Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 June 2016 to 02 September 2016
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:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous
Details on test material:
Appearance: Brown viscous liquid
Storage: Room temperature in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males animals were 69 to 75 days old. Females animals were 62 to 68 days old.
- Weight at study initiation: Males animals were 254 to 296 g. Females were 165 to 198 g.
- Housing: Animals were housed in cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. The cages were distributedon the racking to equalize as far as possible environmental influences amongst the groups.
The nuumber of animals per cage were as follows:
Pre-pairing up to five animals of one sex
Pairing one male and one female
Males after mating up to five animals
Gestation one female
Lactation one female + litter
- Diet: Ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of pairing. Spare animals were removed from the study room after treatment commenced.

WATER AND FOOD QUALITY
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 40-70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated (15 air changes per hour).
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Correction factor: A correction factor (including purity) of 1.205 was used when calculating quantities of test item used during dose preparation.
- Method of preparation: Starting with the lowest concentration, the required amount of test item was weighed into a suitable container. Vehicle was added to the mixture (to achieve approximately 50 % of the final volume) and stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous.
- Frequency of preparation: Weekly.
- Storage of formulation Refrigerated (nominally 2-8 °C).

VEHICLE
- Concentration in vehicle: Nominal concentrations of 20, 60 and 200 mg/mL and Formulated concentrations of 24.1, 72.3 and 241 mL/kg.
- Amount of vehicle: 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined.
Homogeneity and stability of the test item in the vehicle at concentrations of 2 mg/mL and 200 mg/mL was achieved following refrigerated storage (2-8 °C) for up to 15 days. Formulations were also stable at ambient temperature (nominally 21 °C) for up to two days.

- Achieved concentration: Samples of each formulation prepared for administration for the first formulation occasion and the last formulation occasion were analyzed for achieved concentration of the test item.

ANALYTICAL PROCEDURE
The samples were analyzed in accordance with a validated method. The analytical method involved extraction and dilution in propan-2-ol followed by liquid chromatographic analysis with tandem mass spectrometry detection (LC-MS/MS). Sample concentrations were determined with reference to single bracketing standards. Procedural recovery samples were prepared concurrently with samples and results were corrected for the mean recovery value at each level at analysis. Samples and standards were matrix matched by adding appropriate amounts of the initial control vehicle extract as necessary.

CONCENTRATION OF DOSE FORMULATIONS
The formulations for the First formulation occasion and the Last formulation occasion were sampled.
Formulations were sampled (4 × 1 mL, accurately weighed) from the middle of the formulation by Pharmacy personnel. Duplicate samples were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. For the First formulation occasion, for Group 2, individual results were initially >5 % from the mean results. For Group 4, the mean result was <-15 % from the nominal concentration. Investigation of the original results for Groups 2 and 4 showed that this was caused by analytical error and contingency samples had been analyzed as a precaution. Samples were disposed of once satisfactory results were achieved.

RESULTS AND CONCLUSION
The mean concentrations were within applied limits +10/-15 % of the nominal concentration, confirming the accuracy of preparation. Precision of individual samples (% difference for n=2, coefficient of variation for n=4) were within 3 % with the exception of Group 2 for the First Formulation occasion which was 5.53 %. Procedural recovery samples analyzed concurrently with the samples were within acceptance criteria (80-120 %).
Details on mating procedure:
- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks. Male/female separation occurred the day mating was detected.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- After successful mating each pregnant female was caged: Individually

Pairing commenced after a minimum of two weeks treatment. Day 0 of gestation was taken as when positive evidence of mating was detected. The pre-coital interval was calculated for each female as the time between first pairing and evidence of mating.
Duration of treatment / exposure:
- Males: Daily for a minimum of four weeks.
- Females: Daily for 15 days before pairing and until Day 6 of lactation.
- Animals of the F1 generation were not dosed.
Frequency of treatment:
Once daily at approximately the same time each day.
Duration of test:
At least 4 weeks.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the 14-day repeat-dose dose range finding study, and in the toxicity study for four weeks followed by a 2-week recovery period doses of 100, 300 and 1 000 mg/kg/day were well tolerated and did not result in any mortality or signs of systemic toxicity.
Based on this information, it was considered appropriate to investigate a high dose level of 1 000 mg/kg/day (the limit dose for this study type). The intermediate and low dose levels were selected to assess any dose-responsiveness of any test substance-related findings on this study.

- Rationale for animal assignment: On arrival and non-selective allocation to cages. On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management. The groups were adjusted to reduce inter- /intra-group variation.

- Other: Dosing was restricted to the F0 generation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk.
Administration was by oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth. Treatment was with constant doses in mg/kg/day. Individual dose volumes were calculated from the most recently recorded scheduled body weight. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A detailed physical examination was performed weekly for males, and for females weekly before pairing, on Days 0, 7, 14 and 20 after mating and Days 1 and 7 of lactation to monitor general health.
Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

- Signs Associated with Dosing
Daily during the first week of treatment, weekly thereafter and for females on Days 0, 7, 14 and 20 after mating and Days 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration:
- Pre-dose observation
- On return of animal to home cage
- At end of dosing the group
- One to two hours after completion of dosing
- As late as possible in the working day

BODY WEIGHT: Yes
The weight of animals was recorded as follows:

- F0 females:
During acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 3, 7, 10, 14, 17 and 20 after mating.
Days 1, 4, and 7 of lactation.
On the day of necropsy.
Group mean values and SD were calculated from individual body weight data on each recorded occasion. For the offspring, litter mean body weight (and SD) was calculated separately for males and females and the group mean values derived from the individual litter values.
Group mean weight changes were calculated from the weight changes of individual animals.
Offspring body weight change was calculated relative to Day 1 of age.
Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.

FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
- F0 females: Weekly before pairing (from first day of treatment until pairing) and on Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating Days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.
Group mean food consumptions and standard deviations were derived from unrounded cage values.

OTHER:
VAGINAL SMEARS
Wet smears were taken daily after pairing until mating using pipette lavage.

PARTURITION OBSERVATIONS AND GESTATION LENGTH
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

PRE-COITAL INTERVAL
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing was calculated.

SACRIFICE
- Male animals: All animals during Week 5 of respective treatment.
- Maternal animals:
Females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 7 of lactation.
- Method of Kill: Carbon dioxide asphyxiation for all adult animals.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The organs weighed, tissue samples fixed and sections examined microscopically for F0 animals are detailed in Table 1.
For females, the number of corpora lutea and uterine implantation sites were recorded for each ovary.
The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique, for females failing to produce a viable litter only.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals. Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

Histology:
Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes (initially preserved in modified Davidson’s fluid).
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- Full list: All animals in Groups 1 and 4 at scheduled termination.
- Abnormalities only: All F0 animals.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light microscopy:
Tissues preserved for examination were examined as follows:
- All adult animals in groups 1 and 4: All tissues specified in Table 1.
- All F0 animals: Abnormalities only.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes. For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Fetal examinations:
- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of 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.
- Individual offspring body weights: Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS: Yes
- Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues were retained in an appropriate fixative.
- Offspring at scheduled termination: Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.
Statistics:
See below.
Indices:
MATING PERFORMANCE AND FERTILITY
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals pairing) x 100

GESTATION LENGTH AND INDEX
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / Number pregnant) x 100

SURVIVAL INDICES
The following indices were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100 % where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number if offspring born) x 100

Viability index (%) = (Number of live offspring on Day 7/ Number live offspring on Day 1 after littering) x 100

Group mean values were calculated from individual litter values.


SEX RATIO
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 7 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or signs associated with dosing seen that are considered related to treatment with the test material at 100, 300 or 1 000 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no clear effect of treatment on bodyweight or bodyweight change for females prior to pairing, during gestation or lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no clear effect of treatment on food consumption for females prior to pairing, during gestation or lactation.
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:
no effects observed
Description (incidence and severity):
There was no clear effect of treatment on female ovary weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item- related macroscopic findings in adult females at necropsy. The incidence and distribution of all findings were considered incidental and unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Qualitative evaluation of the ovaries in females was conducted in animals of the control and high dose groups. No changes related to treatment with test material were seen. All other microscopic findings were considered incidental and unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Description (incidence and severity):
There was no effect on post-implantation survival.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no effect of treatment on survival of the offspring.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation lengths for the majority of females at 100, 300 or 1000 mg/kg/day were within the expected range of 22-23 days and unaffected by treatment. The gestation index was unaffected by treatment.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pre-coital interval was unaffected by treatment with all animals mating in the first four days of pairing at the first estrus. Mating performance and fertility were unaffected by treatment.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment related effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Bodyweight on Day 1 of age was unaffected by parental treatment with the test material at 100, 300 and 1 000 mg/kg/day for both males and females. Bodyweight gains of male and female offspring from Day 1 to Day 7 of age from litters at 300 or 1 000 mg/kg/day appeared to increase with increasing dose; however the differences were marginal and did not attain statistical significance.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of implantations and litter size through to Day 7 of age was similar in females receiving 100 and 1 000 mg/kg/day and slightly low in females receiving 300 mg/kg/day when compared to the Control. This slight reduction was influenced by two females in Group 3 where the number of implantations was found to be low (6 and 5 implantations, respectively). There was no effect of treatment on survival of the offspring.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratios were unaffected by treatment, and the corpora lutea count was unaffected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The number of implantations and litter size through to Day 7 of age was similar in females receiving 100 and 1 000 mg/kg/day and slightly low in females receiving 300 mg/kg/day when compared to the Control. This slight reduction was influenced by two females in Group 3 where the number of implantations was found to be low (6 and 5 implantations, respectively). There was no effect of treatment on survival of the offspring.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
OFFSPRING CLINICAL SIGNS
There was no change in the clinical condition of offspring that was considered related to maternal treatment.

OFFSPRING MACROPATHOLOGY
Necropsy findings observed in offspring dying prematurely or in those killed at scheduled termination revealed no findings that were considered to relate to parental treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Discussion

The oral (gavage) administration of the test material to Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 100, 300 or 1 000 mg/kg/day was well tolerated with no adverse effect of treatment.

Treatment had no effect upon mating performance or fertility and there was no effect of treatment upon mean gestation length or the gestation index.

The number of offspring born, subsequent survival and clinical condition of the offspring were unaffected by treatment at dose levels up to 1 000 mg/kg/day.

The slight reduction in the number of implantations and litter size through to Day 7 of age in females receiving 300 mg/kg/day was influenced by two females in Group 3 where the number of implantations was found to be low (6 and 5 implantations, respectively). The occurrence of these two small litters is within the historical control range for control litters (minimum number of implantations in a study control group is two and five, which covers the 6 and 5 implantations in a treated group on this study).

The mean post implantation survival indices at 300 and 1 000 mg/kg/day were slightly lower than in Controls but the differences did not attain statistical significance. These differences in the 1 000 mg/kg/day group was slightly outside expected control group performance in Han Wistar rats on OECD 421 screening tests (five studies performed in 2015/2016, range 86.4 to 91.8 %); the value in the 300 mg/kg/day group was within the expected range. However, the differences between the mean number of implantations and the mean total litter size were 1.2 and 1.5 conceptuses at 300 and 1 000 mg/kg/day, respectively, compared with 0.9 conceptuses in the concurrent control. These differences in the 300 and 1 000 mg/kg/day are within expected control group performance in Han Wistar rats on OECD 421 screening tests and therefore no effect of treatment is inferred.

At 300 or 1 000 mg/kg/day, growth of males and female offspring from Day 1 to Day 7 of age was marginally greater than in Controls; differences did not attain statistical significance and were considered likely to reflect the slightly smaller litter sizes in these groups. There were no macroscopic findings in offspring that were considered to relate to parental treatment.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, there were no adverse effects of treatment upon reproductive or developmental parameters up to the high dose level of 1 000 mg/kg/day. The NOAEL is therefore 1 000 mg/kg bw/day.
Executive summary:

The potential for the test material to cause reproductive/developmental effects, was investigated in a GLP study conducted in accordance to the standardised guideline OECD 421.

Three groups of ten male and ten female rats received the test material at doses of 100, 300 or 1 000 mg/kg/day by oral (gavage) administration at a dose volume of 5 mL/kg. Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation. Females were allowed to litter and rear their offspring before they were killed on Day 7 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance and fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations of organs of the reproductive system were undertaken. The clinical condition, litter size, survival, sex ratio, body weight and macropathology for all offspring were also assessed.

The oral (gavage) administration of the test material to Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 100, 300 or 1 000 mg/kg/day was well tolerated, with no deaths and no effect of treatment upon the clinical condition of animals. There was no effect of treatment on body weight gain or food consumption at any of the dose levels investigated and pre-coital interval, mating performance and fertility and gestation length were unaffected. The number of offspring born, offspring sex ratio and offspring survival and growth to Day 7

of age were unaffected at dose levels up to 1 000 mg/kg/day and there were no clinical observations or macroscopic necropsy findings amongst offspring that were considered to relate to parental treatment.

There were no treatment-related macroscopic or microscopic findings in parent animals, and no differences in organ weights, which were considered to be related to treatment.

It is concluded that oral administration of the test material to male and female Han Wistar (RccHan™;WIST) for at least 4 weeks at dose levels of 100, 300 or 1 000 mg/kg/day was well tolerated, with no effect of treatment upon reproductive or developmental parameters up to the high dose level of 1 000 mg/kg/day. The NOAEL is therefore 1000 mg/kg/day.