Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Aug 2006 to 14 Nov 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Jan 2001

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Aug 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Directive 87/302/EEC, OJEC, L133, 47-50

Version / remarks:

1988

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HsdRCCHan:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: At least 5 weeks old
- Weight at study initiation: 84 -158 g and females 77 – 122 g for males and females, respectively
- Fasting period before study: not specified
- Housing: The rats were housed, sexes separately, in multiple rat racks. They were housed in litters initially, two males or two females per cage after they had been assigned to experimental groups and during the pre-mating period; one male was housed with one female for mating; females were housed individually during gestation and with their litter during lactation and were provided with shredded paper bedding material. After mating, males were housed up to four per cage. Males and females from the same group were housed in adjacent cages during the period prior to mating, to avoid anoestrus. The animals were transferred to clean cages and racks, as necessary, during the study.
- Diet: RM3 diet, ad libitum
- Water: Mains water, ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes/hr: At least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 14 Aug 2006 To: 14 Nov 2007

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: At the start of the study
- Mixing appropriate amounts of diets: The experimental diets were prepared in 40 kg batches from premixes prepared by triturating the appropriate amount of test substance with 1 kg of milled diet. The premixes were then added to diet and mixed thoroughly. All diets were based on RM3 diet.
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to14 days
- Proof of pregnancy: vaginal smears referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HOMOGENEITY AND STABILITY OF DIET FORMULATION
Samples from all dietary levels (including controls) were taken prior to the start of the study and at intervals throughout the study and analysed quantitatively for test substance by High Performance Liquid Chromatography (HPLC). At the start of the study the homogeneity of test substance in RM3 diet was determined by analysing samples from the low and high dose levels. The chemical stability of the test substance in diet at room temperature and when frozen was determined for the low and high dose levels over a period of 43 days.

Duration of treatment / exposure:

From the treatment of the original parents throughout the study until termination of the last litter of F2 pups, hence 261 days.

Frequency of treatment:

Continuously

Details on study schedule:

- After 10 weeks, the (F0 parents) animals were mated and allowed to rear the ensuing F1 litters to weaning.
- The breeding programme was repeated with the F1 parents selected from the F1A pups to produce the F2A litters after a 10-week pre-mating period.
-The first generation pups (F1) were retained with their dams until selection of pups as parents of the next generation at day 29 post partum. Twenty six males and twenty six females per group were selected from the first generation pups (F1) to become the next generation (F1) parents. They were selected from all appropriate litters (where possible). Their genealogy was recorded and taken into account during the selection procedure. After selection, the pre-mating and breeding programme was followed (as for the F0 generation), until an F2 litter had been produced and weaned.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Remarks:

Group 2: For dietary equivalents, see in Table 1 in 'Any other information on results incl. tables'

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

Group 3: For dietary equivalents, see in Table 1 in 'Any other information on results incl. tables'

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

Group 4: For dietary equivalents, see in Table 1 in 'Any other information on results incl. tables'

No. of animals per sex per dose:

26

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale: The dose levels selected for this study were based on the results of a preliminary study in the same strain of rat carried out in the performing Laboratory..

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: daily
- Cage side observations: during the study, all rats were observed daily for changes in clinical condition and behaviour

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: weekly. Each rat was made at the same time that the body weight was recorded. Any abnormalities or the observation of no abnormality detected were recorded

BODY WEIGHT:
- Time schedule for examinations: The body weights of all animals were recorded at weekly intervals throughout the pre-mating periods. The initial weights for the F0 generation parents were recorded immediately before first feeding the experimental diets and the initial weights for the F1 generation parents were recorded at selection on day 29. The body weights of all animals were recorded at weekly intervals throughout the pre-mating periods. The initial weights for the F0 generation parents were recorded immediately before first feeding the experimental diets and the initial weights for the F1 generation parents were recorded at selection on day 29.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

- Pre-mating: Daily vaginal smears were taken and examined from all F0 and F1 females for 3 weeks prior to mating.
- Mating: Daily vaginal smears were taken and examined from all females to determine when mating had occurred as indicated by the presence of sperm in the smear. A female with a sperm positive vaginal smear was separated from the male and had no further smears taken.
- Termination: A vaginal smear was taken and examined on the day of termination from each F0 and F1 female.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
- Immediately following the death of each male, the right epididymis was removed and weighed.
- The cauda epididymis was removed and the weight recorded.
- Samples of sperm were then evaluated for motion characteristics.
- The number of sperm was determined
- Sperm morphology was assessed using a light microscope and a minimum of 200 sperm per sample were classified as normal or abnormal. The abnormal sperm were classified according to head (detached, double, abnormal shape, abnormal size, or abnormal acrosome), tail (double, coiled/kinked or abnormal size) or multiple (head and tail abnormalities).
- Homogenisation resistant spermatids were examined from F0 and F1 males in the control and 3000 ppm groups only.

Litter observations:

STANDARDISATION OF LITTERS : Yes

PARAMETERS EXAMINED
The following parameters were examined in [F1 andF2] offspring:
- Litters were examined for dead or moribund pups at least once daily and any such pups were subjected to a gross examination post mortem.
- Any clinical abnormalities seen in the pups were recorded.
- A count of all live and dead pups was made within 24 hours of parturition (day 1) and thereafter on days 5, 8, 15, 22 and 29 post partum.
- The sexes of the pups were also recorded at these times.
- Individual pup body weights were recorded within 24 hours of birth (day 1) and on days 5, 8, 15, 22 and 29 post partum. The litters were weaned on day 29 post partum. Since pups were not individually identified, data were recorded by sex and litter. The pups selected to be the parents of the next generation were weighed on day 29 post partum to give the initial F1 generation parent body weight values. Subsequently, they were weighed at weekly intervals for the duration of the pre-mating period.
- Anogenital distance was recorded for F2 pups on day 1 post partum. Anogenital distance was not recorded for pups which were found dead.

SEXUAL DEVELOPMENT OBSERVATIONS:
- For animals selected as F1 parents the age at which vaginal opening (checked daily from day 29 post partum) and preputial separation (checked daily from day 29 post partum) occurred were determined. Body weights were also recorded on the day the landmark was first recorded.

GROSS EXAMINATION OF DEAD PUPS:
- Yes. Pups killed or found dead were subjected to a macroscopic examination post mortem.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals (including those killed intercurrently or found dead) were subjected to a full examination post mortem. Males were sacrificed after completion of the mating period, while females were terminated on day 29 post partum.
- Maternal animals: All animals (including those killed intercurrently or found dead) were subjected to a full examination post mortem.

GROSS NECROPSY
- Gross necropsy consisted of an external observation and an internal examination of all cranial, thoracic and abdominal organs and structures. All surviving rats and those requiring euthanasia were killed by exsanguination under terminal
anaesthesia induced by halothane Ph. Eur. vapour. The number of implantation sites was recorded for all females which had been mated.
- The weights of the following organs were recorded from all parents at scheduled termination: adrenal glands, pituitary gland, brain, prostate gland, right cauda epididymis ,*seminal vesicles (with prostate and coagulating gland), left epididymis (including cauda), spleen, right epididymis (including cauda), left testis, kidneys, right testis, liver, $ thyroid glands, ovaries and uterus (with oviducts and cervix).
* Seminal vesicles weight was derived by subtracting the weight of the prostate from the combined weight of seminal vesicles, prostate and coagulating gland. $ Thyroid glands were weighed after fixing.
- The following were taken from all animals in all groups and preserved in 10 % neutral buffered formol saline except testis and epididymis which were preserved in Bouin’s fixative, abnormal tissue, ovary, adrenal glands pituitary gland, brain*, prostate gland, cervix, seminal vesicle, coagulating gland spleen* left epididymis and cauda left, testis, kidney*, uterus with oviducts, liver and vagina.
Tissues marked with an asterisk (*) were stored. The remaining tissues from each generation, from the control and high dose (0 and 3000 ppm) groups were routinely processed, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. In addition, the liver was processed for F0 and F1 males and females in the 100 and 500 ppm groups. Tissues from suspected infertile animals were routinely processed, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.

HISTOPATHOLOGY / ORGAN WEIGHTS
All submitted tissues (except those stored) from the control and high dose (0 and 3000 ppm) groups, plus those from suspected infertile animals were examined by light microscopy. In addition the liver was examined for F0 and F1 males and females in the 100 and 500 ppm groups. A quantitative evaluation of primordial follicles was conducted for F1 females in the control and 3000 ppm dose groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 29 days of age.
- All surviving rats and those requiring euthanasia were killed by exsanguination under terminal anaesthesia induced by halothane Ph. Eur. vapour.

GROSS NECROPSY
- Gross necropsy: Three male and three female pups per group from each litter, where available, were selected randomly for a macroscopic examination post mortem.
All pups remaining after selection of the next generation or not chosen for the macroscopic post mortem examination described above were killed by an appropriate method and discarded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weights of the following organs were recorded from one male and one female pup per litter from all litters surviving to scheduled termination: brain, thymus, spleen and liver.
Abnormal tissues were taken from all pups given an examination post mortem. The fixatives used were as described above for the parents. In addition the following were taken from 3 males and 3 females per litter in all groups at scheduled termination and stored: abnormal tissues thymus*, brain*, *liver and spleen*.
Tissues marked with an asterisk (*) were weighed and submitted only from pups selected for organ weight determination.

Statistics:

All analyses were carried out in SAS (1999). For Fisher’s Exact Tests the proportion in each treated group was compared to the control group proportion. Analyses of variance and covariance allowed for the replicate structure of the study design. In addition for the F0 generation, with the exception of pre-mating food consumption and food utilisation analyses also allowed for the litter of origin of the parents. Least-squares means for each group were calculated using the LSMEAN option in SAS PROC MIXED. Unbiased estimates of differences from control were provided by the difference between each treatment group least squares mean and the control group least-squares mean. Differences from control were tested statistically by comparing each treatment group least-squares mean with the control group least-squares mean using a Student’s t-test, based on the error mean square in the analysis. All statistical tests were two sided.

Reproductive indices:

From the records of mating and parturition, the reproductive performance of the parents was assessed. The following were examined:
- The success of mating was established. The criterion for a successful mating was the production of a viable litter i.e. a litter in which at least one pup was found alive at day 1.
- Length of gestation was measured in days from the date of the positive smear to date of birth (but only in females fulfilling the criterion above i.e. production of a viable litter).
- Pre-coital interval was the number of days between the date of pairing and the date of the positive smear.

Offspring viability indices:

From the post partum records of the litters the following were examined:
- The percentage of pups live born (number born live / number born live plus number born dead x 100)
- Survival (number of live pups on days 5, 8, 15, 22 or 29 / number of pups born live x 100)
- Pup sex distribution (number of live male pups / total number of live pups per litter x 100)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One F0 female in the control group was killed due to adverse clinical signs in week 14 of the study. Five F0 females in the 100 ppm group and 2 females in the 500 ppm group were killed before scheduled termination as they had not littered or after loss of their litter.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pre-mating: Body weights were statistically significantly lower than control in F0 males and females in the 3000 ppm group throughout the pre-mating period. The maximum difference from control was approximately 10 % in both sexes. Body weights of females in the 500 ppm group were slightly reduced at the end of the pre-mating period; the maximum difference from control was 3 %. There was no effect on body weights of males in the 500 ppm group or either sex in the 100 ppm group.
Gestation: Body weights were lower than control on day 1 of gestation in F0 females in the 3000 ppm group. In addition gestation weight gain was reduced. Although body weights of F0 females in the 500 ppm group were lower than control, there was no effect on weight gain during gestation. There were no effects on body weights during gestation in F0 females in the 100 ppm group. The statistically significantly higher weight of F0 females in the 100 ppm group on day 22 of gestation was considered to be unrelated to treatment as reduced body weights are seen in response to treatment in high dose animals.
Post partum: F0 females in the 3000 ppm group had lower body weights than control post partum. F0 females in the 500 ppm group had lower body weights than control on day 1 post partum but thereafter weights were similar to control. There were no effects on body weights post partum in F0 females in the 100 ppm group. The statistically significantly higher weight of F0 females in the 100 ppm group on day 5 post partum was considered to be unrelated to treatment as reduced body weights are seen in response to treatment in high dose animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Pre-mating: Food consumption was statistically significantly lower than control in F0 males and females in the 3000 ppm group during the pre-mating period. The maximum difference from control was approximately 10 % in both sexes.
- Food consumption was generally slightly lower than control in F0 males and females in the 500 ppm group. There was no effect on food consumption in males or females in the 100 ppm group. Food utilisation was less efficient than control in F0 males and females in the 3000 ppm group. The difference from control was statistically significant in weeks 1 - 4 for both sexes, in weeks 5 - 8 in females and was also reflected in the overall value for both sexes.
- Food utilisation was similar to control in F0 animals in the 100 and 500 ppm groups.
- Gestation: Food consumption during weeks 1 and 2 of gestation in was reduced in F0 females in the 3000 ppm group. There was no effect on food consumption during gestation in females in the 100 or 500 ppm groups
- Post partum: Food consumption post partum was reduced in F0 females in the 3000 ppm group. The difference from control was statistically significant from weeks 2 - 4 in F0 females. There was no effect on food consumption post partum in the 100 or 500 ppm 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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No changes were detected in the reproductive organs of F0 animals which could be attributed to treatment. Minimal or slight centrilobular/diffuse hypertrophy of hepatocytes was observed in the liver of F0 adults receiving 3000 and 500 ppm test substance but not in any animal receiving 100 ppm test substance or in the control group. The incidence and severity of the change was related to dose level in the affected groups. The change was more widely distributed (i.e. diffuse) in animals receiving 3000 ppm than at 500 ppm where it tended to be confined to centrilobular areas. Hypertrophic hepatocytes also exhibited prominent cytoplasmic basophilia and areas of fine cytoplasmic vacuolation giving them a very distinctive appearance. No changes were observed in any other tissue which could be attributed to treatment with the test substance.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean cycle length of F0 females was approximately 4 days, as expected for the HsdRCCHan:WIST rat.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm measures: There were no effects on epididymal sperm number or motility. The number of homogenisation resistant spermatids in the right testis of F0 males in the 3000 ppm group was lower than control in both generations. The mean values obtained, were well within the range of historical control mean values, therefore, the differences are considered to reflect normal variation and are unrelated to treatment with test substance.
There were no effects on sperm morphology.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The majority of rats in all groups mated within the first 4 days of pairing and there was no evidence of an effect of the test substance on pre-coital interval. The majority of females in all groups and in both generations littered 22 days following detection of a sperm positive smear, and there was no evidence of an effect of the test substance on the length of gestation. The criterion for a successful mating was the production of a viable litter i.e. a litter in which at least one pup was found alive on day 1. The mating success in all group and both generations was good and there was no evidence of an effect of the test substance. In the F1A litter there were 1, 3, 1, 0 whole litter losses in the control, 100, 500 and 3000 ppm groups respectively. There was clearly no evidence of an effect the test substance on the number of whole litter losses.
When whole litter losses are included, the percentage of pups live born was at least 94% in all groups and both generations and was not affected by administration of the test substance.
When litter size of F1A litters excluding whole litter losses was analysed statistically, the number of pups in the 3000 ppm group was lower than that in the control group. However this difference was no longer evident when the control litter which was a whole litter losses (all pups born dead) was excluded. There were no F1A litter losses in the 3000 ppm group. This difference was not reproduced in the F2A litter where the number of pups in the 3000 ppm group was similar to that of the control group, including or excluding whole litter losses. Therefore it is considered that the difference in the F1A litter size was not related to the test substance.
There was no effect of treatment with the test substance on post implantation loss. The number of implants and the number of live + dead pups was lower than control in the 3000 ppm group for the F1 females. The mean values obtained, were well within the range of historical control mean values, therefore, the differences are considered to reflect normal variation and are unrelated to treatment with the test substance.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other: Mean dietary equivalent of 9.3 mg/kg/day

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

> 3 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Mean dietary equivalent of 279 mg/kg bw/day

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two F1 males in the 100 ppm group were found dead and an additional male in this group was killed due to adverse clinical signs between weeks 13 and 16. Two F1 females in the control group and 1 female in the 500 ppm group were killed before scheduled termination as they had not littered or after loss of their litter. An additional control female was killed before scheduled mating as she was found to have been placed in a male cage in error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of F1 animals in the 3000 ppm group were lower than control at selection and this difference from control was maintained throughout the pre-mating period. The difference in unadjusted body weight was 13 - 14 and 10 – 11 % in males and females respectively. Weight of females in the 500 ppm group were similar to control at selection but were reduced from week 2 for the remainder of the pre-mating period; the maximum difference from control was 9 %. There was no effect on body weights of males in the 500 ppm group or either sex in the 100 ppm group.
Gestation: Body weights were lower than control on day 1 of gestation in F1 females in the 3000 ppm group. In addition gestation weight gain was reduced. Although body weights of F1 females in the 500 ppm group were lower than control, there was no effect on weight gain during gestation. There were no effects on body weights during gestation in F1 females in the 100 ppm group.
Post partum: F1 females in the 3000 ppm group had lower body weights than control post partum. There were no effects on body weights post partum in F1 females in the 100 or 500 ppm groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Food consumption was statistically significantly lower than control in F1 males and females in the 3000 ppm group throughout the pre-mating period. The maximum difference from control was approximately 16 % in both sexes.
- Food consumption was also statistically significantly lower than control in F1 females in the 500 ppm group where maximum difference from control was approximately 13 %. There was no effect on food consumption in males in the 100 or 500 ppm groups or females in the 100 ppm group.
- Food utilisation was less efficient than control in F1 females in the 500 and 3000 ppm groups for the 5 - 8 week period only but there was no effect on the overall food utilisation. Food utilisation was similar to control in F1 males in all treated groups and in F1 females in the 100 ppm group. animals in the 100 and 500 ppm groups.
- Gestation: Food consumption during weeks 1 and 2 of gestation in was reduced in F1 females in the 3000 ppm group. There was no effect on food consumption during gestation in females in the 100 or 500 ppm groups
- Post partum: Food consumption post partum was reduced in F1 females in the 3000 ppm group. The difference from control was statistically significant from weeks 2-3 in F1 females. There was no effect on food consumption post partum in the 100 or 500 ppm 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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were increased in F1 females in the 500 ppm group and males and females in the 3000 ppm group. In addition liver weights were increased in F1 females in the 100 ppm group and F1 males in the 500 ppm group. Weights of ovaries and uterus with cervix were lower than control before and after adjustment for bodyweight F1 females in the 3000 ppm group. There were no other differences in organ weights that were considered to be related to treatment with the test substance. In F1 animals kidney weights adjusted for body weight were slightly higher than control in males in the 3000 ppm group and all treated female groups. With the exception of F1 females in the 100 ppm group, the mean values for absolute organ weights are within the range of historical control mean values.
Adrenal weights adjusted for body weight were statistically significantly higher than control in F1 females in the 500 and 3000 ppm groups. However absolute values were not different from the concurrent control. Effects on the weights of the seminal vesicles observed in the F0 generation were not observed for the F1 animals.
The weights of a number of other organs were statistically significantly lower in the 3000 ppm group than in the control group but the difference was no longer evident after adjustment for the lower body weight of the treated rats. These were brain, epididymides, and spleen in males and pituitary and spleen in females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The number of decedent F1 animals was very low and the incidence unrelated to dose level; the reasons for illness or death were considered to be incidental to treatment in all cases.
A few macroscopic findings were observed at low incidences in F1 animals at termination; none was considered to be related to treatment with the test substance. There was no effect of treatment on the number of mated F1 pairs failing to produce litters or suffering total litter loss in the neonatal period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No changes were detected in the reproductive organs of F1 animals which could be attributed to treatment. The number of F1 females receiving 3000 ppm test substance with vaginal histology characteristic of lactational anoestrus was increased compared with F1 control females. However the incidence was comparable to that observed in F0 control females and therefore considered not to be related to treatment. Minimal or slight centrilobular/diffuse hypertrophy of hepatocytes was observed in the liver of F1 adults receiving 3000 and 500 ppm test substance but not in any animal receiving 100 ppm test substance or in the control group. The incidence and severity of the change was related to dose level in the affected groups. The change was more widely distributed (i.e. diffuse) in animals receiving 3000 ppm than at 500 ppm where it tended to be confined to centrilobular areas. Hypertrophic hepatocytes also exhibited prominent cytoplasmic basophilia and areas of fine cytoplasmic vacuolation giving them a very distinctive appearance. There was no evidence of an effect of treatment with the test substance on small follicle counts in the ovaries of F1 females receiving 3000 ppm. No changes were observed in any other tissue which could be attributed to treatment with the test substance

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The mean cycle length of F1 females was approximately 4 days, as expected for the HsdRCCHan:WIST rat. The number of cycles in the 21 day period was statistically significantly higher in F1 females in the 100 and 500 ppm groups than control, but in the absence of a similar finding in the high dose group and as the number of cycles in all groups was generally 4 or 5, this is considered to be an incidental finding.
No significant difference in small follicle counts were detected in the ovaries of F1 females receiving 3000ppm test substance and control F1 females

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm measures: There were no effects on epididymal sperm number or motility. The number of homogenisation resistant spermatids in the right testis of F1 males in the 3000 ppm group was lower than control in both generations. The mean value for the treated F1 males was very close to the control F0 value and, in addition, the mean values obtained, were well within the range of historical control mean values, therefore, the differences are considered to reflect normal variation and are unrelated to treatment with test substance.
There were no effects on sperm morphology.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The majority of rats in all groups and in both generations mated within the first 4 days of pairing and there was no evidence of an effect of the test substance on pre-coital interval. The majority of females in all groups littered 22 days following detection of a sperm positive smear, and there was no evidence of an effect of the test substance on the length of gestation. The criterion for a successful mating was the production of a viable litter i.e. a litter in which at least one pup was found alive on day 1. The mating success in all group and both generations was good and there was no evidence of an effect of the test substance. In the F2A litter there were 3, 1, 0, 0 whole litter losses in the control, 100, 500 and 3000 ppm groups respectively. There was clearly no evidence of an effect the test substance on the number of whole litter losses.
When whole litter losses are included, the percentage of pups live born was at least 94% in all groups and both generations and was not affected by administration of the test substance.
There was no effect of treatment with the test substance on post implantation loss. The number of implants and the number of live + dead pups was lower than control in the 3000 ppm group for the F1 females. The mean values obtained, were well within the range of historical control mean values, therefore, the differences are considered to reflect normal variation and are unrelated to treatment with the test substance.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other: Mean dietary equivalent of 9.3 mg/kg bw/day

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

> 3 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Mean dietary equivalent of 279 mg/kg bw/day

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical observations,

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of F1A pups in the 3000 ppm group were reduced from day 15 onwards, with the maximum difference from control on day 22 where unadjusted body weight values were 16 and 14 % below control in males and females respectively. There was no effect on pup body weight in the 100 or 500 ppm groups.
Total litter weights were reduced in the F1A litters in the 3000 ppm group. This difference was evident from day 1 in the F1A litters. There was no effect on total litter weight in the 100 or 500 ppm groups.

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, non-treatment-related

Description (incidence and severity):

Sexual maturation (F1): F1 males in the 3000 ppm group had a delay in preputial separation (2.3 days). This difference is considered to reflect the decreased body weight of this dose group. There was no effect on the time of preputial separation in the 100 or 500 ppm groups.
F1 females in the 3000 ppm group had a delay in the time of vaginal opening (2 days). This difference is considered to reflect the decreased body weight of this dose group. There was no effect on the time of vaginal opening in the 100 or 500 ppm groups.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were increased in F1A pups in the 500 and 3000 ppm groups. Thymus weights adjusted for bodyweight were statistically significantly higher than control in F1A females in all treatment groups. However all mean thymus weights were within the historical control mean range and therefore these differences are considered not to be related to administration of the test substance. The weights of a number of other organs were statistically significantly lower in the 3000 ppm group than in the control group in both generations but the difference was no longer evident after adjustment for the lower body weight of the treated rats. These were spleen (F1A) and thymus (F1A males). All these differences are considered to be unrelated to treatment with the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings observed in F1A pups which could be related to treatment with the test substance.

Histopathological findings:

not examined

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

Generation:

F1

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Mean dietary equivalent of 9.3 mg/kg bw/day

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of F2A pups in the 3000 ppm group were reduced from day 15 onwards, with the with the maximum difference from control on day 22 where unadjusted body weight values were 18 and 21 % below control in males and females respectively. There was no effect on pup body weight in the 100 or 500 ppm groups.
Total litter weights were reduced in F2A litters in the 3000 ppm group. This difference was evident from day 8 in the F2A litters. There was no effect on total litter weight in the 100 or 500 ppm 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

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no effects on anogenital distance in the F2A pups.

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Liver weights were increased in F F2A pups in the 500 and 3000 ppm groups. The weights of a number of other organs were statistically significantly lower in the 3000 ppm group than in the control group in both generations but the difference was no longer evident after adjustment for the lower body weight of the treated rats. These were brain (F2A only), spleen (F2A) and thymus (F2A males and females). All these differences are considered to be unrelated to treatment with the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings observed in F2A pups which could be related to treatment with the test substance.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

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

Generation:

F2

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Mean dietary equivalent of 9.3 mg/kg bw/day

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

HOMOGENEITY AND STABILITY OF THE TEST SUBSTANCE

The homogeneity of the test substance in diet preparations at concentrations of 100 ppm and 3000 ppm, (for a batch size of 40 kg), was determined and considered satisfactory, percentage deviations from the overall mean were within 2 %.

The reanalysis of the test substance in diet preparations at concentrations of 50 ppm, 100 ppm and 3000 ppm when stored at room temperature was shown to be satisfactory for 43 days, covering the period of dosing. The reanalysis of the test substance in diet preparations at concentrations of 50 ppm, 100 ppm and 3000 ppm when stored in a freezer was shown to be satisfactory for 43 days.

TEST SUBSTANCE INTAKE

Dose rates (based on nominal dietary levels of test substance) were calculated in terms of mg test substance/kg bodyweight/day. As expected these were at a maximum in the early phase of the pre-mating period in each generation and declined during the period of rapid growth. For more details see Table 1.

Table 1 Overall mean dose received (mg/kg/day)

Dietary concentration test substance (ppm)
	100	500	3000	100	500	3000
	Males			Females
F0 parents pre-mating	8.3	41.2	250.1	9.3	46.6	276.6
F1 parents pre-mating	9.5	47.8	288.5	10.2	50.1	301.3
F0 females during gestation				7.4	37.2	217.4
F1 females during gestation				8.1	40.6	239.1
F0 females post partum				25.2	118.9	699.6
F1 females post partum				24.4	129.1	774.0

Note: Food consumption post partum includes food consumed by the dam and pups, when the pups start to consume diet.

Table 2. Body weights (g) - selected findings (adjusted findings after week 1) - F0 and F1

Generation/Sex	Dietary Concentration of test substance (ppm)
	0		100		500		3000
Premating	Males	Females	Males	Females	Males	Females	Males	Females
F0 generation: week 1	121.5	97.6	118.3	97.7	118.6	96.9	117.6	97.8
Week 2	158.7	121.4	159.2	121.0	159.5	121.0	153.5**	119.4*
Week 4	240.4	156.7	237.8	157.1	236.6	153.7	224.6**	150.0**
Week 6	294.4	181.4	288.7	181.8	287.3	178.6	271.4**	170.1**
Week 9	339.2	207.0	334.6	206.4	329.7	201.5*	310.1**	189.2**
Week 11	362.8	216.3	357.5	215.6	352.4	211.1	331.7**	198.5**
F1 generation: week 1	78.1	72.1	75.5	69.6	74.9	70.2	66.8**	64.5**
Week 2	113.4	101.5	113.6	100.9	113.8	99.5*	110.8	100.1
Week 4	196.3	144.0	198.9	143.1	195.3	136.9**	188.1*	141.5
Week 6	263.1	173.5	267.7	171.3	262.4	162.9**	251.0*	167.8
Week 9	323.7	208.7	330.1	203.3	320.9	191.1**	309.8	194.8**
Week 11	351.3	218.7	359.8	215.5	346.4	202.0**	334.4*	205.4**
Gestation	Females
F0 generation: Litter A day 8	232.5		231.6		232.1		228.5**
day 22	301		307.0*		301.4		293.0*
F1 generation: Litter A day 8	235		235.3		234.3		230.5**
day 22	235		308.8		303.6		291.6**
Post Partum	Females
F0 generation: Litter A day 1	228.0		227.2		219.1		207.4**
day 15	277.0		279.6		271.9		250.2**
day 22	273.9		276.7		273.2		255.8**
F1 generation: Litter A day 1	230.8		230		210.8**		202.3**
day 15	281.2		279.3		273.5		253.4**
day 22	280.6		280.5		275.9		257.8**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 3: Food consumption (g/rat/day)/food utilisation (g growth/100g food eaten) - selected findings

Generation/Sex	Dietary Concentration of test substance (ppm)
	0		100		500		3000
Premating	Males	Females	Males	Females	Males	Females	Males	Females
F0 generation food consumption
Week 1	17.3	13.6	17.0	13.6	17.1	13.5	16.0**	13.4
Week 3	22.1	15.3	21.3	15.3	21.2	14.6*	20.0**	14.3**
Week 8	22.3	16.5	21.6	16.3	21.2*	15.8	20.2**	14.8**
Week 10	21.3	15.9	20.9	15.7	20.3*	15.4	20.0**	14.2**
F0 generation food utilisation
Weeks 1 - 4	26.43	17.24	26.18	17.56	26.12	17.15	24.77**	15.74**
Weeks 5 - 8	10.85	8.19	11.01	7.84	10.74	7.69	10.13	6.88**
Overall (Weeks 1 - 10)	16.22	10.78	16.21	10.77	16.10	10.61	15.27**	9.90**
F1 generation food consumption
Week 1	14.5	12.8	13.9	12.2	13.8	11.8**	12.4**	11.7**
Week 2	18.4	14.8	17.9	14.3	17.5	13.8**	15.5**	13.4**
Week 5	24.1	16.7	23.7	16.3	23.2	15.1**	20.7**	14.8**
Week 9	23.6	18.2	23.5	17.7	23.2	16.6**	20.8**	16.0**
Week 10	23.0	17.4	22.9	17.3	22.5	16.1*	20.5**	15.2**
F1 generation food utilisation
Weeks 1 - 4	30.85	22.30	31.53	22.38	31.05	21.73	30.97	22.85
Weeks 5 - 8	13.65	10.00	13.92	9.35	13.57	8.95**	13.68	9.08*
Overall (Weeks 1 - 10)	18.62	12.96	19.09*	13.04	18.52	12.63	18.61	13.21
Gestation F0 generation: Litter A food consumption week 2 week 3 F1 generation: Litter A food consumption week 2 week 3	Females
	20.2 17.4 23.0 19.6		20.2 17.6 21.5 19.4		19.6 17.2 21.9 17.7		17.1** 16.8 19.7* 18.1
Post Partum F0 generation: Litter A food consumption week 3 F1 generation: Litter A food consumption week 3	Females
	69.2 73.7		69.5 72.1		65.4 69.9		58.4** 625*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 4 Selected organ weights (g)

Generation/Sex	Dietary Concentration of test substance (ppm)
	0		100		500		3000
Organ Liver:  F0    - absolute - adjusted for body weight F1     - absolute - adjusted for body weight	M	F	M	F	M	F	M	F
	13.9 13.4 12.9 12.2	14.2 13.6 14.6 13.6	13.2 13.0 13.0 12.5	14.7 14.0 15.6 14.9*	13.6 13.6 13.5 13.4**	14.8 14.7* 15.2 15.6**	15.1** 15.8** 14.3** 15.5**	16.4** 17.6** 16.6** 17.8**
Ovaries: F0    - absolute - adjusted for body weight F1   - absolute - adjusted for body weight	- - - -	0.095 0.093 0.107 0.105	- - - -	0.105 0.102 0.101 0.099	- - - -	0.105 0.105* 0.101 0.102	- - - -	0.077** 0.081* 0.074** 0.077**
Uterus with cervix: F0 - absolute - adjusted for body weight  F1 - absolute - adjusted for body weight	- - - -	0.550 0.560 0.599 0.610	- - - -	0.527 0.527 0.650 0.659	- - - -	0.520 0.512 0.637 0.631	- - - -	0.427** 0.407** 0.389** 0.372**
Kidneys: F0     - absolute - adjusted for body weight  F1     - absolute - adjusted for body weight	2.42 2.35 2.39 2.28	1.96 1.91 1.96 1.88	2.45 2.42 2.39 2.31	2.02 1.96 2.05 1.99*	2.36 2.36 2.40 2.37	1.97 1.96 1.95 1.98*	2.29* 2.39 2.23* 2.43**	1.93 2.02 1.97 2.07**
Adrenals: F0     - absolute - adjusted for body weight  F1     - absolute - adjusted for body weight	0.061 0.060 0.067 0.067	0.085 0.084 0.091 0.085	0.057 0.056 0.063 0.062	0.089 0.086 0.095 0.090	0.058 0.058 0.065 0.065	0.087 0.087 0.095 0.097**	0.063 0.063 0.066 0.067	0.091 0.094 0.087 0.095*
Seminal vesicles: F0     - absolute - adjusted for bodyweight F1     - absolute - adjusted for body weight	1.85 1.81 1.86 1.81	- - - -	1.85 1.83 1.78 1.74	- - - -	1.76 1.76 1.79 1.78	- - - -	1.56** 1.62** 1.74 1.83	- - - -

M = male; F = female

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 5 Selected mean litter and pup weights (g) – adjusted weights after day 1 - F1 and F2

Observation	Dietary Concentration of test substance (ppm)
	0	100	3000
F1A
Mean male pup weight:
Day 1	5.8	5.4*	5.5
Day 15	25.5	26.9	23.6**
Day 29	75.8	77.3	66.9**
Mean female pup weight:
Day 1	5.5	5.1*	5.3
Day 15	25.2	26.4	22.9**
Day 29	71.3	72.2	64.1**
Mean litter weight:
Day 1	62.1	62.6	52.9**
Day 15	284.2	296.1	212.4**
Day 29	822.3	843.9	606.7**
F2A
Mean male pup weight:
Day 1	5.5	5.8*	5.4
Day 15	26.1	26.6	22.4**
Day 29	76.3	76.1	64.5**
Mean female pup weight:
Day 1	5.2	5.4*	5.1
Day 15	26.2	26.0	22.0**
Day 29	73.2	71.2	61.3**
Mean litter weight:
Day 1	64.2	60.0	57.4
Day 15	288.2	278.9	215.7**
Day 29	833.5	782.3	620.1**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 6 Selected organ weights (g) - F1 and F2

Generation/Sex	Dietary Concentration of test subtance (ppm)
	0		100		500		3000
Organ Liver:  F1A    - absolute - adjusted for bodyweight F2A     - absolute - adjusted for bodyweight	M	F	M	F	M	F	M	F
	3.98 3.76 4.03 3.84	3.76 3.61 3.64 3.38	3.86 3.76 4.08 3.77	3.65 3.57 3.92 3.58	4.17 4.08** 4.27 4.19*	3.89 3.84* 3.90 3.84**	4.35* 4.74** 4.19 4.73**	4.29** 4.54** 3.89 4.46**
Thymus:  F1A    - absolute - adjusted for bodyweight F2A     - absolute - adjusted for bodyweight	0.312 0.294 0.309 0.300	0.289 0.273 0.305 0.293	0.305 0.294 0.316 0.293	0.313 0.307* 0.323 0.306	0.311 0.308 0.293 0.288	0.317 0.308* 0.304 0.299	0.270** 0.301 0.256** 0.293	0.284 0.315* 0.252** 0.286

M = male; F = female

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 7 Intergroup comparison of pre-mating vaginal smears F0 and F1

Mean cycle length: F0 parents F1 parents No of cycles in 21 day period: F0 parents F1 parents	Dietary concentration test substance (ppm)
	0	100	500	3000
	4.56 4.10 4.19 4.00	4.21 3.96 4.19 4.31*	4.06 3.99 4.35 4.46**	4.07 4.07 4.42 4.19

Table 8 Intergroup comparison of homogenisation resistant spermatids - F0 and F1

	Dietary Concentration of test substance (ppm)			Historical control data
	0	100	500		3000	Study A, B
Millions	F0 parents F1A litter
Number sperm per g right testis	69				60**	58, 66
	F1 parents F2A litter
Number sperm per g right testis	61				55**	36, 63

Table 9 Reproductive performance - F0 and F1 Observation Dietary Concentration of test substance (ppm)   0 100 500 3000 F0 Parents Litter F1A Mean pre-coital interval (days) 3.23 2.50 2.69 3.15 Proportion of successful matings (%) 96.2 92.3 96.2 100.0 Mean gestation length (days) 22.1 22.1 22.1 22.1 Number of viable litters 26 24 25 26 F1 Parents Litter F2A Mean pre-coital interval (days) 2.42 2.56 3.80* 2.92 Proportion of successful matings (%) 92.0 100.0 92.3 100.0 Mean gestation length (days) 22.1 22.1 22.2 22.1 Number of viable litters 24 26 24 26

 

Applicant's summary and conclusion

Conclusions:

Administration of 3000 ppm test substance via the oral (dietary) route for two successive generations did not result in any effects on reproductive performance. There were no adverse effects at a dose level of 100 ppm in adults or pups. This was equivalent to a received dose of 8.3 to 10.2 mg/kg bw/day during the pre-mating period, mean value 9.3 mg/kg bw/day. The no observed adverse effect level (NOAEL) for effects on reproduction was 3000 ppm, equivalent to dietary intake of 250 to 301 mg/kg bw/day during the pre-mating period, mean value 279 mg/kg bw/day.

Executive summary:

This study was performed in accordance with OECD TG 416 and in compliance with GLP, with the purpose to investigate the effect of diets containing the substance on the propagation of two generations of the HsdRCCHan:WIST rat. The fertility and reproductive performance of each generation of parental animals and the clinical condition, survival and growth of their offspring were determined

Groups of 26 male and 26 female (F0 parents) weanling HsdRCCHan:WIST rats were fed diet containing 0 (control), 100, 500 or 3000 ppm test substance. After 10 weeks, the animals were mated and allowed to rear the ensuing F1 litters to weaning. The breeding programme was repeated with the F1 parents selected from the F1A pups to produce the F2A litters after a 10- week pre-mating period. Test diets were fed continuously throughout the study. For each parental generation the following were assessed: growth clinical condition, reproductive function, mating behaviour, conception, gestation, parturition and lactation. For pups growth and development of the pup were determined.

The number of F0 and F1 animals failing to survive to scheduled termination was very low and the incidence was unrelated to administration of test substance. There were no treatment-related clinical observations. Body weights were statistically significantly lower than control in F0 and F1 animals in the 3000 ppm group throughout the pre-mating, gestation and post partum periods. The maximum difference was 10 - 14 % below control values. Food consumption was also reduced. There were no effects on the length or pattern of oestrus cycle, pre-coital interval, gestation length or the proportion of successful matings in F0 or F1 parents. There were no effects on proportion of pups live born, whole litter losses, pup survival, anogenital distance, pup sex distribution or pup clinical observations. The number of implantations and litter size were lower than control in the 3000 ppm group during production of the F1 litter. However there was no evidence of a similar effect in the F2 litters. Therefore the differences observed in the F1 litter were considered to be unrelated to administration of test substance. F1 and F2 male and female pup body weights were reduced from day 15 onwards in the 3000 ppm group. The maximum difference from control was day 22 where values were between 14 and 21 % below control. There was no effect on pup body weight in the 100 or 500 ppm groups. F1 males and females in the 3000 ppm group had a slight delay in preputial separation (2.3 days) or vaginal opening (2 days) which was considered to reflect the lower body weights in this group. Hepatocyte hypertrophy was observed in the liver of F0 and F1 adults receiving 3000 or 500 ppm test substance but not in any animal receiving 100 pm test substance. Liver weights were increased in F0 and F1 males and females receiving 500 or 3000 ppm.

There were no adverse effects on sperm motility, epididymal sperm number or sperm morphology. The number of homogenisation resistant spermatids in the right testis of F0 and F1 males in the 3000 ppm group was lower than control, however the mean values obtained were well within the historical control mean values. Liver weights were increased in F1 and F2 pups in the 500 and 3000 ppm groups.

Dietary administration of 3000 ppm test substance for two successive generations did not result in any effects on reproductive performance. There were no microscopic changes seen in the reproductive system that could be related to the test substance administration. There was clear evidence of toxicity at a dose level of 3000 ppm seen as decreased body weights and food consumption. The liver was identified as the target organ and hepatocyte hypertrophy and increased liver weights were observed in the liver of F0 and F1 animals. Liver weights were also increased in F1 and F2 pups. At a dose level of 500 ppm body weights and food consumption were decreased in adult females, hepatocyte hypertrophy and increased liver weights were observed in the liver of F0 and F1 animals and liver weights were increased in F1 and F2 pups. There were no adverse effects at a dose level of 100 ppm in adults or pups. This was equivalent to a received dose of 8.3 to 10.2 mg/kg bw/day during the pre-mating period, mean value 9.3 mg/kg bw/day. The no observed adverse effect level (NOAEL) for effects on reproduction was 3000 ppm, the highest dose used. This was equivalent to a received dose of 250 to 301 mg/kg bw/day during the pre-mating period, mean value 279 mg/kg bw/day.