues in imported and domestic commodities -
1983 1981 to 1983. Data submitted to FAO from The Government of
Sweden.