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Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29th April to 12th September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conducted in a GLP compliant laboratory according to recognised regulatory Guidelines
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: reputable commercial supplier
- Age at study initiation: ca 10 wks males and females
- Weight at study initiation: (P) Males: 333-386 g; Females: 228-265 g;
- Fasting period before study:
- Housing: Cages comprised of a polycarbonate body (and floor) with a stainless steel mesh lid; changed at appropriate intervals. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet. Non-restricted access
- Water (e.g. ad libitum): ad libitum public supply
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12h light: 12h Dark

IN-LIFE DATES: From: To: 12th June to 22nd July (Males) or 30th July-5th August (Fermales and litters)
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):The homogeneity and stability of the test material in arachis oil was assessed and confirmed at concentrations of 10 mg/mL and 250 mg/mL.
- Concentration in vehicle: 0, 25, 75, 250 mg/mL
- Amount of vehicle (if gavage):4 mL/kg body weight
- Lot/batch no. (if required): N/a
- Purity: Not measured
Details on mating procedure:
- M/F ratio per cage: 1:1 from within the same treatment groups
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not required
- After successful mating each pregnant female was caged (how): one female per cage
- Any other deviations from standard protocol: None
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Week 1 and the last week of treatment were analysed for achieved concentration of the test substance.
Duration of treatment / exposure:
Males - for a minimum of four consecutive weeks, including two weeks prior to pairing.
Females - two weeks prior to pairing, throughout mating and gestation and until Day 6 of lactation.
Frequency of treatment:
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). Detailed physical examinations were performed on each animal according to the following
schedule: Males - weekly throughout the treatment period; Females - weekly until mated, then Days 0, 7, 14 and 20 of gestation and Days 1 and 7 of lactation

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Males Day that treatment commenced (Week 0), Each week, At necropsy
F0 Females Day that treatment commenced (Week 0), Each week until mating was detected, Days 0, 3, 7, 10, 14, 17 and 20 after mating, Days 1, 4 and 7 of lactation



OTHER: Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded was recorded as follows:
F0 males Weekly before pairing. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
F0 females Weekly until paired for mating. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage
Following pairing, on Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6, of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, litter mean body weight (+ SD) was calculated separately for males and females, organs weights at necropsy

GROSS EXAMINATION OF DEAD PUPS:
yes for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals during week 5 of respective treatment
- Maternal animals: All surviving animals day 7 of lactation or day 25 after last day of pairing for F0 females failing to produce a viable litter

GROSS NECROPSY
- All F0 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively:
Abnormalities, Epididymides (caput, corpus and cauda), Ovaries, Pituitary, Prostate , Seminal vesicles (with coagulation gland), Testes, Uterus with cervix and oviducts, Vagina
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Tissues from the F1 offspring were not prepared for microscopic examination.
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including pre-coital interval, and mating performance and fertility the similarity of the data was such that analyses were not considered to be necessary:
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other analyses t/The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared with Control, overall body weight gain in females receiving 300 or 1000 mg/kg/day, during the two week pre-mating period was low (X 0.77 or 0.67, respectively).
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs recorded for the offspring identified occasional pups with findings, but the type of findings and incidence were typical and showed no relationship to parental treatment.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
The predominant macropathology finding among the small number of decedent offspring on the study was no milk in stomach. This is a common finding and, in the absence of an effect upon offspring survival, is considered to be of no toxicological significanc
Histopathological findings:
not specified
GROSS PATHOLOGY (OFFSPRING)The predominant macropathology finding among the small number of decedent offspring on
the study was no milk in stomach. This is a common finding and, in the absence of an effect
upon offspring survival, is considered to be of no toxicological significance.

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Reproductive effects observed:
no
Conclusions:
For general toxicity, the no-observed-effect level (NOEL) was considered to be 1000 mg/kg/day and for reproductive /developmental toxicity the NOEL was 1000 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on fertility via oral route:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) study conducted in a GLP compliant laboratory according to recognised regulatory Guidelines .

It is concluded that oral administration of S-900 to female Sprague-Dawley rats for at least four weeks at dose up to and including 1000 mg/kg/day was well- tolerated and there was no effect on reproductive performance, including mating performance, fertility and offspring survival and development up to Day 7 of age.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

It is concluded that oral administration of S-900 to female Sprague-Dawley rats for at least four weeks at dose up to and including 1000 mg/kg/day was well- tolerated and there was no effect on reproductive performance, including mating performance, fertility and offspring survival and development up to Day 7 of age.

For general toxicity, the no-observed-effect level (NOEL) was considered to be 1000 mg/kg/day and for reproductive /developmental toxicity the NOEL was 1000 mg/kg/day.

Justification for selection of Effect on developmental toxicity: via oral route:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) study conducted in a GLP compliant laboratory according to recognised regulatory Guidelines .

It is concluded that oral administration of S-900 to female Sprague-Dawley rats for at least four weeks at dose up to and including 1000 mg/kg/day was well- tolerated and there was no effect on reproductive performance, including mating performance, fertility and offspring survival and development up to Day 7 of age.

Justification for classification or non-classification