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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Oral: NOAEL for systemic and developmental toxicity = 100 ppm (equivalent to mean dietary concentration of 9.3 mg/kg bw /day); NOAEL for reproduction = 3000 ppm (equivalent to a mean value 279 mg/kg/day), male/female, rat, OECD TG 416, Milburn 2008.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Jan 2001

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Aug 1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1988

GLP compliance:

yes

Limit test:

no

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

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.

Dose / conc.:

100 ppm

Remarks:

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

Dose / conc.:

500 ppm

Remarks:

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

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..

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)

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: 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.

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

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

Key result

Critical effects observed:

no

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: 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.

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

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

 

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

297 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Guideline study OECD TG 416 performed in compliance with GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A 2-generation reproduction study was performed in accordance with OECD TG 416 and in compliance with GLP (Klimisch 1), with the purpose to investigate the effect of diets containing the substance on the propagation of two generations of the HsdRCCHan:WIST rat (Milburn 2008). 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 andpost partumperiods. 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/day during the pre-mating period, mean value 9.3 mg/kg/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/day during the pre-mating period, mean value 279 mg/kg/day.

Effects on developmental toxicity

Description of key information

- Oral: NOAEL for maternal toxicity is 20 mg/kg bw/day, the NOAEL for developmental toxicity is 20 mg/kg bw/day, rat, OECD TG 414, Pössnecker 2008.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Oct 2007 to 22 Nov 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jan 2001

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Apr 2004

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 - Nousan 8147, Teratology (2-1-18)

Version / remarks:

Nov 2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HanRcc: WIST(SPF Quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11 weeks (day 0 post coitum):
- Weight at study initiation: 192 - 231 g (day 0 post coitum):
- Housing: Animals were housed individually in Makrolon cages (type-3) with wire mesh tops and standardised granulated softwood bedding.
- Diet: Pelleted standard rat/mouse maintenance diet, ad libitum
- Water: Community tap water in water bottles, ad libitum.
- Acclimation period: Minimum 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
Humidity (%): 30 - 70
Air changes (per hr): 10 - 15
Photoperiod: 12 hours light/12 hours dark
Music: background music played at a centrally defined low volume for at
least 8 hours during the light period

IN-LIFE DATES: From: 17 Oct 2007 To: 22 Nov 2007

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% w/v aqueous CMC high viscosity

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: once a week.
- Mixing appropriate amounts with vehicle: The test item was weighed into a plastic tray on a tared precision balance. The test item was then transferred into a mortar and ground with a pestle. Then the vehicle was added (w/v) in portions to suspend the test item and clean the mortar. The preparation was transferred into a glass beaker and homogenised using an ultra-turrax before subdividing. The test item formulation was prepared once a week. The preparation was subdivided into separate aliquots for dosing each day. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
- Storage temperature of food: Dose formulations were stored refrigerated (2 - 8 °C)

VEHICLE
- Concentration in vehicle: 2, 7.5 and 20 mg/mL
- Amount of vehicle: 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY OF TEST SUBSTANCE FORMULATIONS
Samples for determination of concentration, homogeneity and stability (7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration were taken during the last week of the administration period. For homogeneity, three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into amber glass vials. For content and stability, samples were taken from the middle only. The samples were frozen (-25 °C to -15 °C) pending analysis. Analysis was performed using a method provided by the Sponsor (capillary GC).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: yes, in special automatic mating cages i.e. with synchronised timing to initiate the nightly mating period.
- M/F ratio per cage: 1/1
- Length of cohabitation: nightly mating period until evidence of copulation was observed
- Proof of pregnancy: A copulation plug was observed, and/or the daily vaginal smear was sperm positive, referred to as day 0 of gestation.

Duration of treatment / exposure:

Day 4 to 20 p.c.

Frequency of treatment:

Daily

Duration of test:

Untill Day 21 p.c.

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

Low dose. Group 2

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

Mid dose. Group 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

High dose. Group 4

No. of animals per sex per dose:

24

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels were selected based on results of a previous dose range finding toxicity study in Han Wistar rats, using dose levels of 0, 60, 125, and 250 mg/kg/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Once daily, during acclimatisation and up to day of necropsy

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Twice daily for viability / mortality

BODY WEIGHT:
- Time schedule for examinations: daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION:
- 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
Food consumption was recorded at following intervals: Days 0 - 4, 4 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day: on day 21 post coitum, the scheduled necropsy, females were sacrificed by CO2 asphyxiation and the foetuses removed by Caesarean section. The necropsy was carried out in random manner.
- Organs examined: Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of foetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Foetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). At least one half of the foetuses from each litter were fixed in Bouin's fixative (one foetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerine/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerine for preservation and storage. The skeletons were examined and all abnormalities and variations were recorded. The specimens were preserved individually in plastic bags. If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites.
Foetuses with abnormalities were photographed

Statistics:

The following statistical methods were used to analyse maternal, reproduction and skeletal examination data:
- Means and standard deviations of various data were calculated and included in the report.
- All statistical tests were two-sided.
- Statistical significance between groups was evaluated by Analysis of Variance (ANOVA). In the case where variances were non-homogeneous, appropriate transformations were applied (e.g. log, square root, or double arcsine) to stabilise the variances before the ANOVA. The Dunnett many-one t-test was then used to compare each group to control based on the error mean square in the
ANOVA.
- Fisher's exact test was applied if the variables could be dichotomisd without loss of information.
- For statistical tests on foetal data, comparisons were made between groups for number of foetuses affected and number of litters affected, for completeness. The litter was considered the proper unit of measurement for overall study evaluation.

Indices:

- From the on-line recorded reproduction data, the following parameters were calculated: Pre- and post-implantation losses, embryonic and foetal deaths, live and dead foetuses, foetuses with abnormalities and/or variations, foetal sex ratios and foetal body weights.
- For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis.
- Mean foetal weights were calculated from the individual weights on a per litter basis.

Historical control data:

Historic Control Data are provided for all reproductive and foetal findings observed in the current study. These historic control data were taken from 6 studies in the same strain of rat, evaluated by similar foetal examination criteria, in this laboratory between 2006 and 2007.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day, ventral recumbency and sedation were noted in all 24 dams between day 4 to day 9 p.c. Starting again on day 13 p.c., ventral recumbency was noted in a small number of females in this group up to day 18 p.c. Sedation and ruffled fur were noted in a few dams up day 21 p.c., the end of treatment
There were no clinical signs or symptoms noted at 20 and 75 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All female animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 75 and 200 mg/kg/day, mean body weight was statistically significantly reduced compared to controls from day 7 and day 5, respectively, through the end of study. Mean body weight gain in these groups was also statistically significantly reduced starting on day 6 and 5, respectively, again until the end of study. When corrected for gravid uterine weights, females at 200 mg/kg/day lost an average of 6 g of body weight during treatment compared to a body weight gain in controls of approximately 34 g. At 75 mg/kg/day, body weight gain corrected for gravid uterine weight was decreased approximately 41% when compared with controls.
At 20 mg/kg/day, mean body weight and mean body weight gain were not influenced by treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption at 75 and 200 mg/kg/day was statistically significantly reduced for the total treatment period (days 4 to 21 p.c.).
Mean food consumption at 20 mg/kg/day was considered to be unaffected by treatment

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:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were noted in any dam at scheduled necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on pre-implantation loss and post-implantation loss

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on the number of living foetuses.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on implantation rate.

Key result

Dose descriptor:

NOAEL

Remarks:

General systemic toxicity

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal developmental toxicity

Effect level:

> 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Calculated on litter basis, the mean body weight of foetuses was statistically significantly lower at 75 and 200 mg/kg/day.
There were no effects on mean foetal body weight at 20 mg/kg/day.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratios were considered to be not influenced by treatment with the test substance.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Variations were noted in all groups. These variations were known to be common in this strain of rat, and/or were within the range of the historical reference data. Thus, they were considered as not related to the treatment with the test substance.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- Skeletal examination: There were no treatment-related differences in the incidence of bone and cartilage abnormalities. The sporadic incidences noted (vertebra absent, cervical vertebral arch short, misshapen and / or reduced thoracolumbar vertebrae [thoracolumbar scoliosis]) were without dose-relationship and therefore considered to be of incidental nature. There were no treatment-related statistical differences in the incidence of bone and cartilage variations. The incidences of the variation “costal cartilages asymmetrically aligned at sternum” were slightly higher in the dosed groups compared to the control group, but were within the range of the historical reference data (based on litter) and thus considered to be of incidental nature. The other incidences of bone and cartilage variations were noted mostly without dose relationship, and were therefore considered to be of incidental nature.
- Bone examinations - ossification stage / supernumerary ribs: Skeletal examination (ossification stage) of foetuses did not reveal any direct test substance-related findings. Due to the statistically significantly reduced body weight of the foetuses at 75 and 200 mg/kg/day, the increased incidence of non-ossified or incompletely ossified bones were considered to be delayed development, and related to the maternal toxicity observed. At 200 mg/kg/day, statistically significantly higher incidences of a number of variations such as non-ossified vertebral bodies, incompletely ossified proximal phalanges, talus, and metatarsalia were observed. At 75 mg/kg/day, only the incidence of non-ossified cervical vertebral body 3 was statistically increased on a litter basis. All other incidences were similar in the test item treated groups and the control group and / or were within the range of historical reference data.
- Cartilage examinations: The noted incidence in common cartilage variations was dose-independent, not statistically significant in all but one finding on a litter basis, and therefore considered to be of incidental nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

During the visceral examination of foetuses, abnormalities were noted in 5 foetuses. Abnormalities observed included thoracic and abdominal situs inversus, heart interventricular septal defect, and diaphragmatic hernia. As they were noted without dose-relationship, they were considered to be of incidental nature.
Variations were noted in all groups. These variations were known to be common in this strain of rat, and/or were within the range of the historical reference data. Thus, they were considered as not related to the treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: skeletal defects

Description (incidence and severity):

Poor ossification

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Dose verification

The identity of the test substance was confirmed by the retention times of syn-isomer and anti-isomer which were similar to those measured in the working standards. The test item concentration in all samples was found to be within 86.4% and 102.9% of the nominal content. In addition, the homogenous distribution of the test item in 0.5% aqueous carboxymethylcellulose solution was demonstrated. The dosing formulations were considered to be stable for at least 7 days in the refrigerator

Table 1 Mortality and clinical signs or observations

	Test substance (mg/kg/day)
Observation	0 (control)	20	75	200
Number of females	24	24	24	24
Ventral recumbency	0	0	0	24
Sedation	0	0	0	24
Ruffled fur	0	0	0	6
Number of animals with signs	0	0	0	24
No clinical signs or observations	24	24	24	0

Table 2 Intergroup comparison of maternal body weight (g)

	Dose level test substance (mg/kg/day)
Maternal body weight	0 (control)	20	75	200
Day 0 (Pre-dosing)	212	211	211	208
Day 4 (Pre-dosing)	226	224	225	223
Day 5	229	226	227	218**
Day 7	236	231	226**	212**
Day 10	249	243	239**	221**
Day 12	257	252	247*	228**
Day 15	272	267	261**	240**
Day 20	330	323	315*	275**
Day 21	341	333	325*	282**
Maternal body weight gain on day 21	114.6	108.6	99.8*	59.5**
Weight gain on day 21 corrected for gravid uterus weight (g)	34.2	26.0	20.2**	-5.7**

*/**: Dunnett-Test based on pooled variance significant at 5 % (*) or 1 % (**) level

Table 3 Intergroup comparison of food consumption(g/rat/day)

		Dose level test substance (mg/kg/day)
Food consumption		0 (control)	20	75	200
Pre-dosing:	Days 0 - 4	21.9	21.7	21.9	22.1
During dosing	Days 4 - 6	22.9	23.4	19.4**	13.7**
	Days 6 - 9	22.9	21.5	19.4**	13.5**
	Days 9 - 12	24.7	23.8	22.2**	17.1**
	Days 12 - 15	24.6	23.5	22.3**	17.7**
	Days 15 - 18	26.3	25.3	24.0**	17.6**
	Days 18 - 21	25.2	24.6	22.3**	16.7**

*/**: Dunnett-Test based on pooled variance significant at 5 % (*) or 1 % (**) level

Table 4 Caesarean section observations for all pregnant females

Observation	Test substance (mg/kg/day)
	0 (control)	60	125	250
Animals Assigned (Mated)	24	24	24	24
Animals Pregnant	23	23	24	24
Nonpregnant	1	1	0	0
Corpora Lutea/Dam	14.4	14.8	14.4	14.2
Implantations/Dam	13.7	13.9	13.9	13.2
Gravid Uterus Weight (g)	80.4	82.6	79.6	65.2
Total Litters (viable)	23	23	24	24
Foetuses/Dam	12.4	13.1	13.1	12.2
Number of Intra-uterine deaths (litters affected)	29 (15)	18 (12)	18 (13)	25 (14)
Early deaths (Number of litters affected)	26 (13)	16 (11)	16 (11)	25 (14)
Late deaths (Number of litters affected)	3 (3)	2 (2)	2 (2)	0
Mean Foetal Weight (g)	4.8	4.7	4.5*	4.0**
Males (g)	5.0	4.8	4.7*	4.1**
Females (g)	4.7	4.6	4.4*	3.9**
Sex Ratio (% Males per litter)	47.9	51.8	46.3	47.9
Pre-implantation Loss (%)	4.8	6.2	3.5	7.0
Post-implantation Loss (%)	9.2	5.9	5.4	7.9

  * / ** : Dunnett-Test based on pooled variance significant at level 5% (*) or 1% (**)

Table 5 External/visceral examinations

	test substance (mg/kg/day)
	0 (control)	20	75	200
Number of foetuses (litters)	150 (23)	156 (23)	163 (24)	151 (24)
Number of abnormal foetuses (litters)	0	1 (1)	3 (3)	1 (1)
Number of foetuses with variations (litters)	52 (21)	55 (22)	63 (23)	76 (23)

Table 6 Skeletal examinations - bone and cartilage abnormalities and variations

	test substance (mg/kg/day)
	0 (control)	20	75	200
Number of foetuses (litters)	136 (23)	145* (23)	152* (24)	141 (24)
Number of abnormal foetuses (litters)	1 (1)	0	2 (2)	0
Number of foetuses with variations (litters)	23 (12)	31 (18)	38 (17)	34 (18)
Costal cartilages asymmetrically aligned at sternum foetuses (litters)	1 (1)	3 (3)	6 (5)	7 (5)

* One less fetus at cartilage examination.

Table 7 Bone examinations - ossification stage / supernumerary ribs

	test substance (mg/kg/day)
Selected findings	0 (control)	60	125	250
Number of litters examined	23	23	24	24
Non ossified: cervical vertebral body 1 cervical vertebral body 2 cervical vertebral body 3	7 3 3	13 7 6	12 9 10#	17## 19## 13##
Non ossified: toe 2 proximal phalanx, left toe 3 proximal phalanx left toe 4 proximal phalanx left toe 5 proximal phalanx left toe 5 proximal phalanx right	3 3 3 10 10	5 5 5 12 10	5 3 3 14 11	10# 10# 10# 21## 18#

# / ##: Fisher's Exact Test significant at level 5% (#) or 1% (##)

Conclusions:

The NOAEL for maternal toxicity was 20 mg/kg/day beased on reduced body weight food consumptions and clinical signs. The NOAEL for developmental toxicity was considered to be 20 mg/kg/day as a result of reduced body weight and ossification.

Executive summary:

The purpose of this OECD TG 414 study in compliance with GLP was to assess the effects of test substance on the pregnant rat and the embryonic and foetal development when administered orally, by gavage, once daily to mated female rats from day 4 through to day 20 post coitum. Each group consisted of 24 mated female rats. The test substance was administered at dose levels of: 0 (vehicle control, 20, 75 and 200 mg/kg/day, for respectively Group 1, 2, 3 and 4. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (0.5 % w/v aqueous CMC high viscosity). All females were sacrificed on day 21 post coitum and the foetuses were removed by Caesarean section. Examination of dams and foetuses was performed in accordance with international recommendations.

All female animals survived until scheduled necropsy. Ventral recumbency and sedation were noted in 24/24 dams, and ruffled fur in 6/24 dams at 200 mg/kg/day. There were no findings in dams at 20 or 75 mg/kg/day. Mean food consumption at 75 and 200 mg/kg/day was statistically significantly reduced. Mean body weight and mean body weight gain was statistically significantly reduced starting on day 7and day 6 at 75 mg/kg/day and on day 5 post coitum at 200 mg/kg/day. There were no treatment-related effects on pre-implantation loss, implantation rate, post-implantation loss, or the number of living foetuses. No treatment related findings were noted in any dam at scheduled necropsy. At scheduled caesarean section, no abnormal live foetus was noted at external examination at 0, 20, 75, and 200 mg/kg/day, respectively.

Sex ratios were considered not to be influenced by treatment with the test item. Mean foetal body weight was statistically significantly reduced at 75 and 200 mg/kg/day.

As the abnormalities in foetuses of test item-treated dams were noted without dose-relationship, they were considered to be of incidental nature. There were no treatment-related visceral variations. There were no treatment related bone and cartilage abnormalities noted.

Skeletal examination (ossification stage) of foetuses did not reveal any direct test item-related findings. Due to the statistically significantly reduced foetal body weight at 75 and 200 mg/kg/day, the increased incidence of non-ossified or incompletely ossified bones were considered to be delayed development, and probably related to the maternal toxicity observed.

There were no treatment-related increases in the incidences of cartilage variations in any dose group when compared with controls. In this prenatal developmental toxicity study the doses of 20, 75, and 200 mg/kg/day were administered to pregnant Han Wistar rats.

Maternal toxicity was noted at 75 and 200 mg/kg/day with statistically significantly decreased food consumption and corresponding statistically significantly decreased body weight gain. Clinical signs were only noted at 200 mg/kg/day. At 75 and 200 mg/kg/day, the only treatment related foetal effects noted were decreased body weight and delayed ossification. These effects were considered to be secondary to the maternal toxicity observed at these dose levels.

In conclusion, there was no evidence of maternal and foetal toxicity at 20 mg/kg/day, which was established as a no observed adverse effect level (NOAEL).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study OECD TG 414 performed in compliance with GLP.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Two developmental toxicity studies in rats and one in rabbits are available. The study by Pössnecker represents the study with the worst-case findings and was therefore included as key study. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

Developmental toxicity in rats (key study)

 The purpose of this OECD TG 414 study in compliance with GLP was to assess the effects of test substance on the pregnant rat and the embryonic and foetal development when administered orally, by gavage, once daily to mated female rats from day 4 through to day 20 post coitum (Pössnecker 2008). Each group consisted of 24 mated female rats. The test substance was administered at dose levels of: 0 (vehicle control, 20, 75 and 200 mg/kg/day, for respectively Group 1, 2, 3 and 4. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (0.5 % w/v aqueous CMC high viscosity). All females were sacrificed on day 21 post coitum and the foetuses were removed by Caesarean section. Examination of dams and foetuses was performed in accordance with international recommendations.

 All female animals survived until scheduled necropsy. Ventral recumbency and sedation were noted in 24/24 dams, and ruffled fur in 6/24 dams at 200 mg/kg/day. There were no findings in dams at 20 or 75 mg/kg/day. Mean food consumption at 75 and 200 mg/kg/day was statistically significantly reduced. Mean body weight and mean body weight gain was statistically significantly reduced starting on day 7and day 6 at 75 mg/kg/day and on day 5 post coitum at 200 mg/kg/day. There were no treatment-related effects on pre-implantation loss, implantation rate, post-implantation loss, or the number of living foetuses. No treatment related findings were noted in any dam at scheduled necropsy. At scheduled caesarean section, no abnormal live foetus was noted at external examination at 0, 20, 75, and 200 mg/kg/day, respectively. Sex ratios were considered not to be influenced by treatment with the test item. Mean foetal body weight was statistically significantly reduced at 75 and 200 mg/kg/day.

 As the abnormalities in foetuses of test item-treated dams were noted without dose-relationship, they were considered to be of incidental nature. There were no treatment-related visceral variations. There were no treatment related bone and cartilage abnormalities noted. Skeletal examination (ossification stage) of foetuses did not reveal any direct test item-related findings. Due to the statistically significantly reduced foetal body weight at 75 and 200 mg/kg/day, the increased incidence of non-ossified or incompletely ossified bones were considered to be delayed development, and probably related to the maternal toxicity observed. There were no treatment-related increases in the incidences of cartilage variations in any dose group when compared with controls. In this prenatal developmental toxicity study the doses of 20, 75, and 200 mg/kg/day were administered to pregnant Han Wistar rats. Maternal toxicity was noted at 75 and 200 mg/kg/day with statistically significantly decreased food consumption and corresponding statistically significantly decreased body weight gain. Clinical signs were only noted at 200 mg/kg/day. At 75 and 200 mg/kg/day, the only treatment related foetal effects noted were decreased body weight and delayed ossification. These effects were considered to be secondary to the maternal toxicity observed at these dose levels.

 In conclusion, there was no evidence of maternal and foetal toxicity at 20 mg/kg/day, which was established as a no observed effect level (NOEL).

   

 Further developmental toxicity studies in rats

 In another OECD TG 414 study, performed in compliance with GLP (Klimisch 1), groups of 24 time-mated, female HsdRccHan:WIST rats were dosed orally, by gavage, with 0 (control), 20, 75 or 250 mg test substance/kg bw/day (using 0.5 % w/v aqueous carboxymethylcellulose as a vehicle) on days 5 - 21 (inclusive) of gestation (Moxon 2007). The day of confirmation of mating was designated day 1 of gestation. The rats were terminated on day 22 of gestation.

 Oral administration of 250 mg/kg/day was associated with clear evidence of maternal toxicity; a significant reduction in body weight gain and food consumption. These effects together with clinical signs resulted in the premature termination of one of the 24 females. In addition to these maternal effects, there was an effect of 250 mg test substance/kg bw/day on post-implantation loss i.e. there was an increased number of early intra-uterine deaths and therefore a reduced number of live foetuses in this group. Mean foetal weight was also reduced in this treatment group. There was no evidence of maternal toxicity with either 75 or 20 mg test substance/kg bw/day and there was no effect on the number, growth or survival of the foetuses in utero. There was no association with test substance and foetal abnormality. There was an effect of 250 mg test substance/kg bw/day on foetal skeletal variation with clear evidence of reduced ossification. Changes in ossification are transient in nature and are considered not to represent an adverse effect of test substance on foetal development. A small number of reduced ossification centres were seen in foetuses in the 75 mg/kg/day group which were considered to be minimal and of no toxicological significance. There wasno evidence of an effecton skeletal variation at 20 mg/kg/day.

 In conclusion, there was no association with test substance and foetal abnormality. Oral administration of 250 mg test substance/kg bw/day was associated with clear evidence of maternal toxicity: a significant reduction in body weight gain and food consumption and the premature termination of one animal. In addition, this dose level was associated with an increased incidence of early intra-uterine death, a reduction in foetal weight and reduced foetal ossification.There was no evidence of maternal andfoetaltoxicity at the dose level of75 or 20 mg/kg bw/day. The maternal and developmental NOAEL was 75 mg/kg bw/day.

 

 Developmental toxicity in rabbits

 In a GLP compliant OECD TG 414 study, the test substance was administered using the vehicle, 0.5 % (w/v) aqueous medium viscosity carboxymethylcellulose (CMC), orally by gavage to 3 groups of 25 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from gestation days 7 through 28 (Sawhney 2008b). Dose levels were 30, 150 and 500 mg/kg/day administered at a dose volume of 10 mL/kg. A concurrent control group of 25 time-mated females received the vehicle (0.5 % medium viscosity CMC) on a comparable regimen. On gestation day 29, a laparohysterectomy was performed on each surviving female. Examination of dams and foetuses was performed in accordance with international recommendations.

 Maternal toxicity was manifested in the 500 mg/kg/day group by the death of 1 female on gestation day 24 following periods of body weight losses, reduced food consumption and decreased defecation. Decreased defecation was observed in a higher number of animals and at an increased incidence in the 500 mg/kg/day group when compared to the control group. Test substance-related reductions in mean food consumption were observed in the 500 mg/kg/day group during gestation days 7 - 17, with frequent statistically significant differences in the daily intervals. These reductions resulted in slightly lower (not statistically significant) mean food consumption in the 500 mg/kg/day group when the overall treatment period (gestation days 7 - 29) was evaluated. In addition, statistically significantly increased liver weights, correlated with hepatocellular hypertrophy and centrilobular hepatocellular vacuolation, were observed in the 150 and 500 mg/kg/day groups.

 Intrauterine growth and survival at 30, 150 and 500 mg/kg/day were unaffected by test substance administration. There were no treatment-related developmental variations observed in any dose group. A single incidence of microphthalmia was observed at 500 mg/kg/day, which is within the range of values in the historical control database. There were no other indications of any adverse foetal effects in the current study. However, based on the data from the range-finding study, an association between treatment and the single incidence of microphthalmia at 500 mg/kg/day in the current study cannot be excluded.

 Based on the mortality and reduced food consumption at 500 mg/kg/day and increased liver weights accompanied by hepatocellular hypertrophy and centrilobular hepatocellular vacuolation observed at 150 and 500 mg/kg/day, a dose level of 30 mg/kg/day was considered the NOAEL for maternal toxicity. Based only on the single incidence of microphthalmia observed at 500 mg/kg/day, a dose level of 150 mg/kg/day was considered the conservative estimate of a NOAEL for foetal development.

   

 Further developmental toxicity studies in rabbits

 Two dose range finding studies were performed using Himalayan rabbits. In the first dose-range finding study (Gerspach 2008a), the test substance was administered at doses of 0, 100, 200 and 400 mg/kg bw/day (10 females/group). There were no deaths, clinical signs of toxicity, or statistically significant differences in body weight or food consumption related to test substance administration. However, at 400 mg/kg/day, food consumption was sporadically decreased (~12% compared to control) from gestational days 3 to 28. There were no treatment-related macroscopic or necropsy findings. There was no effect on litter size, pre- or post- implantation loss, or foetal body weights. However, two foetuses from two litters at 400 mg/kg/day were found to have small eyes (approximately 75% of normal size), which was considered possibly related to treatment.

 In a subsequent study (Gerspach 2008b), 5 animals per group were administered the test substance via gavage at 0, 600, 800 and 1000 mg/kg/day from gestation days 4 to 27. There were no deaths, clinical signs of toxicity, or discernible differences in body weight related to administration with the test substance. Food consumption was slightly decreased at 1000 mg/kg/day. There was no effect on pre- or post- implantation loss or litter size. There was an increased incidence of “eyes – small” or “eyes – slightly small” across all treated groups, with no dose-response relationship.

 Based on the absence of historical database for intraocular findings at this testing facility, concerns arose regarding the interpretation of these findings. These concerns led to a change in contract laboratory (which meant also changing rabbit strain), with the preliminary and definitive developmental toxicity study conducted in New Zealand White rabbits.

 In the preliminary study (Sawhney 2008a), New Zealand White rabbits were administered the test substance via gavage at 0, 400, 700 or 1000 mg/kg/day from gestational day 7 through 28. Significant maternal toxicity was observed at all dose levels, manifest as marked reduced body weight and food consumption, which was of sufficient magnitude to result in moribundity and/or abortions (≥338 g body weight loss and ≤8 g/day food consumption). Increased liver weight and hypertrophy with corroborative dose-dependent effects on gamma-glutamyl transferase (GGT) and alkaline phosphatase (AP) were also noted at doses ≥400 mg/kg/day. Developmental toxicity was restricted to the 1000 mg/kg/day dose level, as evidenced by decreased foetal growth, survival, and changes in foetal morphology. Two and three foetal incidences of microphthalmia were observed in the litters of 2 dams administered 1000 mg/kg/day. The higher incidence of microphthalmia in the 1000 mg/kg/day group was considered to be related to administration of the test substance. However, the maternal toxicity evident at this dose level was considered excessive, and a top dose of 500 mg/kg/day was selected for the definitive developmental toxicity study with the test substance in New Zealand White rabbits.

 

Table 1. Collated incidence of “small eye” and microphthalmia in Himalayan and New Zealand White rabbits, respectively.

Studies | dose levels in mg/kg bw/day:	0	30	100	150	200	400	500	600	700	800	1000
DRF Himalayan rabbit (Gerspach 2008a) eye variant: slightly small	1(1)*		0		0	2(2)
DRF Himalayan rabbit dose (Gerspach 2008b) eye malformation: small, ~50-75% of normal eye variant: slightly small, ~75-<100% of normal	0 0							2(1) 9(3)		0 5(2)	5(3) 10(3)
DRF NZW rabbit (Sawhney 2008a) Microphthalmia	1(1)					0**			0**		5(2)
Main NZW rabbit (Sawhney 2008b) Microphthalmia	0	0		0			1 (1)

* Foetal incidence, with litter incidence in parentheses.

**Whilst liver toxicity was not specifically examined in the Himalayan preliminary dose-range finding studies, increased liver weight and centrilobular hepatocyte hypertrophy were noted in NZW rabbits, with a LOAEL of 150 mg/kg/day. Dark shaded cells reflect dose levels which were considered to be excessive. Light shaded cells reflect a clear demonstration of maternal toxicity.

 

 Microphthalmia observations and interpretation

 In the main OECD TG 414 study in rabbits and in three dose range finding studies (briefly reported above) incidences of small eye(s) and microphthalmia were observed at high dose levels of the test substance. 

 The studies in Himalayan rabbits reported inconsistent descriptions of small eyes, suggesting some confusion as to how to record these minor and variant findings, which were not of sufficient severity to be considered microphthalmia. Small eyes had not previously been described by the lab where the first DRF was performed and were not in the laboratory’s glossary of foetal effects. Microphthalmia was however listed as being a size reduction in excess of 50%. 

 Albinism of laboratory strains has been associated with a predisposition to microphthalmia due to the expression patterns of genes, which differ from wildtype (pigmented) animals (Searle 1990; Treisman & Land 2002; Hever et al. 2006; Manuel et al. 2008). This condition is therefore the second most common major malformation in New Zealand White rabbits, with a maximum incidence of 1.23% per litter (0.07 ± 0.2%, mean ± SD) from 2007 to 2017. Demonstrating a propensity for this abnormality, the test lab at which the main OECD TG 414 in rabbits was performed reported 20 incidences from 17 litters over the same time period (2018). The minor and variant findings reported in dose range finders performed with Himalayan rabbits were not considered in their foetal glossary, consequently, there was no historical control data available to contextualise the findings. There are significant differences in the calling criteria for microphthalmia and “small eye”.

 The induction of microphthalmia has been associated with the clinical condition of the dam (Millicovsky & DeSesso 1980a; 1980b; Clark et al. 1984; Beyer et al. 2010; Smith et al. 2013; Rutland et al. 2009). Consequently, the interpretation of microphthalmia in rabbits is not clear-cut, and it is vital to consider both the species inherent propensity for the malformation and confounders such as maternal toxicity.

 High incidences of microphthalmia or “small eyes” were only reported in preliminary studies and litter effects were evident, the number of foetuses presenting with microphthalmia clustered in a few litters, rather than being more evenly distributed (Table 1). In the definitive regulatory rabbit study, there were no occurrences of malformations outside of the laboratories historical control data range, and there were no developmental findings of relevance to hazard classification in the rat.

 A higher incidence of microphthalmia was observed in one strain of rabbit in the preliminary study (Sawhney 2008a), at a markedly maternally toxic dose level (1000 mg/kg/day). The single incidence of microphthalmia in the definitive study was within concurrent and historical control data and could therefore be plausibly considered as incidental to administration of the test substance. No major malformations were observed in the absence of maternal toxicity. As previously alluded to, the clinical condition of the dam has been associated with microphthalmia in the literature. With respect to the studies with Himalayan rabbits, it cannot be clearly stated which doses in the Himalayan rabbit studies were maternally toxic because liver parameters were not measured, so it cannot categorically be said that the minor and variant findings of “eyes of smaller than expected size” occurred at non-maternally toxic doses in this strain.

 For these reasons, the registrant considers that microphthalmia / small eyes in one species at dose levels exhibiting excessive toxicity and marked perturbations in liver function, can only constitute some evidence. The evidence is not sufficiently clear for classification as Developmental toxicant Category 1b ‘Presumed human reproductive toxicant’ (H360D). The lack of any evidence of a treatment related increase in major malformations in the definitive regulatory studies should be considered the most significant factor in the judgement that there is insufficient evidence to support classification in Category 1b. Category 2 ‘suspected of damaging the unborn child’ (H361d) is considered the only suitable remaining category.

 

References

Beyer BK, Chernoff N, Danielsson BR, et al. (2010) ILSE/HESI Maternal Toxicity Workshop Summary: Maternal toxicity and its impact on study design and data interpretation. Birth Defects Research (Part B) 92: 36-51.

Clark RL, Robertson RT, Minsker DH, et al. (1984) Diflunisal-induced maternal anaemia as a cause of teratogenicity in rabbits. Teratology 30: 319-332.

Hever AM, Williamson KA, van Heyningen V (2006) Developmental malformations of the eye: the role of PAX6, SOX2 and OTX2. Clin. Genet. 69: 459-470.

Manuel M, Pratt T, Lui M, et al. (2008) Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance. BMC Developmental Biology 8: 59.

Millicovsky G, DeSesso JM (1980a) Cardiovascular alterations in rabbit embryos in situ after a teratogenic dose of hydroxyurea: an in vivo microscopic study. Teratology 22: 115-124.

Millicovsky G, DeSesso JM (1980b) Differential embryonic cardiovascular responses to acute maternal uterine ischemia: an in vivo microscopic study of rabbit embryos with either intact or clamped umbilical cords. Teratology 22: 335-343.

Rutland CS, Jiang K, Soff GA, et al. (2009) Maternal administration of anti-angiogenic agents, TNP-470 and Angiostatis4.5, induces foetal microphthalmia. Molecular Vision 15: 1260-1269.

Searle AG (1990) Comparative genetics of albinism. Ophthalmic Paediatric Genet. 11: 159- 164

Smith F, Hu D, Young NM, et al. (2013) The effects of hypoxia on facial shape variation and disease phenotypes in chicken embryos. Disease Models & Mechanisms 6: 915-924.

Treisman J, Lang R (2002) Development and evolution of the eye: foundation des Treilles, September 2001. Mech. Dev. 112: 2-8

Justification for classification or non-classification

Based on the available data, the registrant considers classification for toxicity to reproduction as Category 2, H361D: Suspected of damaging the unborn child, appropriate in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008. However, the classification in the RAC opinion as Category 1B, H360D: May damage the unborn child, is adopted nonetheless.