Chlorothalonil

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Sep 1988 to 22 Dec 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorothalonil

EC Number:

217-588-1

EC Name:

Chlorothalonil

Cas Number:

1897-45-6

Molecular formula:

C8Cl4N2

IUPAC Name:

tetrachlorobenzene-1,3-dicarbonitrile

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD-VAF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) x 27 days old upon receipt and 40 days old at the initiation of the test material administration;
- Housing: The rats were housed two per suspended stainless steel cage upon receipt and for seven days thereafter in order to aid in their acclimation to the automatic watering. After randomization and for the remainder of the study, the animals were housed individually except during mating and lactation. A solid stainless steel bottom vas placed in the cages of the females from Day 18 of gestation through lactation. Ground corncob bedding vas provided during the period when the cage bottom vas in place.
- Diet: rodent chow ad libitum during the acclimation period. This rodent chow vas also used in the preparation of the diets for the study. Fresh diets were provided as often as necessary to ensure an adequate food supply, but routinely on a weekly basis. Feeders designed to minimize soiling and scattering were used and any incidence of excessive spillage vas noted. The feeders were sanitized weekly.
- Water: fresh potable water was available ad libitum by means of an automatic water system during both the acclimation and study periods

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 40 - 60
- Air changes: 11 or more fresh air changes per hour
- Photoperiod: alternating 12 hour light/dark cycle

IN-LIFE DATES: From: 20 Sep 1988 To: 22 Dec 1989

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
For each dietary level, appropriate amounts of the test material were mixed fresh weekly with a small amount of basal diet in a mortar and pestle in order to make a premix. The premix was then blended with the remainder of the basal diet for a 10 minute period in a Marion ribbon blender. The prepared diets were stored at room temperature in the dark. Diets were prepared at a constant concentration (ppm)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: two weeks
- Proof of pregnancy: a copulation plug in the vagina and/or the presence of sperm in a vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: A solid stainless steel bottom vas placed in the cages of the females from Day 18 of gestation through lactation. Ground corncob bedding vas provided during the period when the cage bottom was in place.
- Females that did not exhibit evidence of mating were cohabited with the same male each night until evidence of mating was observed or until the two week mating period vas completed. In some instances, there were more females than males in a group due to the death of one or more males. When this occurred, selected males that had been sucessfully mated the first night were placed with the females that lacked mates on the second day of mating.
- All animals were mated during the second mating period regardless of whether they had delivered a litter following the first mating period. Different pairs of rats were mated for the second mating periods. During mating for the F1 generation animals care was taken to avoid brother-sister matings.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity
Prior to the initiation of the in life phase of the study, diets at concentrations of 500 ppm and 3000 ppm were prepared using the same methods of mixing as would be used subsequently in the study. Five samples at each concentration were assayed to evaluate homogeneity of mixing.

Stability
Prior to the initiation of the in life phase of the study, samples of diet at 500 ppm and 3000 ppm, which had been prepared for the homogeneity study were analyzed after sitting for 7 or 14 days under study room conditions.

Weekly Diet Samples
The concentration of the test in diets was measured weekly during the first 10 weeks of the study and biweekly for the remainder of the study.

Duration of treatment / exposure:

15 months

Frequency of treatment:

F0 parental animals were treated continuously throughout the 10 week growth period and also throughout the mating, gestation, lactation and rest phases of the study. All offspring and the F1 parents were exposed to the test material throughout their time on study.

Details on study schedule:

- F1 parental animals not mated until 14 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

P0 Group 2: 36.1 and 40.9 mg/kg bw for males and females, respectively

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

P0 Group 3: 110 and 124 mg/kg bw for males and females respectively

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

P0 Group 4: 225 and 255 mg/kg bw for males and females, respectively

No. of animals per sex per dose:

35 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

The parental, animals from each generation were treated continuously during the growth period prior to mating and then throughout the mating, gestation,
lactation and resting phases of the study. The growth period was ten weeks for the F0 animals and fourteen weeks for the F1 animals. Each generation of parental animals was mated twice to produce the F1a, F1b, F2a and F2b litters. The offspring was exposed to the test diets throughout the lactation periods. Thirty-five F1b males and females per group were selected to become the F1 generation after weaning.

Selection of Offspring for the F1 Generation
On Day 21 of lactation the size of each F1b litter was adjusted by random selection. For Group 1, one half of the litters was adjusted to yield three males and two females when possible and the other half of the litters was adjusted to yield two males and three females when possible. For the other groups (2, 3 and 4), each litter was adjusted to yield two males and two females when possible. The extra animals were selected from Group 1 because the number of litters available for selection necessitated having one additional animal in each litter to ensure sufficient animals for selection of the F1 generation.
When the number of male or female pups per litter was less than necessary to yield the selected number of pups, a partial adjustment was made; for example, a litter of six males and one female was adjusted to yield two male and one female pups. No adjustments were made for litters of two or less male and female pups.
After the last F1b litter was weaned (Day 21 of lactation), thirty-five male and thirty-five female pups were randomly selected from each group to produce the F1 generation. When possible at least one male and one female was selected from each litter.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Throughout the period of compound administration the animals were checked early in the morning and in the afternoon for overt signs of toxicity, moribundity and mortality. Prior to the initiation of treatment the animals were checked once a day.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Complete physical examinations were carried out weekly for all male animals throughout the study and for all females throughout the growth period prior to mating and during the two week resting period.

BODY WEIGHT:
- Time schedule for examinations: Individual body weights were recorded weekly from one week prior to initiation of treatment for the F0 animals and from the day of selection for the F1 animals. For males body weights were recorded each week until termination except during the mating periods when no weights were taken. Body weights for females were recorded during the growth periods and during the two week rest periods.
Body weights of females which showed positive signs of mating were recorded on Days 0, 7, 14 and 20 of gestation. Following parturition, body weights were recorded on Days 0, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Individual food consumption was measured weekly prior to mating and during the two week rest period. Food consumption was not recorded for males or females during the mating, gestation and lactation periods. Food was provided ad libitum during those periods.
Mean compound consumption (mg/kg/day) was calculated at weekly intervals prior to mating and during the two week rest period using the body weight, food consumption data and nominal dietary concentrations.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
Date delivered, number, weight and sex of live and stillborn pups and any abnormalities observed in the offspring were recorded. Each litter was examined twice daily for survival and overt clinical signs of toxicity throughout the lactation period. Number and sex of live pups were recorded on Day 0, 1, 4 (pre- and post-cull), 7, 14 and 21 of lactation. Total litter weights for live pups were measured on Days 0, 4 (pre- and post-cull), 7 and 14 of lactation. Individual weights for live pups were measured on Day 21 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
All surviving F0 and F1 adults were killed after the F1b or F2b litters were weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY
- The following tissues were prepared for microscopic examination:
adrenal glands (2), bone (femur), bone marrow (sternum), brain, esophagus, eyes (2), gonads, testes with epididymides and seminal vesicles, ovaries with uterus and vagina, heart with aorta, intestines (duodenum, jejunum, ileum, cecum), colon, rectum, kidneys (2), liver (left and median lobe), lungs - fixed by inflation, pancreas, pituitary, prostate, salivary glands with cervical lymph nodes, skeletal muscle with peripheral nerve, skin with mammary tissue, spinal cord (cervical), spleen, stomach, thymus, thyroid and parathyroid glands, trachea, urinary bladder, all gross lesions

Postmortem examinations (offspring):

SACRIFICE
Those pups culled on Day 4 of lactation were killed on that day. All remaining offspring were killed on Day 21 of lactation, with the exception of the F1b offspring which were saved for selection to be F1 parents. Those F1b offspring not selected to be F1 parents were killed at the time of selection of F1 parents.

GROSS NECROPSY
All grossly abnormal offspring were necropsied and preserved in 10% neutral buffered formalin.

Statistics:

Contingency table analyses were conducted on gestation length, Day 4 pup viability indices and lactation indices instead of the proposed analyses. This course was chosen because for each set of data almost all of the observations were identical, so that contingency table analyses were more appropriate than those proposed. For the contingency table analysis the Hantel-Baenszel test for a trend in the proportions was conducted instead of the Armitage test for trend. The change was made because the computer programming for the Armitage test was not readily available and the analyses give similar results. Except for Bartlett's test and the test for lack-of-fit, the statistical analyses were reported at the 1% level in addition to the 5% level of significance.

Reproductive indices:

Male Indices:
Mating index = (Number of males producing females with positive signs of mating/Total number of males mated) x 100

Male fertility index = (Number of males producing pregnant females/Number of males producing females with positive signs of mating) x 100

Female Indices:
Mating Index = (Number of females showing positive signs of mating/Total number of females mated) x 100

Female Fertility Index = (Number of pregnant females/Number of females showing positive signs of mating) x 100

Offspring viability indices:

Sex Ratio Index = (Live born males/Total live born pups) x 100

Live Born Index = Number of pups live born/Total number of pups born) x 100

Stillborn Index = (Number of pups stillborn/Total number of pups born) x 100

Day 4 Pup Viability Index = (Number of pups alive Day 4 (pre-cull)/Total number of pups born alive) x100

Lactation Index = (Number of pups alive Day 21/Number of pups alive Day 4 (post-cull)) x 100

Litter Indices:

Live Born Index = (Number of litters with live pups Day 0/Total number of litters) x 100

Stillborn Index = (Number of litters with stillborn pups/Total number of litters) x 100

Day 4 Viability Index = (Number of litters with live pups Day 4 (pre-cull)/Number of litters with live pups Day 0) x 100

Lactation Index = (Number of litters with live pups Day 21/Number of litters with live pups day 4 (post-cull)) x 100

Viability Index = (Number of litters with live pups Day 21/Number of litters with live pups Day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only observation which could have been treatment related was an observation of loose stools in seven male rats from the high dose group. For six of the animals the observation of loose stools was noted only once and so was not considered a significant finding.There were no observation of clinical signs which were considered to be treatment related during gestation or lactation.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a clear treatment-related effect on body weight in both sexes.
For the high dose males the mean body weight was approximately 10% lower than the controls throughout the P0 study period. This represented 11 and 15% lower body weight gain by week 10 and 30 respectively. Both the body weight and the body weight gain showed a statistically significant difference for every week on study.
For the mid-dose male group, mean body weight gain was statistically significantly lower for Week 1 and for almost every week from Week 5 onwards. Mean body weights for the mid-dose group reached statistical significance after Week 18. At this time the difference from the control group was approximately 5%. Although the mean body weight and mean body weight gain of the low dose group were slightly lower than the control values throughout the study period, statistical significance vas not reached at any time for either parameter. The no observable effect level (NOEL) for body weight of the F0 males was considered to be 500 ppm.
For the females at the high dose level there were statistically significant differences in mean body weight and mean body weight gain throughout the growth and rest periods. The difference in body weight from the control group vas approximately 5% through to Week 10 and then increased to 7 or 8% during the rest period between the F1a and F1b littering. The body weight gain for the high dose was 13% lower than the controls by the end of the growth period. Although the mean body weight and mean body weight gains of the mid- and low dose groups for the F0 females were consistently lower than the control values, there were no statistically significant differences between groups. It can be concluded that 1500 ppm is the NOEL for weight effects on F0 females.

At Day 0 of gestation the mean body weight of the high dose group vas 7% lower than the control group and the mean body weight of the mid-dose group vas 5% lower than the control group. Both values were statistically significant when compared with the control group. The low dose group was comparable with the control group. During the gestation period the differences from the control group narrowed slightly so that the difference from control was reduced to 5% at the high dose and 3.5% at the mid-dose. On Day 20 of gestation only the high dose mean body weight was statistically lower than the control. The mean body weight of the low dose group was comparable with controls throughout gestation.

At Day 0 of lactation the mean body weight of the high dose group was 6.5% lower than the control group and the mean body weight of the mid-dose group was 5% lower than the control group. Both differences were statistically significant from control group. During lactation the differences between the groups disappeared and at Days 14 and 21 the mean body weights for all groups were comparable.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For males and females the mean absolute and relative food consumptions were statistically reduced for Week 1 for the high dose groups. There were no significant differences in absolute food consumption after Week 1 for the high dose males and females which were considered treatment related.
Since absolute food consumption was similar to controls but body weight was lower than the controls, food consumption relative to body weight was higher for most study weeks for the high dose male and female F0 animals. Absolute and relative food consumption for the mid- and low dose levels for both sexes shoved sporadic incidences of statistically significant differences between groups but no consistent pattern which could be considered treatment related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, treatment-related effects were observed in the stomachs of male and female rats at all dose levels. These alterations to the stomach were characterized microscopically as squamous epithelial hyperplasia and hyperkeratosis.
Treatment-related effects were observed microscopically in the kidneys from male rats at all dose levels and from females at the mid- and high dose level. The effects increased in incidence and severity in a dose-related fashion and the findings were increased in incidence and severity in the males compared to the females.
Significant incidences of tubular epithelial hyperplasia and tubular hypertrophy were observed in both sexes and foci of clear cell hyperplasia and pigmentation were present in the kidney cortex of males as well as one Group 4 female.
In males the number of animals with cervical lymph nodes which were described as enlarged at necropsy was higher in the mid- and high dose groups than in the control group animals but most of the observations were estimated as only 2 to 5 times the normal size. Microscopically the enlarged lymph nodes showed slight to moderately severe lymphoid hyperplasia. Since the severity and incidence of the finding vas not related to dose and enlarged lymph nodes are a common finding, the increase was not considered treatment related.
No effects which were considered treatment related were observed in the primary or accessory sex organs for male or female rats. All of the findings in the sex organs were either seen in comparable severity and incidence in the controls and treated groups or were considered common in rats of this strain and age.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Two males in the high dose group had primary renal neoplasms, one a tubular adenoma and one a tubular carcinoma. The neoplasms were probably associated with the dietary administration of the test substance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The Day 0 mean litter size (total pups and liveborn pups) shoved no statistically significant difference between groups although the litter size and number of live offspring were slightly lower for the high dose group compared with the other groups.

The number of litters with stillborn pups in the treated groups were comparable and slightly lower than the number in the control groups. The mean index of stillborn pups was slightly higher in the control group than in the treated groups.

The mating and fertility indices showed no adverse effect of treatment at any dose level. Out of the 35 females mated from each group, 18, 23, 27 and 27 produced litters from the control, low, mid- and high dose groups, respectively. This represents a pregnancy rate of 51%, 66%, 77% and 77%, respectively. The pregnancy rates for all groups and especially the control group was lower than the pregnancy rate in the F1a
littering. The difference may have been due to the age or size of the females at the time of the F1b littering.

There were no significant differences between the mean number of days of gestation in the treated groups and the control group. The majority of animals delivered on Days 22 or 23 of gestation.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

< 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: Not determinable due to absence of adverse effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 ppm

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four male and two female F1 parental rats died while on study. The males were from each of the three treatment groups and the females were both from the mid-dose group. The deaths were not considered treatment related. There were no clinical observations during the F1 period which were considered to be treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

On Week 0 of the F1 generation the mean body weights of the male and female control groups were 6% higher than the mean weights of the low dose groups. The F1 animals were selected from the F1b pups. At weaning of the F1b pups there was no significant difference between the control and the low dose group. Since the F1 animals were selected randomly from the F1b pups with no restrictions imposed on mean body weight, it is possible that the lower body weight at the beginning of the F1 generation for the low dose groups was due to a fortuitous
choice of heavier and lighter pups from the litters of the control and low dose groups. For males, the mean body weight of the high dose group was 9% lower than the control value at the beginning of the growth period and the difference, which was statistically significant each week, varied from 6 to 11% during the F1 period. In addition, the mean body weight gain of the high dose group was statistically significantly lower than the control group for almost every week. The difference over the entire 34 week period was 11%. The mean body weights and body weight gains of the mid-dose group males shoved no significant differences from the control values at any time during the F1 period but both the mean body weight and body weight gain were slightly lower than the means for the controls towards the end of the F1 period. Since the mid-dose level had a clear effect on body weight and body weight gain in the Fo generation which developed with time, it is possible that the differences in mean body weight and body weight gain in the F1 males, which were 5% and 6% respectively by Week 34, were, in fact, treatment related.
At Week 0 of the F1 growth period the mean body weight of the low dose group males was 6% lower than the control group. Because of the difference in the mean body weights at the beginning of the F1 period, it is considered more relevant to compare body weight gain data rather than body weight data to study the possibility of there being an effect on body weight for the low dose group males during the F1 growth period. There were no statistically significant differences between the mean body weight gains for the low dose group and the control group after Week 5. The differences up to Week 5 were not considered treatment related because the mid-dose group body weight data was comparable to the control data for Weeks 0 to 5. The low dose level, 500 ppm in the diet, is considered a no observable effect level (NOEL) for male body weight effects during the F1 period.
For the females the mean body weight and mean body weight gains for all treatment groups were lower than the·values for the control group for most of the F1 study period.
For approximately the first ten weeks of the F1 period, the mean values for all three dose groups were quite similar and the differences from the control group did not show a dose response. From Week 10 and Week 12 onwards for mean body weight and mean body weight gain respectively, the statistical analysis for trend showed a fit. At Weeks 24 and 25 there were no statistically significant differences in body weight gain between the controls and the low dose group. At the end of the F1 growth period, the percentage difference in mean body weight gain for the
high dose group and control group was similar in the F0 generation (14%) and the F1 generation (15%) which suggests the data for the high dose from the F1 females is valid.
The available information suggests that the difference in body weight data during the growth period for the low dose level was probably not related to treatment but that the lover body weight data at the high dose was treatment related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For males there vas no treatment-related effect on absolute food consumption during the F1 period. For several weeks the absolute food consumption was significantly lower in the low dose group but these differences were not considered to be treatment related since there were no consistent differences in the high or mid-dose groups.
Since the mean body weights of the high and mid-dose group males were lower than the control group but the mean absolute food consumption remained comparable with the control group, the food consumption relative to body weight was higher than for the control group and it is possible that utilization of food was affected by the test substance.
For the F1 females the mean absolute food consumption values for the treated groups, especially the low dose group, were frequently lower than the values for the control group but the data did not form a dose-response pattern. The differences, therefore, in absolute food consumption were not considered treatment related. The high dose F1 females showed an increase in relative food consumption compared with the controls almost every week food consumption was measured. The difference which was similar to that observed in the mid- and high dose male groups was considered to be treatment related. There were no statistical differences for the mid- or low dose groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed in the stomach and kidney both by gross and microscopic pathology.
At necropsy, there were a few observations of raised areas or foci in the mucosa of the forestomach in the male and female rats which had received the test material, but the incidences did not increase in a treatment-related manner for either sex.
There was also a slight increase in the incidence of mottled kidneys in the mid- and high dose groups for both males and females relative to the control and low dose groups, but the observations were more common in the males. There was also a slight increase in the incidence of mottled kidneys in the mid- and high dose groups for both males and females relative to the control and low dose group.
There were two, one and three observations of small testes at necropsy in the low, mid- and high dose groups of male rats. The small testes correlated with the microscopic observations of spermatogenesis.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, treatment-related effects were observed in the stomachs of male and female rats at all dose levels. The alterations to the stomach were characterized microscopically as squamous epithelial hyperplasia and hyperkeratosis. Both the incidence and severity of the microscopic lesions increased in a dose-related manner.
There was an increase in the number of observations made at necropsy in the kidney in the mid- and high dose level groups for both males and females, but the observations were more common in the males. The treatment-related observations were enlarged kidney, granular cortical surface and alteration of colour to pale or green.

Treatment-related effects were observed microscopically in the kidneys from male rats at all dose levels and from females at the mid- and high dose level. The effects, which increased in incidence and severity in a dose-related fashion, were more severe in the males. Tubular epithelial hyperplasia, tubular hypertrophy, foci of clear cell hyperplasia, pigmentation and karyomegaly were the treatment-related findings. In females only, a treatment-related increase in regenerative epithelium was also observed. It is likely that regenerative epithelium was not observed in the males because it was masked by the more severe lesions present in the kidneys of the male rats.
At the high dose level there was one rat with bilateral spermatogenesis, three rats with unilateral spermatogenesis and two with bilateral focal spermatogenesis. Since for most of the rats the finding was focal or unilateral, the incidence of spermatogenesis did not form a dose response and the incidence was considered normal by the pathologist, the spermatogenesis was not considered treatment related. There were no abnormal findings in female reproductive organs which were considered treatment related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

> 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F1a: The Day 0 mean litter size (total pups and liveborn pups) showed no statistically significant difference between groups although the litter size and number of live offspring were slightly lower for the high dose group compared with the other groups.

F1b: The Day 0 mean litter size (total pups and liveborn pups) showed no treatment-related differences. The number of litters with stillborn pups and the mean index of stillborn pups were slightly lower in the control group than in the treated groups. The slight difference were not considered treatment related since the control group from the F1b littering had less stillborns than the control groups in the other three litterings and the values for treated animals were within the range of control values.
No treatment-related effect on offspring viability was observed when the litter indices for the F1b reproduction were evaluated. All pregnant females delivered litters with live pups on Day 0 of lactation. There was one total litter loss from the high dose group but originally there was only one pup in the litter.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1a: The only mean pup weight which was statistically different from the control values during the lactation period was the Day 21 weight of the high dose group which was 92.9% of the control value.

F1b: The mean pup weights on Day 0 were comparable and showed no dose-related reduction. On Day 4, prior to culling, the pup body weights of the mid-dose group were significantly lower than the controls but post-cull the difference was not significant. On Days 7, 14 and 21 of lactation, the mean pup weights for the mid- and high dose groups were significantly lower than the mean weight of the control group. When the pup weights were analyzed by analysis of covariance with litter size as the covariate, there were no statistical differences in mean pup weights between groups on Day 4 precull or Day 7. On Day 21 the differences between the control and the mid- and high doses were 7.7% and 14.1%, respectively. The mean pup weigh ts of the low dose group were comparable with the mean weights of the control group throughout the lactation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1a: Very few abnormal observations were made at the necropsies of the stillborn, spontaneous deaths or culled pups and the incidences do not suggest a treatment-related effect.
The only unusual finding in F1a pups was a single observation of a stillborn pup from the high dose group with an abnormal head which was described as having a small anterior portion with no eye lids, only one nostril apparent and no visible external opening for the oral cavity.
Dilation of the renal pelvis was the only observation from the necropsy of the pups terminated at 21 days of age which was higher in the treated groups. The percentage of litters with at least one finding of dilated renal pelvis was 24% in the control group, 34% in the low dose group, 26% in the mid-dose group and 38% in the high dose group. The percentage of pups with the finding followed the same pattern.
The only incidences of extremely dilated renal pelvises in F1a pups were from the mid- or high dose groups which might suggest a treatment-related effect; however, the data from the F1b, F2a and F2b litterings did not confirm this finding and the finding was not considered treatment related.
F1b: Very few abnormal observations were made at the F1b necropsies and the incidences do not suggest a treatment related effect

Histopathological findings:

no effects observed

Other effects:

not examined

Description (incidence and severity):

F1a: There were no significant differences between the groups for mean ratio of live born males to total live born pups
F1b: There were no significant differences between the groups for mean ratio of live born males to total live born pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Generation:

F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F2a: The Day 0 mean litter size (total pups and liveborn pups) showed no statistically significant difference between groups although the litter size and number of live offspring were slightly lower for the treatment groups compared with the control group. The data did not fall into a dose response pattern, and the mean size of the litter for each group was within the range of the mean control values from the other litterings in the study. The difference was not considered to be treatment related.
The numbers of litters with stillborn pups in the treated groups were comparable and, for the high dose group, slightly lower than the number in the control group.
No treatment-related effect on offspring viability was observed when the litter indices for the F2a reproduction were evaluated. All pregnant females delivered litters with live pups on Day 0 of lactation except for one dam which died during delivery. There was only one total litter loss throughout the lactation period, which was from the low dose group.

F2b: The Day 0 mean litter size and the mean number of live offspring on Day 0 showed no treatment-related differences. The number of litters with stillborn pups in the treated groups were comparable and slightly lower than the number in the control group. The mean index of stillborn pups was slightly higher in the control group than in the treated groups.
No treatment-related effect on offspring viability was observed when the litter indices for the F2b reproduction were evaluated. All pregnant females delivered litters with live pups on Day 0 of lactation and there were no total litter losses throughout the lactation period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F2a: When standard statistics were used, the only statistically significant difference in mean pup weight for the F2a pup was for the high dose group at 21 days, which had a mean pup weight 8.6% lower than for the control group. When litter size was used as a covariant in the statistical analysis, the low- dose group showed a significantly lower mean pup weight. Because the mid-dose group pup weight was comparable with the control group, the low dose difference was not considered treatment related.

F2b: The mean pup weights from Day 0 up until Day 14 showed no treatment-related reduction. On Day 21 the mean pup weights in the high and lov dose groups were lower than the controls and the differences were significant at the 0.01 level for the high dose and the 0.05 level for the low dose. The mean pup weights on Day 21 for the mid-dose vas close to that of the control group. The statistical test for trend on Day 21 was significant; however, there was a lack of fit because the mid-dose group had a value close to the control value;

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2a: Very few abnormal observations were made at the necropsies of the stillborn, spontaneous deaths or culled pups and the incidences do not suggest a treatment-related effect. The only unusual finding in F2a pups was two male pups from a control litter which were joined from the thoracic region up to the head.
The necropsy of the pups terminated at 21 days of age showed a high background incidence of dilated renal pelvis that vas not treatment related.

F2b: No abnormal observations were made at the necropsies of the stillborn, spontaneous deaths or culled pups. The necropsy of the pups terminated at 21 days of age showed several incidences of dilation of the renal pelvis but there was no significant difference in incidence between groups.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

F2a: There were no significant differences between the groups for mean ratio of live born males to total live born pups.
F2b: There were no significant differences between the groups for mean ratio of live born males to total live born pups.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Generation:

F2

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Diet Analysis
The pre-test evaluation of homogeneity and stability of the test substance in the diets demonstrated that a homogeneous dispersion of the test material in the feed was prepared and that the test material was adequately stable in the feed for 7 or 14 days under study room conditions. During the study the overall mean analyzed concentrations of chlorothalonil in the diets were 98%, 99% and 99% of the nominal concentrations for the 500 ppm, 1500 ppm and 3000 ppm
groups, respectively. The results indicated that the diets were prepared at the intended concentrations throughout the study

Table 1. Mean body weights and body weight gain (g) – selected timepoints

	Dietary concentration (ppm)
	F0 Males				F0 Females
Week	0	500	1500	3000	0	500	1500	3000
0	159.1	158.1	161.6	158.6	140.7	138.5	137.8	138.3
5	387.5	378.5	375.1	357.8*	231.9	228.8	225.6	218.4**
10	492.5	478.4	473.2	457.0**	273.7	268.8	262.3	253.3**
20	604.6	577.5	571.8*	540.8**	312.3	308.4	303.7	291.0**
30	650.2	621.9	614.4	577.1**	-	-	-	-
0-10	333.4	320.3	311.6*	298.3**	133.1	130.3	124.5	115.0**
	F1 Males				F1 Females
	0	500	1500	3000	0	500	1500	3000
0	126.4	118.5	125.5	115.3**	112.1	104.9	107.5	102.3**
5	394.5	373.5*	395.3	367.2**	244.9	223.3**	226.7**	223.3**
10	510.9	490.9	505.3	469.7**	294.9	268.0**	274.5**	261.9**
20/24	628.4	601.5	613.4	573.4**	343.8	322.9	323.9	308.3**
30	676.7	638.2*	649.7	608.4**	-	-	-	-
0-10	384.5	372.4	379.9	354.4**	182.9	163.1**	166.9*	159.5**

* Significantly different from control P < 0.05
** Significantly different from control P < 0.01

 

Table 2. Mean food consumption (g/rat/day) – selected timepoints

	Dietary concentration (ppm)
	F0 Males				F0 Females
Week	0	500	1500	3000	0	500	1500	3000
1	23.6	22.4	22.3*	19.3**	17.6	16.2**	16.6	14.5**
5	27.1	26.3	25.9	25.5*	18.7	18.2	17.9	17.9
10	27.9	27.1	27.2	27.0	19.4	18.5	18.7	18.5
Week 10 (g/kg/day)	56.6	56.2	57.2	59.2**	70.8	68.5	70.9	72.1
	F1 Males				F1 Females
	0	500	1500	3000	0	500	1500	3000
1	22.0	19.7*	22.1	21.2	18.3	17.5	17.7	18.2
5	30.4	29.2	31.3	29.5	22.0	20.3*	20.7	21.1
10	29.8	27.9*	30.5	28.4	20.4	19.7	19.9	19.4
Week 10 (g/kg/day)	58.1	56.9	60.0	60.4**	69.3	73.0	72.4	73.9**

* Significantly different from control P < 0.05
** Significantly different from control P < 0.01

 

Table 3. Reproductive Performance (selected parameters)

	Dietary concentration (ppm)
	F1a litter				F1b litter
Observation	0	500	1500	3000	0	500	1500	3000
Number mated	35	35	35	35	35	35	35	35
Number of matings	26	34	34	35	27	32	32	32
Number of pregnancies	25	29	31	32	18	23	27	27
Fertility index (%)	96	85	91	91	67	72	84	84
Mean gestation length (days)	22.1	22.3	22.1	22.0	22.5	22.3	22.0	22.1
Mean pups still born	0.4	0.1	0.2	0.2	0.1	0.1	0.4	0.1
Mean live litter size at birth	13.1	12.9	12.9	12.1	12.3	12.6	13.5	12.9
Mean live day 4 (pre-cull)	13.0	12.4	12.8	11.9	12.2	12.5	13.5	12.8
Mean live day 21	8.0	7.7	7.9	7.7	7.8	7.7	8.0	8.0
Live born index (%)	97.6	99.3	98.7	98.4	99.2	97.8	97.4	98.7
Viability index days 0-4 (%)	99.4	96.1	99.3	98.3	98.8	99.3	99.7	99.5
Lactation index days 4-21 (%)	100	99.6	100	100	100	100	100	96.3
	Dietary concentration (ppm)
	F2a litter				F2b litter
Observation	0	500	1500	3000	0	500	1500	3000
Number mated	35	35	34	35	35	34	33	34
Number of matings	29	29	28	27	31	30	22	27
Number of pregnancies	23	27	24	22	14	23	17	17
Fertility index (%)	79	93	86	81	45	77	77	63
Mean gestation length (days)	22.3	22.2	22.5	22.1	22.6	22.3	22.5	22.4
Mean pups still born	0.7	0.5	0.7	0.1	0.8	0.1	0.1	0.1
Mean live litter size at birth	13.2	12.4	12.4	12.7	12.2	13.0	12.2	13.1
Mean live day 4 (pre-cull)	13.0	12.2	12.7	12.4	12.0	12.9	12.2	13.1
Mean live day 21	8.0	7.8	7.7	7.8	7.7	7.9	7.6	7.8
Live born index (%)	95.0	96.3	94.2	99.1	94.1	99.0	99.1	99.2
Viability index days 0-4 (%)	98.5	94.6	98.2	97.4	97.5	99.7	100	99.6
Lactation index days 4-21 (%)	100	100	99.5	100	100	100	100	99.3

 

Table 4. Pup body weights (g)

	Dietary concentration (ppm)
	F1a				F1b
	0	500	1500	3000	0		500	1500	3000
Mean pup weight at birth	6.2	6.2	6.3	6.3	6.6	6.3		6.2	6.3
Mean pup weight day 7	15.4	15.7	15.5	15.5	17.3	16.9		16.0*	15.9*
Mean pup weight day 14	31.0	31.1	30.8	30.2	33.8	33.0		31.4*	29.5**
Mean pup weight day 21	49.6	49.2	47.9	46.1*	56.2	54.6		51.9*	48.3**
F2a	F2b
Mean pup weight at birth	6.3	6.1	6.3	6.3	6.3	6.1		6.5	6.2
Mean pup weight day 7	16.2	15.7	16.4	15.8	16.5	15.7		16.8	16.0
Mean pup weight day 14	32.5	31.0	33.2	31.1	32.8	31.2		32.2	31.2
Mean pup weight day 21	53.3	50.2	52.5	48.7**	55.0	50.4*		52.1	48.6**

* Significantly different from control P < 0.05
** Significantly different from control P < 0.01

Applicant's summary and conclusion

Conclusions:

The NOAEL for parental toxicity is 500 ppm (equal to 22.6 mg/kg bw/day) based on the decreased body weight in the F1 animals and the results of the macroscopic and microscopic examination in both males and females (effects on kidneys and stomach). Given the decrease in body weight of the F1 pups (day statistically significant on days 7, 14 and 21), the NOAEL for developmental effects is established at 500 ppm (equal to 22.6 mg/kg bw/day). All reproductive parameters investigated showed no treatment-related effects except for pup weights at Day 21 which had a no observable effect level of 1500 ppm.

Executive summary:

In this two generation reproduction study (EPA OPP 83 -4, under GLP) with two litters per generation, the test substance was administered to Crl CD rats by dietary admixture at concentrations of 500, 1500 and 3000 ppm. There were 35 rats of each sex in the three groups. An additional group of 35 rats of each sex received untreated basal diet. The parental animals from each generation were treated continuously during the growth period prior to mating and then throughout the mating, gestation, lactation and resting phases of the study. The growth period was ten weeks for the F0 animals and Fourteen weeks for the F1 animals. Each generation of parental animals were mated twice to produce the F1a, F1b, F2a and F2b litters. The offspring were exposed to the test diets throughout the lactation periods. Thirty-five F1b males and females per group were selected to become the F1 generation after weaning. All animals were observed twice daily for overt signs of toxicity, moribundity and mortality. Complete physical examinations, body weights and Food consumption were recorded at regular intervals. All animals were necropsied and selected organs were preserved after completion of the F1b and F2b lactation periods. Histopathological examinations of F0 and F1 adults were conducted on reproductive organs as well as the target organs of test substance toxicity. All litters were examined daily for survival and overt clinical signs of toxicity throughout the lactation period. The date delivered, number, weight and sex of live and stillborn pups and any abnormalities observed in the offspring were recorded. On Day 4 of lactation the size of litters with nine or more pups was reduced to eight pups. The number, sex and weight of live pups were recorded at regular intervals during lactation. All surviving pups were sexed, weighed and necropsied at the end of each 21 day lactation period, except for selected F1b pups which continued as the F1 parental generation.

The study report describes the No Effect Levels (NOELs) for Parent, reproductive and developmental effects. Following assessment of the study data and the observed effects, the following No Adverse Effect Levels (NOAELs) are derived.

No mortalities or clinical signs of toxicity associated with administration of test substance were observed in the F0 or F1 animals. There was a treatment-related effect towards a lowered body weight in the F0 and F1 adults. The no observable adverse effect level for body weights was 500 ppm. The body weight effect was comparable in the F0 and F1 generations for both males and females. Increased relative Food consumption was observed in the groups that showed lower body weights. The anticipated treatment-related lesions in the kidneys and stomachs of adult animals were observed by gross and microscopic pathology. Chronic studies have shown that the no observable adverse effect levels for stomach and kidney effects were at lower treatment levels than the lowest dose achieved in this study. The effects were observed in the kidney at all dose levels in males and at 1500 and 3000 ppm in females. Stomach effects were observed at all dose levels in both sexes. The stomach and kidney effects were comparable in the F0 and F1 generations for both sexes.

Reproductive parameters in F0 and F1 animals, including mating and fertility indices and gestation length, were not affected by treatment of test substance. No gross malformations which were considered treatment related were observed in offspring in any of the groups throughout the study. No effects on the number of live and stillborn pups, pup sex ratio, pup survival and physical condition of the pups during lactation were observed. No findings which were considered treatment related were observed during necropsy of the pups. The only effect observed in pups in this study was lowered body weight compared to the control on Day 21 of lactation. The no observable adverse effect level for this effect was considered to be 1500 ppm. 

In summary, body weight, kidney and stomach effects were observed in parental animals for this reproduction study with test substance. The no adverse observable effect level (NOAEL; = NOEL) for body weight effects was 500 ppm but the stomach and kidney effects were observed at all dose levels. All reproductive parameters investigated showed no treatment-related effects except for pup weights at Day 21 which had a no observable adverse effect level of 1500 ppm.