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:
18 May 2016 to 17 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (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:
liquid: viscous
Details on test material:
Apperance: Viscous gold coloured liquid
Storage conditions: At ambient temperature, protected from light in a sealed container

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST was used because of the historical control data available.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: The males were 69 to 75 days old and the females 62 to 68 days old.
- Weight at study initiation: Males animals weighed 265 g to 310 g and female animals weighed 164 g to 193 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. Cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups. Solid bottom cages contained softwood bark-free fibre betting, which was changed at appropriate intervals.
Number of animals per cage -
Pre-pairing: Up to gfive animals of one sex.
Pairing: One male and one female.
Males after mating: Up to five animals.
Gestation: One female.
Lactation: One female plus litter.
- Diet: Ad libitum. The diet contained no added antibiotic or other themotherapeutic 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 treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 40-70 %.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated. Minimum 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:
A correction factor (including purity) of 1.429 was used when calculating quantities of test item to be used during dose 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.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- Frequency of preparation: Weekly.
- Storage of formulation: Refrigerated (nominally 2-8 °C).

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: 4.57, 11.43 and 28.58 mg/mL
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
MATING PROCEDURE
- M/F ratio per cage: 1:1 from within the same treatment group.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear; checked daily.
- After successful mating each pregnant female was caged: Individually during gestation.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 (2.858 mg/mL and 285.8 mg/mL as supplied) was achieved following re-suspension of the formulations (by 20-fold inversion and magnetic stirring for a minimum period of 20 minutes) following refrigerated storage (2-8 ºC) for up to 19 days. Formulations were also stable at ambient temperature (nominally 21 °C) for up to 48 hours.
Achieved concentration Samples of each formulation prepared for administration for the first formulation occasion (Week 1), during Weeks 5 and 6 and the last formulation occasion (Week 7) were analysed for achieved concentration of the test item.

ANALYTICAL PROCEDURE
The analytical method involved extraction and dilution in propan-2-ol followed by liquid chromatographic analysis with mass spectrometric detection (LC-MS/MS). Sample concentrations were determined with reference to single matrix matched bracketing standards at 500 ng/mL. Calibration standards and samples were matrix matched. Procedural recovery samples were prepared concurrently with samples and results were corrected for the mean recovery value at each level at analysis.

CONCENTRATION OF DOSE FORMULATIONS
The formulations for the First Formulation occasion, Week 5, Week 6 and the Last Formulation occasion were sampled (4 x 1 mL, accurately weighed) by Pharmacy personnel. Duplicate samples were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. On the First Formulation occasion and for Week 5, contingency samples for Groups 2, 3 and 4 were analysed due to problems with the initial analysis. Samples were disposed of once analysis was complete.

RESULTS AND CONCLUSION
For the Week 1 samples, the procedural recovery results were originally outside acceptance criteria. This was investigated by re-diluting recovery and formulation samples in duplicate. Because the results from this investigation would not be available until the contingency samples had passed their valid stability period it was decided to analyse the contingency samples as a precautionary measure.
For the Week 5 samples, the original analysis could not be reported due to analytical errors which meant the recovery samples were outside the acceptance criteria and in some cases were as high as 200 %. Contingency samples were therefore analysed for this occasion. The mean concentrations were within applied limits +10 %/-15 % of the nominal concentration, confirming the accuracy of formulation.
The coefficients of variation for Groups 2 and 4 on the First Formulation occasion were >5 %, this is considered to be due to the variability of the LC-MS/MS method and formulations were deemed to have been prepared correctly. On all other occasions, the differences from the mean of individual results were <±5 % indicating precise analysis.
Procedural recovery results throughout the study were variable on a run by run basis as a result of the inherent variability of the LC-MS/MS method used for analysis. After a review of the completed study data, it is considered that an acceptance limit of 90 – 110 % of the nominal fortification is acceptable for recoveries. This has been applied to all analytical occasions and was reviewed for each run.
Mean analysed concentrations were calculated using unrounded figures. Analysed concentrations are corrected for the mean recovery values at each level.
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.
Details on study schedule:
Dosing was restricted to the F0 generation. The F1 generation received no direct administration of the test item. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through the milk.
Doses / concentrationsopen allclose all
Dose / conc.:
16 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
100 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: Dose levels were selected based on the results of a two-week preliminary toxicity study and a four-week toxicity study conducted on the test material.
In the two-week preliminary toxicity study, doses of 300 and 1000 mg/kg/day were not tolerated resulting in the premature termination of both sexes receiving 1000 mg/kg/day and females receiving 300 mg/kg/day after a few days. A dose of 100 mg/kg/day was well tolerated in both sexes and elicited no clear test substance-related signs, with macroscopic gastrointestinal findings confined to a single female with a thickened non-glandular region of the stomach. In the 4-week repeat dose toxicity study, doses of 16, 40 and 100 mg/kg/day were well tolerated and did not result in any mortality or signs of other adverse systemic toxicity based on the study investigations performed up to the end of the 4-week treatment period and excluding results of the histopathological assessment.
Based on this information, it was considered appropriate to investigate a high dose level of 100 mg/kg/day in this current study. The intermediate and low dose levels were selected to assess any dose-responsiveness of any test substance-related finding.

- 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.
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacements were made before allocation of two males due to body weight range extremes.

Positive control:
Not examined

Examinations

Parental animals: Observations and 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.
Signs associated with dosing were checked 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.
- After completion of dosing the group.
- One to two hours after dosing.
- As late as possible in the working 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.

BODY WEIGHT: Yes
The weight of animals was recorded as follows:
F0 males
- During acclimatization.
- Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
- On the day of necropsy.
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.
- Day 1, 4, and 7 of lactation.
- On the day of necropsy.
Group mean values and standard deviation (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; the group mean values were 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 males
- Weekly before pairing (from first day of treatment until pairing).
F0 females
- Weekly before pairing (from first day of treatment until pairing).
- 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 of durations of 1-4, 5-8, 9-12 and 13-14 days of pairing was calculated.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

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: Individual litter values were tabulated for the number of corpora lutea, implantation sites, total offspring at Day 1 and live at Days 1, 4 and 7 of age. Group mean litter size and SD were calculated from the individual litter values. Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Individual offspring body weights: Body weights determined on 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.


Postmortem examinations (parental animals):
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 below.
For females, the number of corpora lutea and uterine implantation sites were recorded for each ovary.
The number of implantation sites were checked after staining with ammonium sulphide, only for females failing to produce a litter.

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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to post-mortem examinations as follows: 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.

GROSS NECROPSY
- 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.

HISTOPATHOLOGY / ORGAN WEIGTHS
At scheduled kill, only abnormalities were preserved for examination by light microscopy.

Statistics:
See below.
Reproductive 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
Offspring viability indices:
LITTER SIZE
Individual litter values were tabulated for the number of corpora lutea, implantation sites, total offspring at Day 1 and live at Days 1, 4 and 7 of age. Group mean litter size and SD were calculated from the individual litter values.

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: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs observed were minor and not attributed to treatment.
There were no treatment related changes observed in association with the time of dosing. Salivation was observed in males only towards the end of their respective treatment period (Day 25) at an increasing incidence with increasing dose. However, this only appeared on a single day, and the sign itself is most likely to reflect a general distaste of the formulation, therefore it is not considered a toxic effect of treatment.
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):
Bodyweight gain for males throughout their 4 week treatment period, and for females before pairing, and throughout gestation and lactation, was similar to Controls and unaffected by treatment at all dose levels investigated.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect on food consumption before pairing in males or females, or during gestation and lactation for females, at any dose level.
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, non-treatment-related
Description (incidence and severity):
Mean testis and epididymis weights were slightly lower than Controls at 100 mg/kg/day although no statistical significance was attained. Comparing the current data to historical control data for testis and epididymis weights (mean 3.493, range 3.380-3.652 for testis and mean 1.162, range 1.116-1.198 for epididymis) these changes were considered to represent normal biological variation for animals of this strain and ages at this facility and therefore these changes are considered not to be related to treatment. Seminal vesicle weights were slightly lower than Controls at 40 or 100 mg/kg/day, without dose-relationship, and with only values adjusted for terminal body weight attaining statistical significance at both dose levels.
There were no effects of treatment upon male pituitary and prostate weights, or female ovary weights, at any of the dose levels investigated.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings in adult males or females at necropsy that would infer any treatment related changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic findings in adult males or females at necropsy that would infer any treatment related changes.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Pre-coital interval was unaffected by treatment, with only one pairing on study (in the Control group) which did not mate within the first 4 days of pairing. Treatment had no effect upon mating performance or fertility.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no clear effect of treatment upon mean gestation length or the gestation index.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no change in the clinical condition of offspring that was considered related to maternal treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was not effect on post-implantation survival, live birth or viability indices.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight of offspring on Day 1 of age, and subsequent body weight gain during Days 1-7 of age was similar to Controls across all treatment groups and considered unaffected by treatment at all dose levels investigated.
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
Description (incidence and severity):
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.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Litter Size, Sex Ratio and Survival Indices
Mean numbers of corpora lutea were similar across all groups. Mean litter size was similar to controls across all dose levels on Day 1 of lactation, and subsequent litter size to Day 7 of lactation was unaffected by treatment.
There was no effect of test material on sex ratio.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects.

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Formulation Analysis Results

The mean concentrations were within applied limits +10 %/-15 % of the nominal concentration, confirming the accuracy of formulation. The coefficients of variation for Groups 2 and 4 on the First Formulation occasion were >5 %. This was considered to be due to the variability of the LC-MS/MS method and formulations were deemed to have been prepared correctly. On all other occasions, the differences from the mean of individual results were±<5 % indicating precise analysis.

Discussion

The oral (gavage) administration of the test material to Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 16, 40 or 100 mg/kg/day was well tolerated with no adverse effect of treatment. There was a marginal reduction in testis and epididymis weights in males which received 100 mg/kg/day. Compared to historical control data, these changes were considered to represent normal biological variation for animals of this strain and ages at this facility. Slightly lower seminal vesicle weights amongst males receiving 40 or 100 mg/kg/day were also recorded however, no differences in organ weights were apparent at the same doses on the four-week toxicity study. In addition, there was no microscopic correlate from evaluation of the testes, epididymides and seminal vesicles from males which received 40 or 100 mg/kg/day either on the four-week toxicity study, or following evaluation of the testes and epididymides on the current study, and fertility and fecundity (litter size) were unaffected at all dose levels investigated in the current study. Therefore these differences in organ weights were considered not to represent an adverse effect of treatment and likely to be unrelated to treatment.

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

The number of offspring born, subsequent survival and growth to Day 7 of age and clinical condition of the offspring were unaffected by treatment at dose levels up to 100 mg/kg/day. 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, oral administration of the test material to male and female Han Wistar rats was well tolerated, with no effect of treatment upon reproductive or developmental parameters observed up to the highest dose level tested of 100 mg/kg/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 16, 40 or 100 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 16, 40 or 100 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 100 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, 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 16, 40 or 100 mg/kg/day was well tolerated, with no effect of treatment upon reproductive or developmental parameters observed up to the highest dose level tested of 100 mg/kg/day. The NOAEL is therefore > 100 mg/kg bw/day.