Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 February 2010 to 1 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
- Name of test material (as cited in study report): Propylidynetrimethyl trimethacrylate
- Physical state: colourless liquid
- Storage condition of test material: in a sealed container, at room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 301 to 348 g for males and 209 to 240 g for females
- Housing: up to 5 during pre-mating for all animals and after mating for males, individually with litter for females during gestation and littering.
- Diet (e.g. ad libitum): standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Toxicity subgroup animals.
- Water (e.g. ad libitum): potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: From: 10 February 2010 To: 12 April 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: approximately 50% of the final volume of corn oil was added to the required amount of test material. The formulation was mixed using a magnetic stirrer until all of the test material had dissolved and more corn oil was added to make up the required volume. The formulation was then mixed using a magnetic stirrer until homogeneous.
Initially all formulations were prepared freshly on the day of use and used within two hours of completion of preparation. However, following confirmation of the results from a homogeneity and stability study (Huntingdon Life Sciences Study No. RAJ0008) formulations were prepared freshly each week and stored refrigerated (approximately 4°C) until required for use.

VEHICLE
- Concentration in vehicle: 20, 60 and 180 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last weeks of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two assays from each group were analysed. The mean concentrations of CAS 3290-92-4 in test formulations analysed for the study were within applied limits, +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
Five consecutive weeks for Toxicity subgroup males and females and Reproductive subgroup males. Reproductive subgroup females were treated daily for two weeks prior to pairing, throughout pairing and up until the day prior to termination on Day 7 of lactation. Offspring were not dosed.
Frequency of treatment:
Once a day, 7 days a week
Details on study schedule:
None
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 900 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Toxicity subgroup: 5 males and 5 females/dose
Reproductive subgroup: 5 males and 10 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in a preliminary study (Huntingdon Life Sciences Study Number: RAJ0003), dose levels of 80, 250, 750 and 1000 mg/kg bw/day were employed and there was a suggestion of reduced bodyweight gain among males and females receiving 1000 mg/kg bw/day, and liver and kidney weights were high in all groups. There was no evidence of an effect of treatment on clinical condition, food consumption or oestrous cycles at any dose level investigated, and no treatment-related macroscopic abnormalities were observed. For the current study, treatment would continue for approximately 5 weeks and females would be pregnant, therefore, in recognition of the potential for reduced bodyweight gain, the high dose level was set at 900 mg/kg bw/day, the low dose of 100 mg/kg bw/day was chosen as an anticipated No Observed Adverse Effect Level, and the intermediate dose level of 300 mg/kg bw/day at a logarithmic interval between the low and high doses.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded in relation to dose administration. For the Toxicity subgroup animals and for Reproductive subgroup males these were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For Reproductive subgroup females these were recorded daily during the first week of treatment, twice weekly during Week 2 of treatment, on Days 0, 4, 7, 11, 14 and 20 after mating and Day 4 of lactation.

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal (physical condition and behaviour during handling with particular attention to possible signs of neurotoxicity). For the Reproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.

BODY WEIGHT:
Toxicity subgroup males and females and Reproductive subgroup males were weighed weekly throughout the study. Reproductive subgroup females were weighed weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded weekly for all animals for the first two weeks of treatment. Food consumption continued to be recorded weekly for the remaining three weeks of treatment for Toxicity subgroup females. Food consumption was not recorded during Week 3 of treatment for Toxicity subgroup males or Reproductive subgroup males as the animals were in pairing with the Reproductive subgroup females during this period. Food consumption was recorded, however, during Weeks 4 and 5 of treatment for these males.
For each Reproductive subgroup female, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-6, 7-13 and 14-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation.

OTHER:
- SENSORY REACTIVITY:
Sensory reactivity and grip strength assessments were performed (before dosing) on all Toxicity subgroup animals during Week 5 of treatment. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle reflex, tail pinch response and grip strength.

- MOTOR ACTIVITY:
During Week 5 of treatment (before dosing), the motor activity of each Toxicity subgroup animal was measured using a Rodent Activity Monitoring System (Version 2.0.3). Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).

- HAEMATOLOGY:
During Week 5 of treatment (after sensory reactivity, grip strength and motor activity assessments), blood samples were obtained from all Toxicity subgroup animals after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein. The following were measured using a Bayer Advia 120 haematology analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)) and Platelet count (Plt). The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded. Prothrombin time (PT) (using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent) and Activated partial thromboplastin time (APTT) (using an ACL 3000 Plus Analyser and IL APTT reagent) were also measured.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for peripheral haematology, the plasma was examined using a Roche P Modular Analyser for: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile Acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb) (by chemical assay).
Oestrous cyclicity (parental animals):
For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all Reproductive subgroup females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
No data
Litter observations:
PARAMETERS EXAMINED:
All litters were examined at approximately 24 hours after birth and then daily thereafter for clinical signs (evidence of ill health or reaction to treatment), litter size (mortality and consequent changes in litter size from Days 1-7 of age), sex ratio of each litter (recorded on Days 1, 4 and 7 of age) and individual bodyweight (recorded on Days 1, 4 and 7 of age).

GROSS EXAMINATION OF PUPS:
All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined.
Postmortem examinations (parental animals):
SACRIFICE:
Toxicity subgroup males and females and Reproductive subgroup males were killed in Week 6 after completion of the Week 5 investigations.
Reproductive subgroup females were killed on Day 7 of lactation.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For Reproductive subgroup females, the number of uterine implantation sites was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 1 were weighed.
In the Toxicity subgroup, the following organs were fixed for histopathology: Adrenal glands, Peyer’s patch, Brain, Pituitary, Caecum, Prostate, Colon, Rectum, Duodenum, Sciatic nerves, Epididymides (L&R), Seminal vesicles and coagulation gland, Heart, Spinal cord, Ileum, Spleen, Jejunum, Sternum with marrow, Kidneys, Stomach, Liver, Testes (L&R), Lungs, Thymus, Lymph nodes (left axillary and mesenteric), Thyroid with parathyroids, Trachea, Mammary area (caudal), Urinary bladder, Oesophagus, Uterus with cervix and oviducts, Ovaries (L&R) and Vagina.
In the Reproductive subgroup, the following organs were fixed for histopathology: Mammary area (caudal), Testes (L&R), Ovaries (L&R), Uterus with cervix and oviducts, Pituitary, Vagina and Prostate. Samples of any abnormal tissues were also retained and processed for examination.
All tissues preserved for examination were examined for all Toxicity subgroup and Reproductive subgroup animals of Control group and 900 mg/kg bw/day group. Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals.
Postmortem examinations (offspring):
All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were also examined.
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, litter size and survival indices and clinical pathology data: 1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. 2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.
For gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.
Reproductive indices:
Percentage mating : Number animals mating / Animals paired × 100
Conception rate (%) : Number animals achieving pregnancy / Animals mated × 100
Fertility index (%) : Number animals achieving pregnancy / Animals pairing × 100
Offspring viability indices:
Gestation index (%) : Number of live litters born / Number pregnant × 100
Post - implantation survival index (%) : Total number offspring born / Total number uterine implantation sites × 100
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Lactation index (%) : Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One Reproductive subgroup female receiving 100 mg/kg/day was killed for welfare reasons on Day 22 of gestation due to dystocia. This female had shown evidence of parturition for approximately 4 hours in the absence of any pups being born, and showed signs of underactive behaviour, pallor, piloerection, fast respiration and blood discharge from the vagina immediately prior to despatch to necropsy. Macroscopic examination revealed twelve live and three dead fetuses in utero, and dark contents in the lower gastro-intestinal tract. In the absence of similar instances of dystocia, the demise of this female in the lowest dose group was considered incidental and unrelated to treatment.
One Reproductive subgroup Control female was killed for welfare reasons prior to dose administration on Day 4 of lactation; as a consequence the litter was also sacrificed. The female showed signs of underactive behaviour, pallor, dark eyes and gasping respiration immediately prior to despatch to necropsy. Macroscopic examination revealed clear fluid in the thoracic cavity with pale adhesions attached to all surfaces, dark areas on the lungs and trauma to the trachea; these findings would suggest that this animal had been mis-dosed at some point in the previous few days.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Among males receiving 900 mg/kg bw/day, mean bodyweight gain was slightly lower than Control during the two week pre-pairing period, with the difference in weight gain between Week 1 and Week 2 of treatment attaining statistical significance. Thereafter, mean weight gain was essentially similar to Control and overall gain during the treatment period (Week 0 to Week 5) was 92% of Control. The mean bodyweight gain of males receiving 300 or 100 mg/kg bw/day was considered unaffected by treatment.
Among females receiving 900 mg/kg bw/day, mean bodyweight gain was statistically significantly lower than Control during the first week of treatment. Thereafter, mean weight gain was similar or superior to Control, such that overall gain during the treatment period for the Toxicity subgroup females was 114% of Control.
For the Reproductive subgroup females at 900 mg/kg bw/day, mean bodyweight at pairing was slightly low, setting the baseline at the start of gestation. There was no effect of treatment on the mean bodyweight gain of Reproductive subgroup females during gestation or lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food intake of males and females in all dose groups was considered unaffected by treatment throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of haematological parameters for Toxicity phase animals during Week 5 of treatment revealed some minor inter-group differences but no clear evidence of an adverse effect of treatment.
Among females receiving 900 mg/kg bw/day, mean cell haemoglobin concentration was slightly lower than Control and monocyte concentrations were slightly higher than Control, with differences attaining statistical significance. In the absence of any other differences in the erythrocyte or leucocyte parameters, these minor differences were considered to be of no toxicological importance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
During Week 5, slightly high plasma potassium and albumin concentrations were apparent in Toxicity subgroup males receiving 900 mg/kg bw/day, slightly high phosphorous plasma concentration in males receiving 300 or 900 mg/kg bw/day and slightly low sodium among females at 900 mg/kg bw/day. In view of the differences in response between the sexes, the toxicological significance of these minor differences is uncertain.
All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
SENSORY REACTIVITY OBSERVATIONS AND GRIP STRENGTH: Sensory reactivity observations and grip strength values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.

MOTOR ACTIVITY: Motor activity scores for Toxicity subgroup males and females were considered to be unaffected by treatment.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The microscopic examination performed after five weeks of treatment revealed no test substance-related lesions. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.
The incidence and distribution of all findings were consistent with the commonly seen background of microscopic changes.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Oestrous cycle length, pre-coital interval and mating performance and fertility of the Reproductive subgroup females was unaffected by treatment at all dose levels investigated. All animals mated at the first oestrus opportunity after cohabitation. There was a suggestion of a minor shift towards a slightly longer gestation length among Reproductive subgroup females receiving 900 mg/kg bw/day, with no females having a 22-day gestation length compared with 3, 5 and 4 females at 0, 100 and 300 mg/kg bw/day, respectively. The incidence of females with a 23.5 day gestation length was marginally outside the Historical control data range. The gestation index was 100% in all groups, with the exception of the 100 mg/kg bw/day group, where one female was killed for welfare reasons on Day 22 of gestation due to dystocia.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

LITTER SIZE, SEX RATIO AND SURVIVAL INDICES: The mean number of implantation sites, and consequently mean litter size, was lower than Control among Reproductive subgroup females receiving 900 mg/kg bw/day, with 4/10 females having less than 14 implantations compared with 0/10 in the concurrent Control group; mean values were slightly outside the Historical control data range. This statistically significant reduction in the mean number of implantation sites is associated with low litter size were observed at the high dose (900 mg/kg bw/d). In this screening study, the number of corpora lutea present for each animal was not determined. Thus, it cannot be assessed whether this is a spontaneous finding related to a reduction in the number of eggs available for fertilization or indicative of a treatment-related pre-implantation loss.

There was no effect on pre- or post-natal survival at any dose level, and the percentage of males in the litters in all groups was consistent between Days 1 and 7 of age, indicating that there was no preferential mortality of either sex.

CLINICAL SIGNS (OFFSPRING): There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.

BODY WEIGHT (OFFSPRING): At 900 mg/kg bw/day, mean male and female offspring bodyweights on Day 1 of age were higher than Control, with statistical significance achieved for females; mean absolute bodyweight remained higher than Control to Day 7 of age. Mean bodyweight gain of males and females from birth to Day 4 of age was statistically higher than concurrent Control. These differences were attributed to the slightly lower litter size and slight shift in gestation length observed in this group.
Offspring bodyweight and bodyweight gain were unaffected by maternal treatment at 100 and 300 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING): The type and distribution of findings at necropsy of the offspring that survived to scheduled termination on Day 7 of age did not suggest any adverse effect of treatment of the Reproductive subgroup females.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.
Executive summary:

In a GLP study conducted according to OECD guideline 422, three groups each comprising five male and five female rats for the Toxicity subgroup and five male and ten female rats for the Reproductive subgroup received propylidynetrimethyl trimethacrylate at doses of 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and Reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume-dose.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

There were two mortalities during the course of the study. One Reproductive subgroup female receiving 100 mg/kg bw/day was killed for welfare reasons on Day 22 of gestation due to dystocia. In addition, one Reproductive subgroup Control female (and consequently the litter) was killed for welfare reasons prior to dose administration on Day 4 of lactation. These isolated incidents were considered unrelated to treatment.

There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was also no effect of treatment on the food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study.

At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than Control during the two week pre-pairing period; females in this dose group showed similarly low weight gain during the first week of treatment only. Thereafter, for both sexes mean weight gain was essentially similar to Control, and the bodyweight gain of females during gestation and lactation was unaffected by treatment.

Oestrous cycle length, pre-coital interval and mating performance and fertility of the Reproductive subgroup females was unaffected by treatment. There was a suggestion of a minor shift towards a slightly longer gestation length among Reproductive subgroup females receiving 900 mg/kg bw/day.

Among the Toxicity subgroup animals, liver weights were slightly high in males and females receiving 900 mg/kg bw/day, and kidney weights were also high among females in this dose group. There were no associated haematological or biochemical changes, or macroscopic/microscopic abnormalities to explain the difference in the weight of the organs.

There were no macroscopic abnormalities and no test substance-related lesions at microscopic examination.

There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.

A statistically significant reduction in the mean number of implantation sites, associated with low litter size were observed at the high dose (900 mg/kg bw/d). In this screening study, the number of corpora lutea present for each animal was not determined. Thus, it cannot be assessed whether this is a spontaneous finding related to a reduction in the number of eggs available for fertilization or indicative of a treatment-related pre-implantation loss.

There was no effect on pre- or post-natal survival and on sex ratio at any dose level. At 900 mg/kg bw/day, mean male and female offspring bodyweights on Day 1 of age were higher than Control; these differences were attributed to the slightly lower litter size and slight shift in gestation length observed in this group. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment.

Based on the results of this study, it was concluded that the NOAEL for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.