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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
The standard study design has been enhanced by the inclusion of learning and memory tests and hormone analyses (TT3, TT4, TSH, FSH, LH, prolactin, E2 and testosterone) in F1 offspring
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-days premating, during mating, gestation and lactation
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Males were used for mating only and were not exposed to the test substance prior to mating.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
The test guidleline is not specifically stated in the report. The study design has been enhanced to include neurobehavioural and circulating hormone endpoints in F1 offspring.
Deviations:
yes
Remarks:
No exposure of males; inclusion of neurobehavioural and hormone measurements
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR REPRODUCTION SCREENING TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 28 days for females only
- Basis for dose level selection: Based on a 3-month oral study in male and female rats at dose levels of 0, 25, 50, 125 and 312 mg/kg/day. Toxicological effects were recorded in females treated at 50 mg/kg/day and above on thyroid hormone levels, thyroid histopathology and blood chemistry

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Included

- Route of administration: Oral gavage
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The Han Wistar rat was the strain used on previous repeat-dose oral studies from which dose levels were based; the dosing vehicle of 0.25% aqueous Methocel K4M premium was chosen as a standard vehicle for this type of solution; the number of animals per group was the minimum recommended in the guideline.

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
HanBrl:WIST SPF rats from RCC Ltd, Biotechnology & Animal Breeding Division, Wolfstrasse 4, CH-4414 Fullinsdorf/Switzerland
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Switzerland
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 13 wks
- Weight at study initiation: (P) Females: 180-216 g
- Fasting period before study: NA
- Housing: Individually housed in Type-3 Makrolon cages except during the mating period when one male/one female were housed in pairing cages
- Use of restrainers for preventing ingestion (if dermal): No
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Eight days under test conditions
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3oC
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light):12/12
IN-LIFE DATES: From: 30 July 2002 To: 18 November 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Hydroxypropyl methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and the vehicle added (w/v). The mixtures were prepared using a homogeniser. During the daily administration period, homogeneity was maintained using a magnetic stirrer. Dose formulations were stored in a refrigerator.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not stated
- Concentration in vehicle: Varied for each dose level
- Amount of vehicle (if gavage): 10 mL/Kg body weight dose volume
Details on mating procedure:
- M/F ratio per cage: One male/one female per cage during pairing period
- Length of cohabitation: Maximum of 14 days
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear, referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged individually
- Untreated male rats of the same source and strain were used for mating only. The fertility of these males had been proven and was continuously controlled.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of determination of concentration, homogeneity and stability (4 days) of the dose formulations were taken during the third week of the administration period, during the first week of gestation and during the last week of the administration period. On each occasion three samples of approximately 2g were taken from the top, middle and bottom of each formulation and transferred to flat-bottomed flasks. The samples were frozen and were analysed as soon as possible using an HPLC method.
Duration of treatment / exposure:
Parent females were treated during a 28-day prepairing period and also during the pairing, gestation and lactation periods amounting to a total of approximately 84 days.
Frequency of treatment:
Daily
Details on study schedule:
- F1 pups were not mated; At weaning, at least half of the pups of each sex of each litter (randomly selected) were selected for futher rearing. The remaining pups were sacrificed and examined for structural abnormalities or pathological changes. The remaining F1 offspring were reared until day 55 post partum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a 90-day rat oral study in which dose levels of 0, 25, 50, 125 and 312 mg/kg/day were used and the dose levels of 50 mg/kg/day and higher in females were shown to induce changes in thyroid pathology and hormone levels and also haematology.
- Rationale for animal assignment (if not random): Randomly grouped using a computer-generated ramdom algorithm. F1 animals were selected for sacrifice at weaning or rearing based on the randomly allocated pup numbers.
Positive control:
No positive control included

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations checked included: Mortality, signs of reaction to treatment and symptoms of ill-health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: P females were weighed twice weekly during the prepairing and lactation periods. During gestation females were weighed daily.

FOOD CONSUMPTION and WATER CONSUMPTION: Yes
- Food/water consumption were measured twice weekly (during prepairing and lactation together with the recoding of body weights), except during the pairing period when no food/water consumption were measured. During the lactation period of the females, food/water consumption were recorded only until day 14 post partum.

BLOOD SAMPLING FOR HORMONE ANALYSIS:
- Blood samples were collected from P females on Day 21 of the prepairing period from the retroorbital sinus (under light isoflurane anaesthesia) and at necropsy via heart puncture (under terminal anaesthesia). The following hormone levels were determined: Triiodothyronine (T3), Thyroxine (T4), Thyroid stimulating hormone (TSH), Estradiol (E2), Testosterone (Testo), Follicle stimulating hormone (FSH), Luteinising hormone (LH), Prolactin (PRL).
Oestrous cyclicity (parental animals):
Daily vaginal smears were taken commencing after two weeks of treatment and during pairing until evidence of mating had been noted.
Sperm parameters (parental animals):
Parameters examined in F1 males sacrificed at weaning or after rearing:
- testis weight, epididymis weight, seminal vesicle weight
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, weight gain, physical or behavioural abnormalities, anogenital distance (AGD)]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: NA

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: NA
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Remained untreated and were only used to impregnate the females
- Maternal animals: All surviving animals at or shortly after weaning of their litters

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination for any structural abnormalities or pathologial changes with special attention directed to the organs of the reproductive system, thyroid gland, adrenal glands, thymus and any suspected target organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues indicated in were weighed and prepared for microscopic examination respectively: uterus, cervix (histopathology only), ovaries, pituitary gland, thyroid gland, adrenal glands, thymus and suspected target organs.
Postmortem examinations (offspring):
SACRIFICE
- At weaning half of the F1 pups of each litter (equally divided as per sex) were sacrificed. On Day 55 post partum the remaining F1 offspring were sacrificed.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination for any structural abnormalities or pathological changes. Special attention was directed to external and internal sex characteristics, the organs of the reproductive system, thyroid gland, adrenal glands, thymus and any suspected target organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
- The following tissues indicated in were weighed and prepared for microscopic examination respectively: uterus, cervix (histopathology only), ovaries, testes, epididymides, seminal vesicles, prostate gland, pituitary gland, thyroid gland, adrenal glands, thymus, suspected target organs.
- Histopathological evaluation was conducted on the ovaries, uterus, testes and prostate of F1 offspring killed at the end of weaning only.
Statistics:
- Means and standard deviations of various data were calculated
- If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group)
- The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomised without loss of information.
Reproductive indices:
- Fertility indices
- Mean precoital time
- Pre and Post implantation losses
Offspring viability indices:
- Embryonic and foetal deaths
- Breeding losses

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Prepairing period:
Increased water intake at 50 mg/kg/day (+28.7%), and also at 25 and 12.5 mg/kg/day (+12.5% and +13.4% respectively)
Gestation period:
Increased water intake at 50 mg/kg/day (+31.5%) and also at 12.5 mg/kg/day (+14.1%)
Lactation period:
Increased wter intake days 1-4 post partum at 50 mg/kg/day (+15.2%)
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
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of one acyclic female at 25 mg/kg/day was considered to be non-treatment-related.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

- Increased water consumption at 40 mg/kg/day throughout the treatment period.
- Reproduction performance: Not affected by treatment
- Fertility and mating performance - neither median or mean precoital time gave any indication for treatment related effects. In all groups all pregnant females gave birth to living pups (gestation index 100%)
- Duration of gestation - Similar in all groups 1-4 (21.7, 21.7, 21.8 and 21.6 days respectively)
- Implantation rate and post-implantation loss - the mean number of implantations per dam was similar in groups 1-4 (13.2, 13.7, 12.5 and 12.3 respectively); The incidence of post implantation loss was similar in groups 1-4 (9.1, 8.9, 6.4 and 11.4% of implantation sites respectively)
- Litter size - Mean number of pups per litter was similar in groups 1-4 (12.0, 12.4, 11.7 and 10.9 respectively); Birth indices were similar in groups 1-4 (90.9, 91.1, 93.6 and 88.6% respectively)
- Postnatal loss - Viability indcies were high and similar in groups 1-4 (99.2, 99.1, 100.0 and 99.1% respectively)
- Breeding loss - Weaning indices were high and similar in groups 1-4 (100.0, 99.1, 100.0 and 100.0% respectively)

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
water consumption and compound intake
Remarks on result:
other: Although reduced water intake was recorded at all treatment levels this was considered not to be an adverse effect. Dose levels up to 50 mg/kg/day did not influence the reproductive function of female rats.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
At birth the sex ratio, percentage of male and female pups, was close to 50% in all groups
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes in serum levels of T3, T4, TSH, FSH, LH, E2, Prolactin and Testosterone
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
F1 weanlings - No effects on pinna unfolding, incisor eruption, onset of coat development, opening of the eye; no signs of nipple retention in male pups.
F1 reared to Day 55 - No effects on descensus of testes or balano-preputial separation or time of vaginal opening.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effects on learning and memory function in the water maze test.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No treatment related changes were observed in F1 weanlings upto Day 21 or Day 55 post partum

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
sexual maturation
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
developmental neurotoxicity

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL in this study for reproductive function of female rats and growth and development of F1 offspring was 50 mg/kg/day.
Executive summary:

Groups of 10 female rats were dosed with the test substance at dose levels of 12.5, 25, or 50 mg/kg bw/day at a standard dose volume of 10 mL/kg of vehicle (hydroxypropyl methylcellulose) for 28 days prior to mating, during pairing, gestation and lactation periods. Males remained untreated (used for pairing only) and F1 offspring were not directly treated with the test item. No clinical signs or adverse reactions to treatment were noted, and there were no effects on mortality, body weight gain or food consumption. A significant increase in water consumption was recorded for females from all treatment groups throughout the various phases of the study with the highest intake recorded for those treated at 50 mg/kg/day.

 

There were no test item related effects on any of the reproduction parameters included in the scope of investigations (estrous cycles, fertility indices, mating performance, duration of gestation, implantation rate, post-implantation loss, litter size and postnatal pup loss). There were no significant effects on the weight or histopathology of reproductive and endocrine organs. No significant differences in thyroid hormones (T3, T4, TSH) or reproductive hormones (FSH, LH, prolactin, E2 and T) were found in parental females or in F1 offspring. Developmental indices (pinna unfolding, incisor eruption, onset of coat development and eye opening) and anogenital distances in treated offspring did not differ from controls. There was no indication of effects on learning or memory function.

 

There were no other changes recorded in the study that were considered to be indicative of a reaction to treatment. Consequently, the recorded increase in water consumption was considered not to be an adverse effect and that the high dose level of 50 mg/kg/day represents the NOAEL for reproductive function of female rats and growth and development of the F1 offspring. This study was assigned a reliability rating of 2: reliable with restrictions, as the study design was similar to OECD 421, but deviated by omitting male exposure.