4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Jul 2016 to 28 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This reproductive toxicity study was generated to meet the data requirements of regulations not related to REACH in non-EEA countries.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

January 2001

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 - Nousan No. 8147

Version / remarks:

November 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Cas Number:

2061933-85-3

Molecular formula:

C23H19Cl2F4N3O4

IUPAC Name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) males 5 - 6 wks, females 4 - 5 wks; (F1) Day 20 of age
- Weight at study initiation: (P) Males: 195 - 269 g; Females: 125 - 159 g; (F1) Males/Females: not specified
- Fasting period before study: not specified
- Housing: The P generation animals and selected F1 generation animals were housed in groups of 3 for males and 4 for females, until pairing and for males post-pairing. For pairing, 1 male and 1 female from the same group (avoiding sibling mating for F1 animals) were housed together in grid-floor cages suspended over paper-lined trays. On confirmation of mating, the males were returned to the group cages and the mated females were housed individually and subsequently with their litter, in solid-floor cages with appropriate bedding material.
- Diet: powdered rodent diet, ad libitum
- Water: mains tap water (in bottles); ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 07 Jul 2016 To 28 Jul 2017

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: The formulated diets were prepared within the known stability period
- Mixing appropriate amounts with: The formulated diets were prepared using powdered diet; an initial premix was made at the highest concentration to be used in the study (300 ppm) by mixing a specified quantity of test substance with basal diet. A sub-sample of the premix was weighed and mixed with a further quantity of basal diet in order to obtain the required nominal concentration in the final mix. Control animals were provided with basal diet only.

Details on mating procedure:

Doses - M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- After successful mating each pregnant female was caged individually.
- Remark: During the pairing period, where both sexes shared a cage, males were given the same dietary concentration as the females. During pairing and gestation, fixed dietary concentrations were the same as those given in the last week of the pre-pairing period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HOMOGENEITY AND STABILITY OF DIET FORMULATIONS
Homogeneity and stability of test substance formulations prepared at concentrations of 5, 50 and 300 ppm, spanning those used in this study (10 to 300 ppm), were examined in an earlier formulation validation study.

HOMOGENEITY AND ACHIEVED CONCENTRATIONS OF DIET FORMULATIONS
Samples were taken from all formulations. Samples taken from the first preparation and approximately once every 2 months thereafter, and before the start of pairing and lactation periods for both generations were analysed for test substance to assess homogeneity and achieved concentrations. Duplicate samples were taken from the basal diet fed to Controls and were analysed to confirm absence of test substance.

Duration of treatment / exposure:

The test substance was administered, freely available, in the diet to P and F1 generation animals for at least 10 weeks before pairing for mating. Formulated diet continued to be freely available to males during pairing and until necropsy and to females during pairing, gestation, lactation and until necropsy.

Frequency of treatment:

Continuously

Details on study schedule:

F1 GENERATION SELECTION:
From Day 20 of age, 24 male and 24 female pups per group were randomly selected (at least 1 of each sex from each of the weaned litters, where possible) for rearing to sexual maturity. A random selection was made of those available litters and 2 pups per sex in the first instance were taken from selected litters to ensure a full complement of 24 males and 24 females per group. Pups were selected on a total randomisation basis

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.5 mg/kg bw/day

Remarks:

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

Doses / concentrations 2

Dose / conc.:

1.5 mg/kg bw/day

Remarks:

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

Doses / concentrations 3

Dose / conc.:

4 mg/kg bw/day

Remarks:

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

Doses / concentrations 4

Dose / conc.:

4 mg/kg bw/day

Remarks:

Group 5: females. For dietary equivalents, see in Table 1 in 'Any other information on results incl. tables'

Doses / concentrations 5

Dose / conc.:

12 mg/kg bw/day

Remarks:

Group 6: males

Doses / concentrations 6

Dose / conc.:

12 mg/kg bw/day

Remarks:

Group 7: females

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

The dose levels were selected on the basis of results from preceding studies. In the 90-day dietary study in the rat the high dose level of 300 ppm (average achieved dose 23.0 mg/kg/day) was considered to exceed the suitable high dose for a 2-generation reproduction study in the rat, due to excessive reductions in body weight gain and specific target organ effects. The average achieved dose of 12 mg/kg/day (dietary inclusion rate of 150 ppm) from the 90-day study was selected as the high dose level for this study. It represents a dose which is 2-fold lower than the overtly toxic dose level of 300 ppm, but allows identification of toxic effects in the principal target organs while avoiding severe toxicity, such as moribundity or death. The low dose level of 1.5 mg/kg/day was selected as this was expected to represent a clear NOAEL, and the mid-dose level of 4 mg/kg/day was selected to provide information on the dose response relationship of test substance-related effects in the target organs for toxicity.

Examinations

Parental animals: Observations and examinations:

P AND F1 GENERATION
For P generation animals, body weights and clinical signs were recorded from the day before the start of dosing. For the F1 generation animals, body weights and clinical signs were recorded from Day 21 of age and once all F1 generation animals had been selected, the recording of body weights, food intake and observations were synchronised for all the F1 animals to start on the same day and this has been reported as Day 0, Week 0 of the F1 generation.

CLINICAL OBSERVATIONS
Animals were examined twice daily for mortality and morbidity. From the start of treatment, all animals were examined daily for clinical signs of toxicity or changes in behaviour and appearance and each animal was given a detailed clinical examination once each week.

BODY WEIGHT
Male body weights were recorded 1 day before the first day of dosing, at weekly intervals throughout the study and then on the day of necropsy.
Female body weights were recorded 1 day before the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation ) and on Days 1, 4, 7, 14 and 21 of lactation and on the day of necropsy. Females that failed to litter were weighed weekly until necropsy, but these data have not been reported.

FOOD INTAKE
The amount of food consumed by each cage of males was recorded at weekly intervals during their pre-pairing and post-pairing periods. Food intake of the females was recorded weekly during the pre-pairing period and over Days 0 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 17 and 17 - 20 of gestation and over Days 1 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 17 and 17 - 21 of lactation. For non-mated females food intake was recorded weekly from the end of the pairing period and for mated, but apparently non-pregnant females, food intake was recorded weekly from Day 26 after mating.

PARTURITION OBSERVATIONS
Females were observed from Day 21 of gestation until the start of the working day when the last pregnant female was on Day 26 after mating. Following completion of parturition, pups were designated as Day 0 of age with the next day classified as Day 1 of age. Dead offspring were not removed from the litter until parturition had been completed.
Females that failed to litter were not killed and were retained until the end of the dosing period for that generation.

SEXUAL DEVELOPMENT OBSERVATIONS
All F1 generation females were examined daily from Day 25 of age for vaginal opening. All F1 generation males were examined daily from Day 35 of age for balano-preputial separation.
Body weights were recorded on the day that these indicators of sexual maturation were observed

Oestrous cyclicity (parental animals):

For 21 days before the start of the pairing periods, vaginal smears were taken daily by lavage. The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present.

Sperm parameters (parental animals):

SPERM EVALUATION
Sperm motility and concentration were assessed for all males killed at scheduled necropsy using the Hamilton Thorn IVOS Computer Assisted Sperm Analysis (CASA) system. The assessment was performed using fluid from one cauda epididymis. The cauda epididymis was weighed separately.
A sample of the epididymal fluid was retained in neutral buffered formalin, a smear was prepared for each Control and high dose male and at least 200 sperm per sample were examined for morphological abnormalities.

HOMOGENISATION RESISTANT TESTICULAR SPERMATID COUNT
After weighing of the testes, the tunica albuginea of 1 testis was removed and the testis was snap frozen in liquid nitrogen and stored at -20 °C until required. For each Control and high dose male, the testis was thawed, homogenised and the resistant spermatids counted using the CASA system.

Litter observations:

OBSERVATIONS OF LITTERING FEMALES
The females were allowed to rear their offspring to weaning on Day 21 of lactation. Abnormalities of nesting or nursing behaviour were recorded.

LITTER SIZE, SEXES AND CULLING
The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded daily from Day 1 of age. On Day 4 of age, the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 males and 4 females. Litters of fewer than 8 pups were not altered. Pups were selected on a total randomisation basis. Non-selected pups were killed using a suitable Schedule 1 method, fixed and retained for possible future examination.
Day 0 of age for the pups (equivalent to Day 0 of lactation for observations assigned to the dams) was defined as the day of completion of littering.

CLINICAL OBSERVATIONS
Animals were examined twice daily for mortality and morbidity. All pups were examined daily for malformations and clinical signs of toxicity.

ANOGENITAL DISTANCE
The anogenital distance of the F2a generation was measured on Day 1 of age.

BODY WEIGHTS
Pups were weighed individually on Days 1, 4, 7, 14 and 21 of age.

Postmortem examinations (parental animals):

NECROPSY
Females with litters were killed on Day 21 of lactation at weaning of their litters. The remaining females, including those apparently non-pregnant and those where all the litter had died before Day 21 of age, were killed after confirmation that a second mating was not required. The males were killed approximately 6 weeks after completion of the mating phase (after successful littering). All animals were killed by exposure to carbon dioxide gas in a rising concentration.
The body weight was recorded and the cranial, thoracic and abdominal cavities were opened and the major organs and uterus were examined. The number of implantation scars/sites for each female was recorded. The uterus of any apparently non-pregnant female was given a visual assessment under magnification to confirm pregnancy status. Organs or tissues showing any macroscopic abnormalities were recorded and retained. For each male, 1 epididymis was processed for sperm evaluation.

ORGAN WEIGHTS
The following organs were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together):
adrenals; pituitary; brain; prostate & seminal vesicles (including coagulating gland); epididymides; epididymis cauda(3); spleen; kidneys; testes(1); liver; thyroids (including parathyroids)(2); ovaries; uterus (including uterine cervix and oviducts).
(1) = weighed separately
(2) = weighed after fixation
(3) = only one weighed

MACROSCOPIC AND MICROSCOPIC PATHOLOGY
For all animals, with the exception of the testes, either whole organs or samples of the tissues listed below, were preserved in neutral buffered formaldehyde. The testes were fixed in Modified Davidson’s solution.
adrenal glands; prostate & seminal vesicles (including coagulating gland); brain* (7 levels); duodenum; spleen*; epididymides; testes; jejunum; thyroids* (including parathyroids); kidneys*; uterus (including uterine cervix and oviducts); liver; vagina; ovaries; all gross lesions, pituitary*
* These tissues were held in fixative and not processed further.

For all animals, the tissues specified in the tissue list were wax embedded, cut at a nominal thickness of 4 µm to 5 µm and stained with haematoxylin and eosin. For all Control and high dose animals and any premature decedents, the tissues specified in the tissue list were examined microscopically. Additionally, reproductive organs of males and females that failed to mate, non-pregnant females, males that failed to sire a pregnancy and females that failed to deliver healthy offspring, were also examined microscopically.
Following microscopic examination and external peer review, microscopic changes were detected in the duodenum and jejunum of the high dose males and females and in the liver and testes of the high dose males of the P and F1 generations. Consequently, the duodenum and jejunum from the low and intermediate dose P and F1 generation males and females and the liver and testes from the low and intermediate dose P and F1 generations males were examined microscopically.
A peer review was performed in accordance with current standard operating procedures. An external peer review was also performed by the Sponsor’s pathologist.

OVARIAN FOLLICLE EXAMINATION
Quantitative evaluation of follicles was performed for the F1 generation only. For all F1 females, the centre of the left ovary was sectioned using the butterfly technique (five sections, approximately 100 µm apart) and stained with haematoxylin and eosin for microscopic examination. The following follicles were then counted for Control, intermediate and high dose females:
- Primordial follicles and naked oocytes (Types 1 and 2 on the Pederson and Peters classification).
- Primary follicles (Type 3a on the Pederson and Peters classification).

Postmortem examinations (offspring):

NECROPSY
With the exception of pups culled on Day 4 of lactation, which were not examined further, a necropsy was conducted on all pups killed or found dead during lactation and all unselected pups on Day 21 of age. The pups were killed by an intraperitoneal injection of sodium pentobarbitone solution (for those up to the age of 14 days) or by exposure to carbon dioxide gas in a rising concentration (for older pups). Where possible, a dead body weight was recorded for pups where organ weights were to be determined. The cranial, thoracic and abdominal cavities were opened and the major organs were examined.
F1a and F2a animals were identified at necropsy; numbers were allocated as the dam number followed by a 2-digit pup identifier.

ORGAN WEIGHTS
For 1 male and 1 female pup from each litter, the following organs were weighed after trimming of fat and other contiguous tissue:
adrenals; spleen; brain; testes; epididymides; thymus; liver.

MACROSCOPIC PATHOLOGY AND MICROSCOPIC PATHOLOGY
For 1 male and 1 female pup per litter, the tissues listed below were retained. All other pups had a gross macroscopic examination and only gross abnormalities were retained.
For all animals, with the exception of the testes, either whole organs or samples of the tissues listed below were preserved in neutral buffered formaldehyde. The testes were fixed in Modified Davidson’s solution.
adrenal glands; liver; brain (including olfactory bulbs); spleen; duodenum; testes; epididymides; thymus; jejunum; all gross lesions.
Due to the age and size of early decedent pups, and to prevent damage to any tissues during removal, the affected tissues were left in situ and the whole carcass was retained in neutral buffered formaldehyde.
For all animals, the tissues specified in the tissue list were wax embedded, cut at a nominal thickness of 4 µm to 5 µm and stained with haematoxylin and eosin. In the first instance, for Control and high dose animals and any premature decedents, the tissues specified in the tissue list were examined microscopically.
Following microscopic examination and external peer review, microscopic changes were detected in the duodenum of the high dose F1a pups (males and females). Consequently, the duodenum from the low and intermediate dose F1a male and female pups were examined microscopically.
A peer review was performed in accordance with current standard operating procedures. An external peer review was also performed by the Sponsor’s pathologist.

Statistics:

Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3, 4, 5, 6 and 7 against Group 1.

Reproductive indices:

Female copulation index (%) = (no. of females mated / no. of females paired) x 100

Male copulation index (%) = (no. of males mated / no. of males paired) x 100

Female fertility index (%) = (no. of pregnant females / no. of females paired) x 100

Male fertility index (%) = (no. of males siring a pregnancy / no. of males paired) x 100

Gestation index (%) = (no. of pregnant females with live pups born / no. of pregnant females) x 100

Post-Implantation Loss (%) = ((no. of implantation scars – no. pups born) / no. of implantation scars) x 100

Offspring viability indices:

Live birth index (%) = (no. pups born alive / total no. pups born) x 100

Viability index 1 (%) = (no. pups alive on Day 4 of age before culling / no. pups born alive) x 100

Viability index 2 (%) = (no. pups alive on Day 7 of age /no. pups alive on Day 4 of age after culling) x 100

Viability index 3 (%) = (no. pups alive on Day 14 of age / no. pups alive on Day 7 of age) x 100

Viability index 4 (%) = (no. pups alive on Day 21 of age / no. pups alive on Day 14 of age) x 100

Lactation index (%) = (no. pups alive on Day 21 of age / no. pups on Day 4 of age after culling) x 100

Cumulative survival index (%) = ((no. pups alive Day 21 / no. pups alive Day 4 after culling) x (no. pups alive Day 4 before culling / no. pups born)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 6 of gestation, one female (12 mg/kg/day) was killed due to a subcutaneous mass in the mammary gland area. The pathology findings showed a mammary tumour (mammary adenocarcinoma). As this was an isolated incident, it was considered unrelated to the test substance. On Day 22 of gestation, another female(4 mg/kg/day) was killed due to dystocia.Dystocia is seen occasionally in this strain of rat and as the remaining females in this group littered successfully, this was considered not to be related to the test substance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no deaths related to the test substance. On Day 6 of gestation, one female (12 mg/kg/day) was killed due to a subcutaneous mass in the mammary gland area. The pathology findings showed a mammary tumour (mammary adenocarcinoma). As this was an isolated incident, it was considered unrelated to the test substance. On Day 22 of gestation, another female (4 mg/kg/day) was killed due to dystocia. Dystocia is seen occasionally in this strain of rat and as the remaining females in this group littered successfully, this was considered not to be related to test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In all groups given the test substance, group mean male body weights and body weight gains were slightly higher compared with Controls throughout dosing, so that over the whole period group mean body weight gain was significantly higher than Control in the groups given 4 or 12 mg/kg/day (p<0.01, p<0.05, respectively). Group mean female body weights and body weight gains during the pre-pairing period were also slightly higher compared with Controls in all groups given the test substance, so that at the end of the pre-pairing period, females in these groups had gained statistically significantly more weight than the Controls, although there was no dose-relationship (p<0.01, p<0.05, respectively).
There were no effects of the test substance on body weight gain of females during the gestation or lactation periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on food intake for males or females given test substance, when compared with Controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes attributed to the administration of the test substance were found in animals given 12 mg/kg/day. These were found in the duodenum/jejunum of males and females and in the liver and testes of males.

DUODENUM AND JEJUNUM
Epithelial vacuolation was found in the duodenum/jejunum in males and females given 12 mg/kg/day. This finding was more frequently seen in the jejunum. See 'Any other information on materials and methods incl. tables' for more details.

LIVER
Centrilobular hepatocyte vacuolation was found in a few males given 12 mg/kg/day but not in females from this dose group. A minimal degree of this finding was also present in one male from the Control group. The finding was considered to be related to test substance.See 'Any other information on materials and methods incl. tables' for more details.

TESTES
Focal degeneration/atrophy of the epithelium in the seminiferous tubules (minimal) was found in 6 of the 24 males given 12 mg/kg/day. See 'Any other information on materials and methods incl. tables' for more details.
Tubular degeneration/atrophy may be seen as a low incidence spontaneous background finding in the rat. A minimal severity of this finding is characterised by the presence of one or a small number of tubules lined only by Sertoli cells. The spectrum of all other microscopic findings was consistent with changes encountered in rats of this age kept under laboratory conditions. In particular, hypoplasia of the testes was seen in a single male given 12 mg/kg/day. This finding was considered to be spontaneous and of no toxicological importance.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of the test item on the mean number or the length of the oestrous cycles recorded during the pre-pairing period.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on sperm motility, concentration or morphology.

At 12 mg/kg/day, group mean sperm motility was slightly decreased and sperm concentration was significantly lower (p<0.05), when compared with Controls. This was mainly due to one male that was azoospermic and was found to have small testes at necropsy and hypoplasia of the testes at microscopic examination, which was considered to be a spontaneous finding. When this male was excluded from the group mean data, the slight difference from Control in sperm motility and sperm concentration were not significant and values for both parameters were within the historical control range.
The mean percentage of abnormal sperm was slightly, but not significantly, higher in males given 12 mg/kg/day compared with Controls but all values were within the normal range.
There was no effect of the test substance on group mean homogenisation resistant spermati

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

FERTILITY AND MATING PERFORMANCE:
- There was no effect of the test substance on fertility or mating performance at any dose level.
One mating pair from the group administered 12 mg/kg/day was excluded from the fertility and mating performance assessment. One female was most likely not pregnant due to hypoplasia of the testes and azoospermic sperm noted in the paired a male; these findings in the male are commonly seen spontaneously and were considered to be unrelated to dosing with the test substance. In addition, another female in the group given 12 mg/kg/day was killed on Day 6 of gestation and although no implantation scars were recorded for this female at necropsy, there is a possibility that this female was at an early implantation stage at a time of kill and could have been pregnant. Therefore, two tables have been produced including and excluding this female. The assessment of fertility and mating of the P-generation was based on 24, 24, 24 and 22 mating pairs in the Control, 1.5, 4 and 12 mg/kg/day dose groups respectively.
With the exception of 1 female in the Control group, all of the paired females mated, typically within 4 days. All mated females were pregnant with the exception of 1 female in the Control group and 3 females in the group given 12 mg/kg/day, all mated females were pregnant.

GESTATION AND PARTURITION
- There was no effect of the test substance on the mean duration of gestation or on parturition.
The day of mating for two females given 4 mg/kg/day, was missed and therefore these females were excluded from the gestation length assessment. Another female given 4 mg/kg/day had no pups in the cage on completion of parturition and therefore, it could not be confirmed whether pups were born alive or dead. All other pregnant females had live litters.
One female given 4 mg/kg/day, showed difficulties during parturition and was subsequently euthanised after giving birth to 9 live pups. Due to the isolated nature of this finding, it was considered not to be associated with the test substance.

PREGNANCY AND LITTER DATA
- There was no effect of test substance on the mean number of pups born alive.
- At 4 or 12 mg/kg/day, the mean numbers of implantations were slightly lower than Control and the incidences of post-implantation loss, slightly higher; this was largely due to 2 females in the 4 mg/kg/day group (two females) and 1 in the 12 mg/kg/day group (another female) with small numbers of implantations. These differences from Control were not statistically significant and there was no clear dose response, and were within the normal range. One female had no pups in the cage on completion of parturition and the number of pups born (alive or dead) could not be confirmed, therefore this female was excluded from pregnancy and post-implantation loss assessment.
- There was a slight decrease in the group mean live birth index at 12 mg/kg/day compared with Controls (100 %, 99.68 %, 98.77 % and 90.59 % for 0, 1.5, 4 and 12 mg/kg/day, respectively). This was largely due to the live birth index for 2 females; the difference from Control was not statistically significant but was marginally outside the historical control range.
- At 4 or 12 mg/kg/day, pup survival between Days 0 and 4 of age, shown as group mean viability index 1, was slightly reduced when compared with Control (97.70 %, 98.86 %, 93.72 % and 93.75 % for 0, 1.5, 4 and 12 mg/kg/day respectively).
- At 4 mg/kg/day this reduction was due to the single incidence of dystocia in one female, which was considered to be unrelated to test substance.
- Viability index 1 values for all groups given test substance were within the historical control range and were not statistically significant.
- There was a slight reduction in the cumulative survival index for the group given 12 mg/kg/day (94.74 %, 98.54 %, 92.42 % and 84.65 % for 0, 1.5, 4 and 12 mg/kg/day respectively), which was due to the lower live birth index and pup survival between Days 0 and 4 of age.
- Pup survival in the group given 1.5 mg/kg/day was similar to Control at all ages.
-There was no effect on the mean pup sex ratio.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

>= 12 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effect of reproductive performance was observed despite the effect on atrophy observed in the testis in parental animals

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There no deaths or clinical signs associated with the test substance. On Day 23 of gestation, one female (Control) was killed during parturition due to prolonged labour (over 12 hrs). At necropsy, large live and dead pups were found in the uterus.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In all groups given the test substance, group mean male body weights and body weight gains were slightly higher compared with Controls throughout dosing, and, at 12 mg/kg/day, the differences from the Control achieved statistical significance between Weeks 8 and 10 (p<0.05). Group mean female body weights and body weight gains during the pre-pairing period were also higher compared with Controls in groups given 4 or 12 mg/kg/day, so that over the whole pre-pairing period, females in these groups gained statistically significantly more weight than the Controls (p<0.05, p<0.01 respectively).
There were no effects of the test substance on body weight gain of females during the gestation or lactation periods.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Group mean food intake was higher than Control for all males given the test substance from Week 6 of dosing, p≤0.05 to p≤0.01, however, overall food intake was unaffected. Group mean food intake was similar to Controls for females at all doses during the pre- pairing, gestation and lactation periods

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related, increase in group mean absolute and adjusted male liver weights compared with Controls, however, only the absolute values attained statistical significance (p≤0.01) at 12 mg/kg/day; this was considered to correlate with the microscopic changes in the liver.
There was an increase in male group mean absolute and body weight adjusted adrenal weights in all groups given the test item compared with Controls; however, there was no clear dose response and the adjusted value achieved statistical significance at 4 mg/kg/day only (p≤0.05).
There was a dose-related increase in group mean absolute, adjusted and body weight-related kidney weights compared with Controls. The increase in adjusted kidney weights attained statistical significance at 4 and 12 mg/kg/day (p≤0.01). Group mean absolute and adjusted spleen weights were higher than Controls for males given 4 or 12 mg/kg/day (p≤0.05 to p≤0.01) although the increases were not dose-related. This was considered to be due to the higher mean body weights for these animals.
There was no effect of the test substance administration on female organ weights.
The increases in liver, kidney and adrenal weights were considered to be test item-related. Other statistically significant inter-group differences were considered to reflect normal biological variation rather than any effect of the test substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic findings. A variety of spontaneous macroscopic changes was noted in Controls and animals given the test item with no indication of an effect of the test substance. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The changes attributed to the administration of the test substance found in the F1 generation adults were similar to those seen in the P generation adults. In animals given 12 mg/kg/day, test item-related changes were found in the duodenum/jejunum of males and females and in the liver and testes of males.

DUODENUM AND JEJUNUM
Epithelial vacuolation was found in the duodenum/jejunum in males and females given 12 mg/kg/day.

LIVER
Centrilobular hepatocyte vacuolation was found in a few males given 12 mg/kg/day but not in females from this dose group. A minimal degree of this finding was also present in one male given 4 mg/kg/day but the change was present in a single Control male in the Parental generation and therefore this single finding was considered unrelated to the test item. The finding was consistent across both generations and was considered to be related to the test substance.

TESTES
There was an increased incidence of focal degeneration/atrophy of the epithelium in the seminiferous tubules in males given 12 mg/kg/day when compared with the Controls. When compared with the findings in the P generation adults, there were more animals affected in the F1 generation (12 rather than 6). The low incidence of this focal change in males given 4 mg/kg/day was considered not to be test item-related.
The appearance of the tubular degeneration in the high dose group in the current study was similar to the spontaneous changes, although one Control animal in this study showed a slight degree of this change rather than minimal. The spectrum of other microscopic findings was consistent with changes encountered in rats of this age kept under laboratory conditions.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No effect on sexual development was attributed to test substance administration.
At 12 mg/kg/day, the group mean day of completion of balano-preputial separation was approximately 1 day later than that of the Control males and they were slightly heavier on the day of sexual maturation. However, the age and body weight ranges were comparable with the Control ranges and these slight, non-significant, differences in group mean values are considered not to be test item-related. For females at all dose levels the day of vaginal opening was comparable with the Controls. There was no effect on sexual development for the groups given 1.5 or 4 mg/kg/day.

There was a statistically significant decrease in the group mean number of primary follicles (p<0.05) in females given 12 mg/kg/day, when compared with the Controls. This finding contributed to a statistically increased number of total small follicles (p<0.01) in this dose group. The number of follicles in the ovaries of rats given 4 mg/kg/day was similar to the Controls. Although the decreased number of primary follicles in the ovaries of the high dose females, when compared with the Controls, achieved statistical significance, the numbers were still considered to be within the normal range. It was concluded that the change was due to inter-animal variability and therefore of no toxicological significance.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of the test substance on the mean number or length of the oestrous cycles recorded during the pre-pairing period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There was no test item-related effect on sperm motility, concentration or morphology.
At 12 mg/kg/day, group mean sperm motility was marginally lower and sperm concentration was slightly lower, when compared with Controls. The mean percentage of abnormal sperm was slightly higher in males given 12 mg/kg/day compared with Controls. However, all mean values were within historical control ranges and the differences from Control did not attain statistical significance.
Mean homogenisation resistant spermatid concentration was marginally, but not significantly, lower in males given 12 mg/kg/day compared with Controls, however, the value was within the historical control range.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

FERTILITY AND MATING PERFORMANCE
There was no effect of the test substance on fertility or mating performance at any dose level.
Most of the paired females mated, typically within 4 days, with the exception of 1 female given 4 mg/kg/day and 2 females given 12 mg/kg/day which did not mate. Furthermore, one Control female, one given 1.5 mg/kg/day, and one given 4 mg/kg/day and two given 12 mg/kg/day, mated successfully but were found to be not pregnant.

GESTATION AND PARTURITION
There was no effect of the test substance on the mean duration of gestation or on parturition. All pregnant females given the test item gave birth to live litters.

PREGNANCY AND LITTER DATA
There was no effect of the test substance on the mean number of pups born alive.
At 12 mg/kg/day, the mean incidence of post-implantation loss was slightly higher than that of the Controls, largely due to one female that had only 5 implantation scars and gave birthto 2 pups resulting in a post-implantation loss incidence of 60 %. Furthermore, the slight increase in the group mean post-implantation loss at 12 mg/kg/day was not statistically significant and there was no effect on the mean number of pups born alive therefore, this was considered to be incidental to dosing with the test item.
There was a slight decrease in the live birth index at 12 mg/kg/day compared with Controls; this was considered due to the same female, which gave birth to 2 pups, 1 of which was dead resulting in a live birth index of 50 % for this female; irrespective of this female the mean live birth index was within the historical control range and therefore this effect in a single female was considered not to be test item-related.
At 12 mg/kg/day, there was a slight reduction in the mean viability index 1 compared with the Control group. Again, this was due to the same female, which gave birth to a single live pup that died before Day 4 of lactation, resulting in a 0 % viability index 1 for this female. The group mean viability index 1 at 12 mg/kg/day was within the historical control range and not significantly different to Control. Therefore, there was no effects of the test substance administration on pups viability.
There was no effect on the mean pup sex ratio.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 12 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs considered to be related to the test substance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no pup deaths considered to be related to the test substance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of the the test substance on pup body weights or body weight gains over Days 1 to 21 of age.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was an increase group mean absolute and body weight adjusted adrenal weights in males given 12 mg/kg/day compared with Controls; these difference were statistically significant (p<0.05).
At 12 mg/kg/day, there was a statistically significant increase in group mean adjusted testes weight compared with Controls (p<0.01); however, as absolute testes weights were unaffected, this was considered not to be an effect of test substance administration.
Other inter-group differences were considered to reflect normal biological variation rather than any effect of the test substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings considered to be related to the test substance.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related microscopic findings in the pups.
Epithelial vacuolation was found in the duodenum of pups from the Control and 12 mg/kg/day groups. Since this finding showed a slight increase in incidence in pups from the group given 12 mg/kg/day when compared with the Controls, the duodenum from low and intermediate group pups were examined. It was considered that the epithelial vacuolation represented small amounts of fat which was physiologically normal in this age of rat; therefore the vacuolation was not recorded as an abnormality.

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs considered to be related to the test substance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no pup deaths considered to be related to the test substance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of the the test substance on pup body weights or body weight gains over Days 1 to 21 of age.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance was unaffected by the test substance.
The mean adjusted anogenital distance for male and female pups was similar across the groups.

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was an increase in group mean absolute, adjusted and body weight-related adrenal weights in males given 12 mg/kg/day compared with Controls; absolute and adjusted differences were statistically significant (p<0.05 to p<0.01).
Group mean adjusted and body weight-related spleen weights were higher than Controls for males and females given 4 or 12 mg/kg/day, attaining statistical significance for adjusted weights in males from these groups and females from the group given 4 mg/kg/day only (p≤0.05 to p≤0.001).
At 12 mg/kg/day the test substance, there was a statistically significant increase in adjusted testes weight compared with Controls (p<0.05); however, as absolute testes weights were unaffected, this was considered not to be an effect of the test substance administration.
Other inter-group differences were considered to reflect normal biological variation rather than any effect of the test substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings considered to be related to the test substance.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings in the pups.
Small amounts of fat (epithelial vacuolation) were present in the duodenum of the Control and high dose animals. This finding was considered to be physiologically normal for this age of rat and was therefore not noted as pathology.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Diet Analysis

Diets containing the test substance were considered to have been accurately prepared and homogeneous as the mean measured concentrations of the test substance were within 10 % of their nominal values with coefficients of variation no greater than 5.1 %, fulfilling the acceptance criteria. No test substance was detected in diet given to Control animal.

Table 1: P Generation: Achieved dosages

Target Dose Level (mg/kg/day)	Achieved dosage (mg/kg/day)
	Males	Females
		Pre-pairing	Gestation	Lactation
	Mean (S.D)	Mean (S.D)	Mean (S.D)	Mean (S.D)
1.5	1.5 (0.06)	1.5 (0.08)	2.0 (0.06)	2.3 (0.60)
4	4.0 (0.13)	4.1 (0.19)	5.2 (0.17)	5.8 (1.31)
12	12.1 (0.41)	12.2 (0.52)	14.9 (0.51)	16.9 (3.77)

Table 2: P Generation: duodenum/jejunum in parental males and females given 12 mg/kg/day

		Males				Females
Group		1	2	4	6	1	3	5	7
Dose Level (mg/kg/day)		0	1.5	4	12	0	1.5	4	12
Number of rats examined		24	24	24	24	24	24	24	24
Duodenum
Vacuolation, epithelium	Minimal	0	0	0	5	0	0	0	3
	Slight	0	0	0	1	0	0	0	0
	Total	0	0	0	6	0	0	0	3
Jejunum
Vacuolation, Epithelium	Minimal	0	0	0	3	0	0	0	8
	Slight	0	0	0	6	0	0	0	4
	Total	0	0	0	9	0	0	0	12

Table 3: P Generation: liver in parental males given 12 mg/kg/day

		Males
Group		1	2	4	6
Dose Level (mg/kg/day)		0	1.5	4	12
Number of rats examined		24	24	24	24
Liver
Vacuolation, hepatocyte, centrilobular	Minimal	1	0	0	1
	Slight	0	0	0	4
	Total	1	0	0	5

Table 4: P Generation: testes in parental males

		Males
Group		1	2	4	6
Dose Level (mg/kg/day)		0	1.5	4	12
Number of rats examined		24	24	24	24
Testes
Degeneration/atrophy, tubular	Minimal	0	0	1	6
	Total	0	0	1	6

 

Table 5: Epithelial vacuolation in the duodenum/jejunum in males and females

		Males				Females
Group		1	2	4	6	1	3	5	7
Dose Level (mg/kg/day)		0	1.5	4	12	0	1.5	4	12
Number of rats examined		24	24	24	24	24	24	24	24
Duodenum
Vacuolation, epithelium	Minimal	0	0	0	5	0	0	0	3
	Slight	0	0	0	0	0	0	0	2
	Total	0	0	0	5	0	0	0	5
Jejunum
Vacuolation, Epithelium	Minimal	0	0	0	4	0	0	0	3
	Slight	0	0	0	2	0	0	0	3
	Moderate	0	0	0	0	0	0	0	1
	Total	0	0	0	6	0	0	0	7

Table 6 Centrilobular hepatocyte vacuolation was found in males given 12 mg/kg/day

		Males
Group		1	2	4	6
Dose Level (mg/kg/day)		0	1.5	4	12
Number of rats examined		24	24	24	24
Liver
Vacuolation, hepatocyte, centrilobular	Minimal	0	0	1	4
	Total	0	0	1	4

Table 7 Focal degeneration/atrophy of the epithelium in the seminiferous tubules in male testis

		Males
Group		1	2	4	6
Dose Level (mg/kg/day)		0	1.5	4	12
Number of rats examined		24	24	24	24
Testes
Degeneration/atrophy, tubular	Minimal	0	0	1	9
	Slight	1	0	1	3
	Total	1	0	2	12

Table 8 Fertility and Mating Data - P Generation - Group Mean Values

Sex: Both		Group: 1 Control 0 mg/kg/day	Group: 2 test substance 1.5 mg/kg/day	Group: 3  test substance 1.5 mg/kg/day	Group: 4  test substance 4mg/kg/day	Group: 5 test substance 4mg/kg/day
Number of Females Paired	N+ve	24	0	24	0	24
Number of Females Mated	N+ve	23	.	24	.	24
Number of Fertile Females	N+ve	22	.	24	.	24
Copulation Index Female % (#)	Mean	95.8	.	100.0	.	100.0
Female Fertility Index % (#)	Mean	95.7	.	100.0	.	100.0
Number of Males Paired	N+ve	24	24	.	24	.
Number of Males Mated	N+ve	23	24	.	24	.
Number of Fertile Males	N+ve	22	24	.	24	.
Copulation Index Male % (#)	Mean	95.8	100.0	.	100.0	.
Male Fertility Index % (#)	Mean	91.7	100.0	.	100.0	.

Sex: Both		Group: 6 test substance 12mg/kg/day	Group: 7 test substance 12mg/kg/day
Number of Females Paired	N+ve	0	23
Number of Females Mated	N+ve	.	23
Number of Fertile Females	N+ve	.	21
Copulation Index Female % (#)	Mean	.	100.0
Female Fertility Index % (#)	Mean	.	91.3
Number of Males Paired	N+ve	23	.
Number of Males Mated	N+ve	23	.
Number of Fertile Males	N+ve	21	.
Copulation Index Male % (#)	Mean	100.0	.
Male Fertility Index % (#)	Mean	91.3	.

  (#) - Dunnett(Arc SineSQRT1000)

Table 8 Fertility and Mating Data - F1 Generation - Group Mean Values

Sex: Both		Group: 1 Control 0 mg/kg/day	Group: 2 test substance 1.5 mg/kg/day	Group: 3 test substance 1.5 mg/kg/day	Group: 4 test substance 4mg/kg/day	Group: 5 test substance 4mg/kg/day
Number of Females Paired	N+ve	24	0	24	0	24
Number of Females Mated	N+ve	24	.	24	.	23
Number of Fertile Females	N+ve	23	.	23	.	22
Copulation Index Female % (#)	Mean	100.0	.	100.0	.	95.8
Female Fertility Index % (#)	Mean	95.8	.	95.8	.	95.7
Number of Males Paired	N+ve	24	24	.	24	.
Number of Males Mated	N+ve	24	24	.	23	.
Number of Fertile Males	N+ve	23	23	.	22	.
Copulation Index Male % (#)	Mean	100.0	100.0	.	95.8	.
Male Fertility Index % (#)	Mean	95.8	95.8	.	91.7	.

Sex: Both		Group: 6 test substance 12mg/kg/day	Group: 7 test substance 12mg/kg/day
Number of Females Paired	N+ve	0	24
Number of Females Mated	N+ve	.	22
Number of Fertile Females	N+ve	.	20
Copulation Index Female % (#)	Mean	.	91.7
Female Fertility Index % (#)	Mean	.	90.9
Number of Males Paired	N+ve	24	.
Number of Males Mated	N+ve	22	.
Number of Fertile Males	N+ve	20	.
Copulation Index Male % (#)	Mean	91.7	.
Male Fertility Index % (#)	Mean	83.3	.

(#) Dunnett(Arc SineSQRT1000)

Table 9 Pregnancy and Litter Data - P Generation Females and F1a Animals - Group Mean Values

Sex: Female		Group: 1 Control 0 mg/kg/day	Group: 3 test substance 1.5 mg/kg/day	Group: 5 test substance 4mg/kg/day	Group: 7 test substance 12mg/kg/day
Total No of Implantation Scars	Mean	13.0	13.0	12.1	12.1
	N	22	24	22	21
Total Pups Born (#)	Mean	11.8	11.6	10.9	10.8
	N	22	24	22	21
Post Implantation Loss % (#1)	Mean	10.3	10.5	12.0	12.3
	N	22	24	22	21
Live Pups on Day 0	Mean	11.8	11.6	10.8	10.2
	N	22	24	22	21
Live Pups on Day 1	Mean	11.7	11.5	10.7	10.0
	N	22	24	22	21
Live Pups Day 4 (Pre Cull)	Mean	11.5	11.4	10.5	9.6
	N	22	24	22	21
Live Pups on Day 7	Mean	7.5	7.6	7.4	7.1
	N	22	24	22	21

(#) - Dunnett(Square Root); (#1) - Dunnett(Arc SineSQRT1000)

Sex: Female		Group: 1 Control 0 mg/kg/day	Group: 3 test substance 1.5 mg/kg/day		Group: 5 test substance 4mg/kg/day		Group: 7 test substance 12mg/kg/day
Live Pups on Day 14	Mean	7.5	7.6		7.4		7.1
	N	22	24		22		21
Live Pups on Day 21	Mean	7.5	7.6		7.4		7.1
	N	22	24		22		21
Live Birth Index (%) (#)	Mean	100.00	99.68		99.17		90.59
	N	22	24		22		21
Viability Index 1 (%) (#)	Mean	97.70	98.86		97.98		93.75
	N	22	24		22		20
Viability Index 2 (%) (#)	Mean	96.59	100.00		99.43		98.29
	N	22	24		22		20
Viability Index 3 (%) (#)	Mean	100.00	100.00	n	100.00	n	100.00	n
	N	22	24		22		20
Viability Index 4 (%) (#)	Mean	100.00	100.00	n	100.00	n	100.00	n
	N	22	24		22		20

(#) - Dunnett(Arc SineSQRT1000): n - Inappropriate for statistics

Sex: Female		Group: 1 Control 0 mg/kg/day	Group: 3 test substance 1.5 mg/kg/day	Group: 5 test substance 4mg/kg/day	Group: 7 test substance 12mg/kg/day
Lactation Index (%) (#)	Mean	96.59	100.00	99.43	98.29
	N	22	24	22	20
Cum Survival Index % (#)	Mean	94.74	98.54	96.63	84.65
	N	22	24	22	21
% Males (#)	Mean	50.83	52.54	42.22	51.48
	N	22	24	22	20

(#) - Dunnett(Arc SineSQRT1000)

Table 10 Pregnancy and Litter Data - F1 Generation Females and F2a Animals - Group Mean Values

Sex: Female		Group: 1 Control 0 mg/kg/day	Group: 3 test substance 1.5 mg/kg/day	Group: 5 test substance 4mg/kg/day	Group: 7 test substance 12mg/kg/day
Total No of Implantation Scars	Mean	11.6	12.9	12.2	12.3
	N	23	23	22	20
Total Pups Born (#)	Mean	10.7	11.8	11.4	10.8
	N	22	23	22	20
Post Implantation Loss % (#1)	Mean	8.7	8.4	7.1	13.4
	N	22	23	22	20
Live Pups on Day 0	Mean	10.6	11.6	11.3	10.7
	N	22	23	22	20
Live Pups on Day 1	Mean	10.5	11.3	11.2	10.4
	N	22	23	22	20
Live Pups Day 4 (Pre Cull)	Mean	10.5	11.0	11.2	10.3
	N	22	23	22	20
Live Pups on Day 7	Mean	7.5	7.9	7.9	7.5
	N	22	23	22	20

  (#) - Dunnett(Square Root), (#1) - Dunnett(Arc SineSQRT1000

Sex: Female		Group: 1 Control 0 mg/kg/day	Group: 3  test substance 1.5 mg/kg/day		Group: 5  test substance 4mg/kg/day		Group: 7 test substance 12mg/kg/day
Live Pups on Day 14	Mean	7.5	7.9		7.8		7.5
	N	22	23		22		20
Live Pups on Day 21	Mean	7.5	7.9		7.8		7.5
	N	22	23		22		20
Live Birth Index (%) (#)	Mean	98.86	98.84		99.30		96.53
	N	22	23		22		20
Viability Index 1 (%) (#)	Mean	97.73	95.74		98.94		92.71
	N	22	23		22		20
Viability Index 2 (%) (#)	Mean	99.43	98.91		100.00		100.00
	N	22	23		22		19
Viability Index 3 (%) (#)	Mean	100.00	100.00		98.86		100.00
	N	22	23		22		19
Viability Index 4 (%) (#)	Mean	100.00	100.00	n	100.00	n	100.00	n
	N	22	23		22		19

(#) - Dunnett(Arc SineSQRT1000): n - Inappropriate for statistics

Sex: Female		Group: 1 Control 0 mg/kg/day	Group: 3 test substance 1.5 mg/kg/day	Group: 5 test substance 4mg/kg/day	Group: 7 test substance 12mg/kg/day
Lactation Index (%) (#)	Mean	99.43	98.91	98.86	100.00
	N	22	23	22	19
Cum Survival Index % (#)	Mean	96.59	93.54	97.20	96.57
	N	22	23	22	19
% Males (#)	Mean	46.92	47.85	50.70	44.41
	N	22	23	22	20

(#) - Dunnett(Arc SineSQRT1000)

Applicant's summary and conclusion

Conclusions:

Administration of the test substance, once daily, via diet at 1.5, 4 or 12 mg/kg/day to male and female Crl:WI(Han) rats for 2 successive generations was generally well tolerated, with no effect on reproductive performance, mating behaviour or conception. Based on these findings, the No-Observed-Adverse-Effect-Level (NOAEL) for effects on reproduction was considered to be 12 mg/kg/day. There were test-substance related higher liver, spleen, kidney and adrenal weights in both generations at 4 or 12 mg/kg/day. In addition, at 12 mg/kg/day there were test substance-related microscopic changes in the duodenum and jejunum of both sexes and in the liver and testes of males. Based on these findings, the NOAEL for systemic toxicity was considered to be 4 mg/kg/day

Executive summary:

This study was performed in accordance with OECD TG 416 and in compliance with GLP, with the purpose to investigate the effects of the test substance on the integrity and performance of the male and female reproductive systems, including gonadal function, the oestrous cycle, mating behaviour, conception, gestation, parturition, lactation and weaning, and the growth and development of the offspring over 2 successive generations in the rat, when administered orally via the diet. This study also assessed the potential effects of the test substance on neonatal morbidity and mortality and provided supplementary data on prenatal and postnatal developmental toxicities.

Four groups of 24 male and 24 female rats of the Crl:WI(Han) strain were dosed orally via the diet at dose levels of 0 (Vehicle), 1.5, 4 or 12 mg/kg/day test substance for 10 weeks before pairing, during pairing, gestation and lactation, and until necropsy. Four groups of 24 male and 24 female F1 generation animals were selected from the weaned P generation litters; these animals were dosed once daily orally via the diet at dose levels of 0 (Vehicle), 1.5, 4 or 12 mg/kg/day from Day 21 of age for approximately 10 weeks before pairing, during pairing, gestation and lactation and until necropsy. All animals were examined for effects on general condition, body weight and food intake. The stage of the oestrous cycle was recorded for 21 days before pairing for P and F1 parental females, and during the pairing period, vaginal smears were taken daily until sperm were found in the smear. The females were allowed to litter and rear their offspring to weaning. The day of sexual development was recorded for all selected F1 animals.

The P and F1 parental males were subjected to macroscopic necropsy once successful littering was completed. The testes and epididymides were removed and weighed and sperm evaluation was conducted. The P and F1 parental females were killed and subjected to necropsy on Day 21 of lactation. A macroscopic necropsy was performed and the number of implantation scars was recorded. For the P and F1 parental males and females, a selection of organs were weighed, fixed and examined microscopically. Unselected P generation pups and all F1 generation pups were killed on Day 21 of age. A gross macroscopic necropsy was performed on all pups and, for 1 male and 1 female pup per litter from the P and F1 generations, the brain, spleen and thymus were weighed. Homogenisation resistant testicular spermatids were counted for the P and F1 parental males and ovarian follicle evaluation was performed for the F1 generation parental females.

There were no test substance-related deaths or clinical signs in the P and F1 generations and no test substance-related clinical signs in the litters. In both generations, in all groups given the test substance, group mean male body weights and body weight gains were slightly higher compared with Controls throughout dosing. Group mean female body weights and body weight gains during the pre-pairing periods were also higher compared with Controls in all groups given the test substance in the P generation and in the groups given 4 or 12 mg/kg/day in the F1 generation. There were no effects of the test substance on body weight gain of females during the gestation or lactation periods in either generation. There was no effect of the test substance on body weight of the P or F1 generation pups. There was no effect on food intake for males or females given the test substance for both generations. There was no effect of the test substance on oestrous cycling, fertility and mating performance or on gestation length for either generation at any dose level. At 12 mg/kg/day there was a slight decrease in the live birth index in the P generation due to an increased incidence of post implantation loss, however, in the absence of a corroborative effect in the live birth index of the F1 generation, this is not considered related to test substance administration. There was no effect of the test substance on the mean number of pups born alive and no effect on the sex ratio.

In the P generation, at 12 mg/kg/day, pup survival between Days 0 and 4 of age was lower than Controls, which was marginally outside of historical control range. However, there was no effect on pup survival in the F1 generation. Therefore, the effects on pup viability were not considered attributable to test substance administration. There was no effect of the test substance on the sexual maturation or the number of ovarian follicles in the F1 generation and there was no effect on anogenital distance of the F2a animals. There was no test substance-related effect on sperm motility, concentration or morphology. Mean homogenisation resistant spermatid concentration was comparable with Controls in both generations. For the males in both generations, there was a dose related increase in group mean absolute and adjusted liver, adrenal, kidney and spleen weights at 4 or 12 mg/kg/day compared with Control. For the F1a animals at 12 mg/kg/day and F2a animals at 4 and 12 mg/kg/day, mean absolute and adjusted adrenal weights were higher than Controls. For the F2a males mean absolute, adjusted and body weight-related spleen weights were higher than Controls at 4 or12 mg/kg/day.

There were no test substance-related macroscopic findings.In the P and F1 generation animals given 12 mg/kg/day, test substance-related changes were found in the liver of males (centrilobular hepatocyte vacuolation), duodenum and jejunum of males and females (epithelial vacuolation) and in testes of males (tubular degeneration/atrophy).There were no test substance-related microscopic findings in the F1a or F2a animals.

Administration of the test substance, once daily, via diet at 1.5, 4 or 12 mg/kg/day to male and female Crl:WI(Han) rats for 2 successive generations was generally well tolerated, with no effect on reproductive performance, mating behaviour or conception. Based on these findings, the No-Observed-Adverse-Effect-Level (NOAEL) for effects on reproduction was considered to be 12 mg/kg/day. There were test-substance related higher liver, spleen, kidney and adrenal weights in both generations at 4 or 12 mg/kg/day. In addition, at 12 mg/kg/day there were test substance-related microscopic changes in the duodenum and jejunum of both sexes and in the liver and testes of males. Based on these findings, the NOAEL for systemic toxicity was considered to be 4 mg/kg/day.