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Administrative data

screening for reproductive / developmental toxicity
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-06 - 2008-07-30
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 22 March 1996
no neurobehavioural examination
GLP compliance:
Limit test:

Test material

Constituent 1
Test material form:
solid - liquid: suspension
dispersion containing micronized ETH50 (nano form)
Details on test material:
Please refer to "Confidential details on test material".

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: 11 weeks old
- Mean group weight at study initiation: 430 g (males, range: 399 to 477 g); 244 g (females, range: 214 to 283 g)
- Fasting period before study: no
- Housing: individually, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm)
- Diet: SSNIFF R/M-H pelleted maintenance diet, ad libitum
- Water: filtered (0.22 µm) tap water, ad libitum
- Acclimation period: 7 or 14 days

- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: about 12 per hour (filtered, non-recycled air)
- Photoperiod: 12 hours light (7:00 - 19:00) and 12 hours dark

IN-LIFE DATES: From: 2007-03-13 To: 2007-05-05

Administration / exposure

Route of administration:
oral: gavage
(purified water, obtained by reverse osmosis)
Details on exposure:
The test item mixture containing 47.6 % of the test item or placebo mix was administered as a suspension in the vehicle. The test item/F or placebo was mixed with approximately 90% of the final volume of the dosage form, before pH adjustment. When pH was adjusted, the remaining quantity of vehicle was added to yield the target concentrations of test item or placebo. The test item dosage forms were prepared daily from day 1 to day 8 inclusive, once from day 9 to day 11 inclusive and once a week from day 12 to day 54 inclusive according to the homogeneity and stability results and were stored at +4°C prior to use.

Since the nominal percentage of the test item envisaged at the beginning of the study was 47.6 %, a correction factor of 2.1 was used to prepare the test item dosage forms from the test item mixture. Thus, dose-levels of the test item mixture of 210, 1050 and 2100 mg/kg bw/d are an equivalent to dose-levels of 100, 500 and 1000 mg/kg bw/d of the test item, respectively. The dose-levels detailed in this study report are expressed in terms of the test item and therefore appear as 100, 500 and 1000 mg/kg bw/d.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Each female was placed with the same male until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually; and with their litter after birth, in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved wood shavings (SICSA, Alfortville, France) as nesting material
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Samples of test item dosage forms were taken during the study for analytical determination of concentration, homogeneity and stability. The analytical results indicated that the doses were accurately formulated during the toxicity study. The results also confirmed that the formulations were homogeneous and stable from the time of preparation to completion of dosing.
Duration of treatment / exposure:
- Males: 14 days before mating, during the mating (2 weeks) and post-mating periods (2 weeks) until sacrifice (maximum of 6 weeks in total)
- Females: 14 days before mating, during the mating period (maximum of 14 days), during pregnancy and lactation, until day 4 post-partum inclusive, or until sacrifice for un-mated and non-pregnant females.
- Day 1 corresponds to the first day of treatment period
Frequency of treatment:
once a day (7 days / week)
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 13 weeks old
Doses / concentrations
Doses / Concentrations:
0 (vehicle), 0 (placebo), 100, 500 and 1000 mg/kg bw/d
nominal conc.
see "Details on exposure"
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
other: -Placebo, containing all excipients without active component
Details on study design:
- Dose selection rationale: based on the results of a 13-week toxicity study performed in the same species with the test item (CIT/Study No. 28341 TCR) in which animals were given 100, 300 or 1000 mg/kg bw/d for 13 consecutive weeks. In this previous study, the test item was not in micronized particle size, and the NOAELwas set at 1000 mg/kg bw/d. Consequently, the test item dose-levels of 100, 500 and 1000 mg test item/kg bw/day were selected for the present study.
Positive control:
not applicable


Parental animals: Observations and examinations:
- Time schedule: twice a day
- Parameters monitored: mortality or signs of morbidity

- Time schedule: once a day

- Time schedule for examinations:
Males: once before group allocation, on the first day of treatment (day 1), then once a week until sacrifice.
Females: once before group allocation, on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 4 post-partum

- Time schedule:
Each male: food consumed was recorded once a week, over a 7-day period, from the first day of treatment until the start of the pairing period
Each animal: food consumed was recorded once a week, over a 7-day period, from the first day of treatment through gestation (days 0 - 7, 7 - 14, 14 - 20 post-coitum intervals) and lactation (days 1 -4 post-partum interval) until sacrifice.
During the mating period, the food consumption was noted for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

- Time schedule for collection of blood: on the day of sacrifice/at terminal sacrifice (week 7)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight period of at least 14 hours)
- How many animals: 5 rats / sex / dose level
- Parameters examined: erythrocytes, reticulocytes, haemoglobin, mean cell volume, packed cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology: neutrophils, eosinophils, basophils, lymphocytes and large unstained cells, monocytes
- Coagulation: prothrombin time, activated partial thromboplastin time, fibrinogen

- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes
- How many animals: see HAEMATOLOGY
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase

- Time schedule for collection of urine: see HAEMATOLOGY
- Metabolism cages used for collection of urine: Yes (in the presence of thymol crystals)
- Animals fasted: Yes (overnight period of at least 14 hours)
- How many animals: see HAEMATOLOGY
- Parameters examined:
Quantitative: volume, pH, specific gravity; semi-quantitative: proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, cytology of sediment: leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells; qualitative: appearance, colour
Oestrous cyclicity (parental animals):
The estrous cycle stage was recorded.
Litter observations:
- Total litter size and numbers of pups of each sex, as soon as possible after birth
- Number of live, dead and cannibalized pups, litters were observed daily
- Clinical signs or abnormal behavior: observed daily
- Body weight of each pup: recorded on days 1 and 4 post-partum

Postmortem examinations (parental animals):
- Males: after the end of the mating period (total treatment period was at least 6 weeks)
- Females: on day 5 post-partum
- Females which did not mate: 24 - 26 days after the last day of the mating period
- Females which had not delivered by day 25 post-coitum: on or after day 25 post-coitum
- Mother whose litter died entirely: as appropriate

- A complete macroscopic post-mortem examination was performed on all study animals: External surfaces, all orifices, the cranial cavity, external surfaces of the brain, thoracic, abdominal and pelvic cavities with their associated organs and tissues and neck with its associated organs and tissues. In all females, the number of implantation sites and corpora lutea was recorded. In the case of delivery in females for which mating was not detected, the implantation sites and the corpora lutea were recorded. In apparently non-pregnant females, the presence of implantation sites on the uterine horns was checked using ammonium sulphide staining technique.

- Body weight of the F0 generation males and females was recorded before sacrifice
- Testes and epididymides were weighed in all males
- Following organs were weighed in the first surviving five male parents, and five surviving female parents which delivered, per group: Adrenals, brain (including medulla/pons, cerebellar and cerebral cortex), heart, kidneys, liver, spleen, thymus

All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).
- In case of all animals the macroscopic lesions were specified and following tissues were preserved: epididymides, ovaries (with oviducts), prostate, seminal vesicles, testes, uterus (horns and cervix)
- In the first surviving five male and five delivery female parent animals per group, following tissues were preserved also: Adrenals, brain (including medulla / pons cerebellar and cerebral cortex), cecum, colon, duodenum, heart, ileum (with Peyer’s patches), jejunum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), rectum, sciatic nerves, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, thymus, thyroids with parathyroids, trachea, urinary bladder
- all macroscopic lesions

- First surviving five male and five female parent animals in the control group, placebo group and test item treated group (1000 mg/kg bw/d): All preserved tissues were examined, except lungs with bronchi
- First surviving five male and five female parent animals per group: Lungs with bronchi were examined
- all macroscopic lesions
Postmortem examinations (offspring):
- The F1 offspring (surviving pups) were sacrificed on day 5 post-partum.
- These animals were subjected to postmortem examinations as follows:

- Macroscopic examination was performed for all pups
- Macroscopic lesions were preserved (appropriate fixative used)

Not performed
Data were expressed as group mean values ± standard deviation (body weight, food consumption, corpora lutea, implantations, fetuses, resorptions, pups, gestation length) or as proportions (pre-implantation loss, post-implantation loss, fetal observations, gestation index, live birth index, viability index). Whenever necessary, the experimental unit of comparison was the litter. Data of non-pregnant females were excluded from group mean calculations. Lactation period data (body weight, food consumption, etc) of F0 females which delivered although had no evidence of mating were included in group mean calculations as were their pups' data.

Statistical tests were carried out for hematology, blood biochemistry, urinalysis and organ weight data:
- placebo group (group 2) versus water group (group 1)
- test item groups 3 (100 mg/kg bw/d), 4 (500 mg/kg bw/d) and 5 (1000 mg/kg bw/d) versus placebo group (group 2)

Number of animals/sex in each group >=5: Yes:
- Kolmogorov-Lilliefors test, log transformation if necessary; normality of distribution
- Bartlett test (3 or more groups) or Fisher test (2 groups); homogeneity of variances
- Bartlett test, if not homogenous: Dunn test (3 or more groups)
- Bartlett test, if homogenous: Dunnett test (3 or more groups)
- Fisher test, if not homogenous: Mann-Whitney / Wilcoxon test (< 3 groups)
- Fisher test, if homogenous: Students t-test (2 groups)

Number of animals/sex in each group >=5: No:
- Mann-Whitney / Wilcoxon test, if < 3 groups
- Dunn test, if 3 or more groups

The other data were compared by one-way analysis of variances and Dunnett test (quantitative values) or by Fischer exact probability test (proportions).
Reproductive indices:
- Mating index
- Fertility index
Offspring viability indices:
- Preimplantation loss
- Postimplantation loss
- Gestation index
- Live birth index
- Viability index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

One male given the placebo was found dead on day 28 without prior clinical signs. At necropsy the spleen was abnormally enlarged and of irregular color. In addition, reddish content was noted in the abdominal cavity. Sarcoma of the spleen was found at microscopic examination and was considered to be the major factor contributing to the death. This death was therefore considered not to be related to treatment. One control female with dead litter (cannibalized litter) was sacrificed on day 1 post-partum. At necropsy, 16 implantation sites were noted and no abnormal findings were observed. Four mated females, one control, two given the placebo and one treated at 100 mg/kg bw/day were sacrificed on day 26 post-coitum after no delivery. They were all confirmed not pregnant at necropsy. At necropsy, the control female showed serous cysts in enlarged ovaries and both uterine horns were dilated, had thickened walls and had a serous content. There were no other unscheduled deaths.

The male mean body weight, mean body weight change and mean food consumption were unaffected by the test item treatment. Lower mean body weights and mean body weight gains associated with slightly low mean food consumption were noted in placebo control females through the pre-mating, gestation and lactation (minimally lower mean body weights only) periods.

None of the clinical pathology parameters evaluated were considered to be affected by the test item treatment or placebo administration in any group.

Reproductive data evaluation showed that neither the mating nor the fertility parameters were adversely affected by the test item treatment or administration of the placebo.

There were no test item-related effects on organ weights.

At the post-mortem examinations of the F0 generation animals, no test item-related macroscopic observations were noted.

Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle. No microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea or in the evaluation of the uterus. Microscopic examination revealed aggregates of large histiocytes in the bronchioles of two females of the 1000 mg/kg bw/d dose-group, an effect attributed to accidental aspiration of the dosing mixture subsequent to administration and not to the test item directly. Slight microscopic changes noted in the lungs of a few females treated at 100 or 500 mg/kg bw/d were considered not to be test item-related.

Effect levels (P0)

Dose descriptor:
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: No signs of test substance toxicity at a dosage of 1000 mg/kg bw/d applied (high dose equals limit dose).

Results: F1 generation

Details on results (F1)

The number of pups dying between days 1 and 4 post-partum was similar between all the groups. One pup at 100 mg/kg bw/d showed pallor of body from day 2 post-partum. At 1000 mg/kg bw/d, one pup presented a hematoma on the head on days 1 and 2 post-partum. Due to the very low incidence of these findings (less than 1%), a relationship with the test item treatment was not established.

Mean pup body weights and body weight gains were similar to those of the controls throughout the lactation period.

The mean percentage of male pups was between 40.5 % and 54.3 % at birth and between 40.0 % and 54.0 % on day 5 post-partum. There were no effects of the test item on the sex ratio of the pups at any dose-level.

There were no grossly observable malformations in pups found dead or sacrificed on day 5 post-partum.

Effect levels (F1)

Dose descriptor:
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: No test substance related effects were observed on pups after birth, at a dosage of 1000 mg/kg bw/d applied (high dose equals limit dose).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion