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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 June to 10 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to internationally recognised guideline
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: a well established supplier
- Age at study initiation: (P) Males: 5 wks; Females: approximately 11 wks
- Weight at study initiation: (P) Males: 96-143 g; Females: 224-268 g
- Housing: P2000 cages (pre-mating and males after mating; RB3 cages for mating; 2154 cages for gestation/lactation. The RB3 cages were suspended over trays covered with absorbent paper which was changed at appropriate intervals. Wood based bedding was used in the cages with a solid floor.
- Diet: free access to standard rodent diet
- Water: free access to potable water taken from the public supply
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): not applicable (each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated).
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 June 2010 (animal arrival) To: 22 October 2010 (necroscopy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% w/v carboxymethylcellulose sodium salt.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported
- Concentration in vehicle: 0, 100, 500, 1000 mg/mL
- Amount of vehicle (if gavage): 10mL/kg
- Purity: 0.5% w/v carboxymethylcellulose sodium salt.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear
- After successful mating each pregnant female was caged: in a polycarbonate cage type P2154
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix.
The analytical method involved dissolution and dilution in methanol/acetonitrile/water (25/25/50 v/v/v) followed by reverse phase high performance liquid chromatographic analysis with ultra-violet detection (254 nm). Sample concentrations were determined with reference to external standards prepared in the concentration range 1 μg/mL – 5 μg/mL.
Duration of treatment / exposure:
Males were treated for 17 weeks, including 10 weeks before pairing.
Females were treated for two weeks before pairing and until Day 20 of lactation (except during parturition).
Dosing was restricted to the F0 generation; animals of the F1 generation were not dosed.
Frequency of treatment:
All animals were dosed once each day at approximately the same time of the day, seven days per week.
Details on study schedule:
Week 1 treatment of F0 males begins
Week 9 treatment of F0 females begins
Week 11-12 F0 mating
Week 13-25 F1 born and litter size adjusted to 10 offspring
Week 16-18 end of study
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500, 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
24
Details on study design:
- Dose selection rationale:

Doses were selected following review of the results of an embryo-fetal main study conducted with the test substance at Huntingdon Life Sciences (HLS, 2010, Rat, oral). In that study, doses of 100, 500 or 1000 mg/kg/day, administered to pregnant animals from conception (Day 1 post coitum) to the end of gestation (Day 19 post coitum) at a volume dosage of 10 mL/kg by the oral gavage route concluded that there was no effect of treatment with the test substance on maternal clinical condition, bodyweight, food consumption, macropathology, mean gravid uterine weight or pregnancy outcome and no effect on embryo-fetal survival growth and development, or incidence of external or internal fetal abnormalities, at any of the doses.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each F0 male was recorded on the day that treatment commenced (Week 0), at weekly intervals throughout treatment, and before necropsy. The weight of each F0 female was recorded on the day that treatment commenced (Week 0), at weekly intervals until mating was detected and on Days 0, 3, 6, 10, 14, 17 and 20 after mating, and on Days 1, 4, 7,
11, 14, 18 and 21 of lactation.

OTHER: a detailed physical examination was performed on each F0 animal to monitor general health. This was performed weekly for males and for females before mating. After mating, the examination was performed on Days 0, 6, 13 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears were taken from all 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):
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.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum pups/litter: 10

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:

Litter size;
Survival indeces;
Sex ratio;
Clinical signs;
Bodyweight;
Macroscopic pathology.










[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]





GROSS EXAMINATION OF DEAD PUPS:
[no / 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: after 17 weeks of treatment, following completion of the necroscopy of the majority of the litters.
- Maternal animals: day 21 of lactation, day 21 after last pairing if no viable litter was produced, or on the day the last offspring died where a litter did not survive.

GROSS NECROPSY
- All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic,
abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. For F0 females, the numbers of implantation sites in each uterine horn was counted. For females failing to produce a viable litter, the number of uterine implantation sites was re-checked after staining with ammonium sulphide

HISTOPATHOLOGY

Those tissues subject to histological processing included the following regions:

Adrenal - cortex and medulla
Ovaries - qualitative evaluation of one section from each ovary
Seminal vesicles - included coagulating glands in section
Uterus - uterine body with cervix section and oviducts

For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.

All adult tissues preserved for examination were examined for males and females of Groups 1 (Control) and 4 (1000 mg/kg/day). Reproductive organs were examined for animals of Groups 2 (100 mg/kg/day) and 3 (500 mg/kg/day) with suspect fertility. Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals.

ORGAN WEIGHTS

The following organs, taken from each F0 animal, were dissected free of adjacent fat and other contiguous tissue and the weights recorded:

Adrenals, Prostate, Seminal vesicles, Epididymides, Spleen, Kidneys, Testes, Liver, Ovaries, Uterus with cervix and oviducts, Pituitary




Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring day 4 or day 21 of age.

GROSS NECROPSY
- All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves.After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ (for animals killed or dying prematurely during the study).
Any abnormal position, morphology or interaction was recorded. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

- Premature deaths before weaning: Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before weaning were examined as detailed above except that the cranial roof was not removed unless required to investigate a cranial abnormality; this also included an assessment for the presence of milk in the stomach, where this was possible
Statistics:
Statistical analyses were performed on the majority of data and results of the study, whether significant or non-significant. For some parameters, including pre-coital interval and mating performance and fertility the similarity of the data was such that statistical analyses were considered not to be necessary.

Statistical tests used for bodyweight, food consumption, oestrous cycles, gestation length and gestation index, litter size and survival indices, organ weights, absoloute and adjusted for terminal bodyweight:

1) A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level.
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.

For gestation length an exact two-tailed Linear-by-linear test, with equally spaced scores, was applied to all groups.
For oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test (Cytel 1995) was applied to all groups.
For litter size and survival indices data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed.
Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect.
For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate.




Reproductive indices:
* Mating performance and fertility: group values were calculated for males and females separately for the following:
Percentage mating = (Number animals mating/Animals paired) x 100
Conception rate (%) = (Number animals achieving pregnancy/ Animals mated) x 100
Fertility index (%) = (Number animals achieving pregnancy /Animals pairing) x 100

* Gestation length: 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 calculated for each group as:
Gestation index (%) = (Number of live litters born /Number pregnant) x 100

* Litter size

Offspring viability indices:
*Survival indices: the following 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 of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day of examination/Number live offspring on Day 4 (after culling)) x 100

Sex ratio (% 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 generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Females

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

There were no deaths, clinical signs, or signs following administration of the substance to males for 17 weeks or in non-mated females or females during gestation or lactation.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body Weight
1) Males: there were no effects on body weight gain throughout the 17 week treatment period at doses up to 1000mg/kg/day.
2) Females: mean bodyweight gain in females receiving 500 or 1000 mg/kg/day was low during the two week pre-mating period (0.68 or 0.55X Control, respectively) with statistical significance during the first week of treatment. On Day 0 of gestation the mean bodyweight of females receiving 500 or 1000 mg/kg/day was 0.95 or 0.94X Control, respectively. Mean weight gain during gestation was low in these animals (0.91 or 0.85X Control, respectively), with statistical significance during the periods Day 6-14 and 17-20 in animals at 1000 mg/kg/day. On Day 1 of lactation, mean bodyweight in animals at 500 or 1000 mg/kg/day were both 0.93X Control. Weight gain during lactation at 500 or 1000 mg/kg/day was significantly high (1.28 or 1.59X Control, respectively) during Days 7-11. Final bodyweight values were similar to Control.

Food consumption
1) Males: there were no effects on food consumption throughout the 10 week before pairing at doses up to 1000mg/kg/day.
2) Females: overall food consumption in females at 1000 mg/kg/day was slightly low (X 0.88 Control) during the two week pre-mating period. Food consumption during gestation was statistically low during the second week of gestation in animals at 500 mg/kg/day and during the first two weeks of gestation at 1000 mg/kg/day and overall consumption was marginally low in both groups of animals.



REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Assessment of oestrous cycles during the two week pre-pairing period revealed that 3/24 females receiving 500 mg/kg/day and 4/24 females at 1000 mg/kg/day had a 4/5 day cycle length, compared with the majority of Control females (23/24) which had regular 4 day cycles; one Control female had a 5 day oestrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
1) Males: there were no effects on reproductive performance
2) Females: mean gestation length for all treated pregnant females was within the expected range of 22-23 days and was similar to Control. However, one litter at 1000 mg/kg/day was born 25 days after mating. Parturition in three animals treated at 1000 mg/kg/day was considered to be slightly longer than expected.
3) Gestation length was prolonged in one animal and parturition was protracted in three animals, at 1000 mg/kg/day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
1) Males: following 17 weeks of treatment, adjusted mean kidney weight was slightly high in males receiving 500 or 1000 mg/kg/day and spleen weight was low in males treated at 1000mg/kg/day.
2) Females: at the end of lactation, adjusted mean ovary weight was slightly low in animals receiving 1000 mg/kg/day.
As there was no histopathological correlation, these differences were considered not to be of biological or toxicological importance.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No toxicity
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on reduced bodyweight gain during the two week pre-mating and gestation.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects on male reproductive performance
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproductive effects: treatment with 1000mg/kg/day to females caused longer gestation length and protracted parturition and treatment with 500mg/kg/day caused extended oestrous cycle

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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
Histopathological findings:
no effects observed

Details on results (F1)

GROSS PATHOLOGY (OFFSPRING)
There were a small number of decedent offspring in treated groups which was considered similar to Control. There was no common macropathology finding and, in the absence of an effect upon offspring survival, was considered to be of no toxicological significance.
Macroscopic findings among offspring examined on Day 21 of age were considered not to be related to treatment with BPS-MAE. Findings were limited to renal pelvic dilatation of one and/or both kidneys in a small number of animals in a few litters that was not related to dose and is generally considered to be transitory or of no long-term detriment to survival.


VIABILITY (OFFSPRING)
Post-implantation survival and offspring survival from birth to Day 21 of age were not affected by parental treatment with the substance.

CLINICAL SIGNS (OFFSPRING)
Clinical signs recorded for the offspring identified occasional pups with findings, but the type of findings and incidence were typical of post-natal offspring and showed no relationship to parental treatment.

BODY WEIGHT (OFFSPRING)
Assessment of offspring bodyweight data showed that parental treatment of the substance at doses up to 1000 mg/kg/day did not have any adverse effects on pup growth.

SEXUAL MATURATION (OFFSPRING)
No data


GROSS PATHOLOGY (OFFSPRING)
There were a small number of decedent offspring in treated groups which was considered similar to Control. There was no common macropathology finding and, in the absence of an effect upon offspring survival, was considered to be of no toxicological significance. Macroscopic findings among offspring examined on Day 21 of age were considered not to be related to treatment with the substance. Findings were limited to renal pelvic dilatation of one and/or both kidneys in a small number of animals in a few litters that was not related to dose and is generally considered to be transitory or of no long-term detriment to survival.

HISTOPATHOLOGY (OFFSPRING)
There were no microscopic findings or macroscopic findings in the offspring.

OTHER FINDINGS (OFFSPRING)
When compared with Control, the percentage of males in the litters in all groups from Day 1 to Day 21 of age was not affected by parental treatment with the substance.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on offspring

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Pre-coital interval mating performance, conception rate and fertility were unaffected by treatment.

Parental treatment with the substnace did not affect the number of implantations, litter size, postimplantation survival, offspring survival to Day 21 of age, sex ratio or offspring clinical signs or bodyweight gain.

There was no effect of treatment on parental organ weight, or macroscopic or microscopic findings or macroscopic findings in the offspring.

An adenocarcinoma of the mammary gland observed in a single female given 100 mg/kg/day was considered to be within background incidental findings in female rats of this age and strain and therefore unrelated to treatment.

Applicant's summary and conclusion

Conclusions:
The extended oestrous cycle in some animals at 500 or 1000 mg/kg/day, longer gestation length in one animal and protracted parturition in three animals at 1000 mg/kg/day were considered to be related to treatment. It was therefore considered within this study, for reproductive function, as measured by gonadal function, mating behaviour and fertility, that the no-overall-effect-level (NOEL) was 100 mg/kg/day. The unsubstantiated association with treatment of both longer gestation period and extended parturition at 1000 mg/kg/day precludes this dose as the no-observed-adverse-effect-level (NOAEL) therefore; it is considered in this study that the NOAEL was 500 mg/kg/day.