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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test the NOAEL for parental toxicity and for reproductive performance (mating and fertility) was considered to be 1000 mg/kg bw/day, which was the highest dose tested.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-06 - 2008-07-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 22 March 1996
Deviations:
yes
Remarks:
no neurobehavioural examination
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- 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
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(purified water, obtained by reverse osmosis)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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.

CALCULATION OF DOSE LEVELS
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:
yes
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
Remarks:
Doses / Concentrations:
0 (vehicle), 0 (placebo), 100, 500 and 1000 mg/kg bw/d
Basis:
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:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Parameters monitored: mortality or signs of morbidity

CLINICAL SIGNS
- Time schedule: once a day

BODY WEIGHT: Yes
- 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

FOOD CONSUMPTION: Yes
- 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.

HAEMATOLOGY: Yes
- 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

CLINICAL CHEMISTRY: Yes
- 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

URINALYSIS: Yes
- 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:
PARAMETERS EXAMINED (in F1 offspring)
- 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):
SACRIFICE
- 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

GROSS NECROPSY
- 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.

ORGAN WEIGHTS
- 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

PRESERVATION OF TISSUES
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

HISTOPATHOLOGY: Yes
- 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):
SACRIFICE
- The F1 offspring (surviving pups) were sacrificed on day 5 post-partum.
- These animals were subjected to postmortem examinations as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed
Statistics:
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
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

HAEMATOLOGY / BLOOD BIOCHEMISTRY / URINALYSIS (PARENTAL ANIMALS)
None of the clinical pathology parameters evaluated were considered to be affected by the test item treatment or placebo administration in any group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related effects on organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At the post-mortem examinations of the F0 generation animals, no test item-related macroscopic observations were noted.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
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).
VIABILITY / CLINICAL SIGNS (OFFSPRING)
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.

BODY WEIGHT (OFFSPRING)
Mean pup body weights and body weight gains were similar to those of the controls throughout the lactation period.

SEX RATIO (OFFSPRING)
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.

GROSS PATHOLOGY (OFFSPRING)
There were no grossly observable malformations in pups found dead or sacrificed on day 5 post-partum.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
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).
Reproductive effects observed:
not specified
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
Additional information

In the chosen key study, i.e. a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG No 422, GLP) three groups of 10 male and 10 female Sprague-Dawley rats received micronized (nano-form) ETH50 daily, by oral gavage administration before mating, through mating and, for the females, through gestation until day 4 post-partum, at the dose-levels of 100, 500 or 1000 mg ETH50 /kg bw/day (CIT 32799 RSR, 2008).

Two other groups of 10 males and 10 females received either the vehicle (purified water) or placebo (respective formulation without ETH50) alone, under the same experimental conditions and acted as control groups. The dosing volume was 5 mL/kg bw/day. Clinical signs and mortality were checked daily. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Hematology and blood biochemistry investigations were performed and urinalysis was carried out at terminal sacrifice. Body weights and principal organ weights were recorded, a complete macroscopic post-mortem examination was performed and microscopic examination was performed on selected organs with particular attention paid to the male gonads. The litters were sacrificed on day 5 post-partum and were carefully examined for gross external abnormalities and a macroscopic post-mortem examination was performed.

At all dose-levels with ETH50, treatment-related mortalities or clinical signs of toxicity did not occur. There were no effects on mean body weight gain or food consumption of males or females compared to the control group given purified water; however, when compared to the placebo control group the females had higher body weight gains and food consumption. Adverse effects did not occur based on hematology, blood biochemistry or urinalysis parameters. Estrous cycling, pairing, mating or fertility were not adversely affected in any dose-group and pups showed no effects after birth in any group. Test item-related effects did not occur on organ weights and macroscopic abnormalities were not apparent. Microscopic examination revealed aggregates of large histiocytes in the bronchioles of two females of the 1000 mg/kg/day dose-group, an effect attributed to accidental aspiration of the dosing mixture subsequent to administration and not to the test item directly.

Under the experimental conditions of this study, it was considered that the reproductive and parental No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg/day according to findings noted in the lungs secondary to the aspiration of formulation.

The substance ETH50 registered represents the form of pure (bulk) non-nano material. As such, however, it is not usable for its intended use in cosmetic products. In order to transform it into suitable cosmetic ingredient, ETH50 has to be micronized in a water-mill. As a consequence of the extreme hydrophobicity of substance, surfactants need to be added to introduce it in the water phase. After milling, rheology modifiers prevent the nano-particles from agglomerating and stabilize the resulting preparation. Thus, the substance is marketed in a 50 % mixture together with different components; it is not available as pure material in nanoform. The key study for reproductive toxicity was performed with a formulation containing micronized ETH50 (relating to the marketed mixture for the use in cosmetics). Although the form of the test substance used does not represent the form of the substance registered (i.e. pure (bulk) non-nano ETH50) these data can be used to assess the endpoint reproductive toxicity for the registered substance satisfactorily.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study the NOAEL was established at 1000 mg/kg bw/day,

which was the highest dose tested.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-12-21 - 2005-09-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The GLP-compliant study is scientifically valid and is acceptable for assessment.
Qualifier:
according to guideline
Guideline:
other: Note for guidance on reproductive toxicology: detection of toxicity to reproduction for medicinal products. Committee for proprietary medicinal products (CPMP/ICH/386/95). European Agency for Evaluation of Medicinal Products, adopted September 1993.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: mean body weight of 268 g (range: 224 g to 306 g)
- Housing: individually housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage.
- Diet: A04 C pelleted maintenance diet, (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: tap water, filtered with a 0.22 µm filter, ad libitum
- Acclimation period: at least 5 days before the beginning of the mating period

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (w/v) aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder using a mortar and pestle. It was then suspended in the vehicle in order to achieve the concentration of 20, 60 or 200 mg/mL. The suspensions were then homogenized using a magnetic stirrer. The test item dosage forms were prepared weekly, and were stored at +4°C prior to use and protected from light, according to the storage conditions of the test item.

VEHICLE
- Concentration in vehicle: 20, 60 or 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first and the last day of treatment, duplicate samples were taken at three levels of the container (top, middle and bottom) from each control and test item dosage form (except for the control dosage form on the last day of treatment) and analyzed for concentration of the test item to evaluate the homogeneity of the suspensions. The mean results of the homogeneity analyses were taken as actual values of the concentration of the test item in the dosage forms.
Details on mating procedure:
Monitoring of estrous cycle:
- during the week of mating, determined periodically from a fresh vaginal lavage

Mating:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight for the duration of the mating period
- Proof of pregnancy: in situ, vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (designated as day 0 post-coitum (p.c.))
- Verification of same strain and source of both sexes: yes, males were from the same strain and the same breeder

Allocation to groups:
- before day 3 p.c., the animals were allocated to the groups, according to a stratification procedure based on body weight, recorded on day 0 p.c. to ensure comparatively similar mean body weight among groups
- A larger number of animals than necessary was paired to permit the selection and/or the replacement of individuals before start of treatment
Duration of treatment / exposure:
Test item was administered on gestation days (GD) 6 through 19.
Frequency of treatment:
once a day
Duration of test:
sacrifice on day 20 p.c.
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
24 mated female rats (only the first 20 pregnant females were taken into consideration for fetal examinations)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose-level selection: The dose-levels (100, 300 and 1000 mg/kg bw/day) were selected based on the results of a previous preliminary embryo-fetal development toxicity study conducted in pregnant rats of the same strain, which received, orally, the test item at 300, 600 or 1000 mg/kg bw/day from day 6 to day 19 of gestation (CIT/Study No. 28342 RSR).
Maternal examinations:
MORTALITY AND MORBIDITY: Yes
- at least twice a day during the treatment period
- at least once a day on the other days

CAGE SIDE OBSERVATIONS (CLINICAL SIGNS): Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes (each female)
- Time schedule for examinations: on days 0, 3, 6, 9, 12, 15, 18 and 20 p.c.

FOOD CONSUMPTION: Yes (each female)
- Time schedule: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 20 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 p.c.
- Macroscopic post-mortem examination of the principal thoracic and abdominal organs
- Organs examined: ovaries and uterus
- Uterine horn(s) without visible implantation site was (were) immersed (when appropriate) in an aqueous solution of ammonium sulphide to reveal the presence of uterine scars.
- Gross evaluation of placentas was undertaken
- Preservation of tissues: macroscopic lesions from all females were sampled and kept preserved in appropriate buffer
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (with at least one live fetus).
- Number of corpora lutea: Yes
- Number and distribution of dead and live fetuses: Yes
- Number and distribution of early and late resorptions: Yes
- Number and distribution of uterine scars: Yes
- Number and distribution of implantation sites: Yes
Fetal examinations:
- Body weight: Yes, all per litter
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, remaining live fetuses per litter
- Head examinations: Yes, remaining live fetuses per litter
- Sex of the fetuses: determined before evisceration or at the time of serial sectioning
The fetal findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformations or variations (Wise et al., 1997; Chahoud et al., 1999).

References:
Chahoud I, et al. (1999). Classification terms in developmental toxicology: need for harminization. Reprod Toxicol 13(1): 77-82.
Wise LD, et al. (1997). Terminology of developmental abnormalities in common laboratory mammals (version I). Teratology 55: 249-292.
Statistics:
Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
Indices:
- Preimplantation loss (in %) [(number of corpora lutea – number of implantations)/number of corpora lutea]
- Post-implantation loss (in %) [(number of implantations - number of live fetuses)/number of implantations]
- Fetal or litter incidence (in %) [total number of fetuses or litters with a particular finding/total number of fetuses or litters examined]
- Mean proportion of affected fetuses (in %) [sum of proportion of fetuses affected in each litter/total number of litters examined]
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Dose-levels of up to and including the high dose level of 1000 mg/kg bw/d did not evoke any test substance related maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no test substance related developmental toxicity observed in the fetuses in any treated group.
Abnormalities:
not specified
Developmental effects observed:
no

MATERNAL DATA:

Mortality

- There were no premature deaths during the study.

Clinical signs

- No treatment-related clinical signs were observed in any test item-treated group.

Body weight and food consumption

- The body weight, the net body weight change from day 6 p.c., the carcass weight and the mean gravid uterus weights were unaffected by treatment with the test item. Treatment had no influence on the mean food consumption in any test item-treated group.

Necropsy findings:

- No treatment-related findings were noted in any test item-treated females.

 

LITTER DATA:

- There was no effect of treatment on pregnancy parameters at any dose-level.

The numbers of corpora lutea and implantation sites were equivalent between test item-treated and control groups.

When compared to control values, the sex ratio was inverted at 100 and 1000 mg/kg/day. As this finding was not observed at 300 mg/kg/day, and as the mean values of percentage of male and female fetuses from all test item-treated animals were within or very close to the range of historical control data this was considered not to be treatment-related.

The mean fetal body weights recorded in the test item-treated groups were similar to those of the control group.

Fetal evaluation:

- Fetal external and visceral examinations: All fetuses appeared normal except one fetus from the high-dose group that showed mandibular micrognathia associated with microglossia; this fetus also presented marked dilated cerebral ventricles. As only this one fetus was affected, it was considered not to be related to treatment.

- Skeletal examination No treatment-related malformations or variations were noted

 

DOSAGE ANALYSIS:

- The results of the analyses demonstrated the homogeneity of each dosage form investigated on the first day and the last day of treatment.

- Satisfactory correspondence was noted between the nominal and the measured concentrations of the test item for each administered dosage analyzed (between -9% and - 1% of nominal values).

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

In the chosen key study, i.e. a prenatal developmental toxicity study (CIT 28343 RSR, 2005) three groups of 24 mated female rats of the Sprague-Dawley strain received non-micronized ETH50 by daily oral administration at 100, 300 or 1000 mg/kg bw/day and at a constant dosage volume of 5 mL/kg bw/day from day 6 to day 19 post-coitum (p.c.). Another group of 24 mated females of the same strain received the vehicle alone (0.5 % carboxymethylcellulose), under the same experimental conditions and acted as a control group. Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals. On day 20 p.c., the dams were sacrificed and subjected to a macroscopic post mortem examination. Only the first 20 pregnant females were taken into consideration for fetal examinations. The gravid uteri were weighed and the fetuses were removed by hysterectomy. The following litter parameters were recorded: numbers of corpora lutea, implantation sites, early and late resorptions, dead and live fetuses. The fetuses were weighed, sexed and subjected to external, soft tissue or skeletal (bones) examinations.

There were no premature deaths during the study. No treatment-related clinical signs were observed in any test item-treated group. The body weight, the net body weight change from day 6 p.c., the carcass weight and the mean gravid uterus weights were unaffected by treatment with the test item. Treatment had no influence on the mean food consumption in any test item-treated group. There was no effect of treatment on pregnancy parameters at any dose-level. At fetal external and visceral examinations, all fetuses appeared normal except one fetus from the high dose group that showed mandibular micrognathia associated with microglossia; this fetus also presented marked dilated cerebral ventricles. As only this one fetus was affected, it was considered not to be related to treatment. No treatment-related malformations or variations were noted at skeletal examination. The test item did not elicit any signs of maternal toxicity. Thus, the NOAEL for maternal and developmental toxicity in this study was found to be 1000 mg/kg bw/day.

In the associated range finding study (CIT 28342 RSR, 2005) non-micronized ETH50 was administered daily by gavage to pregnant female Sprague-Dawley rats, from days 6 to 19 p.c. inclusive, at 300, 600 or 1000 mg/kg bw/day. None of the maternal or litter parameters were affected by treatment. One fetus presented an external malformation. Consequently, the highest dose level of 1000 mg/kg bw/day was selected for the further main developmental toxicity study (CIT 28343 RSR, 2005).

Toxicity to reproduction: other studies

Additional information

Estrogen receptor binding assay:

To investigate whether non-micronized ETH50 can interact with the rat estrogen receptor (ER) a ER competitive binding assay was performed (CTL XR474, 2005).

The study consisted of harvesting the uteri from 40 immature Alderley Park rats. Freshly prepared cytosolic preparations of uterine tissue were then incubated with the test substance at a range of concentrations (0.5 nM to 0.5 mM) and a fixed concentration (5 nM) of the radiolabelled estrogen, 3H-estradiol in order to determine the ability of the test item to displace the 3H-estradiol.

Quantitation of displacement was used to determine the intrinsic activity of the test item to interact with the estrogen receptor (ER).

Two independent assays with duplicates in each assay were performed.

The results for the test item showed a good correlation in both assays and between duplicates in each assay, with no increase in radioactivity displacement at the concentrations of test item tested in both assays.

Inability to displace 3H-estradiol from cytosolic preparations of rat uterine tissue, indicated that, at concentrations up to 0.5 mM, non-micronized ETH50 did not possess intrinsic potential to interact with the rat estrogen receptor in the in vitro estrogen receptor binding assay.

Androgen receptor binding assay:

To investigate whether non-micronized ETH50 can interact with the rat androgen receptor (AR) an androgen receptor competitive binding assay

was performed (CTL XR7473, 2005). The rationale of the assay is that if a test agent is an AR ligand it will compete with the 3H-R1881 (a known AR ligand) for the AR, thus reducing the amount of radioactivity associated with the receptor. The androgen receptor (AR) competitive binding assay involves incubating a prostate cytosolic AR preparation with the androgen 3H-methyl trienolone (R1881) and a putative AR ligand.

Phase I of the study consisted of dosing 8 male Alderley Park rats with the GnRH antagonist (Antarelix) and harvesting the prostate gland cytosol 24 hours later, prior to conducting the in vitro phase of the study (Phase II). In Phase II, cytosolic preparations of prostate gland tissue were incubated with the test substance at a range of concentrations (0.5 nM to 0.5 mM) and a fixed concentration (5 nM) of the radiolabelled androgen, methyl trienolone (3H-R1881) in order to determine the ability of the test item to displace the 3H-R1881. Quantitation of displacement was used to determine the intrinsic activity of the test item to interact with the androgen receptor (AR). Two independent assays with duplicates in each assay were performed.

The results for the test item also showed a good correlation in both assays and between duplicates in each assay, with no increase in radioactivity displacement at the concentrations of test item tested in both assays. Inability to displace 3H-R1881 from cytosolic preparations of rat prostate gland tissue, indicated that, at concentrations up to 0.5 mM, non-micronized ETH50 did not possess intrinsic potential to interact with the rat androgen receptor in the in vitro androgen receptor binding assay.

Uterotrophic assay:

In an uterotrophic assay (CTL RR1054, 2005) groups of 10 immature female Alpk:APfSD (Wistar derived) rats received a single oral dose of 0 (vehicle control), 250, 500 or 1000 mg/kg bw/day of non-micronized ETH50 once a day for 3 consecutive days. As a positive control, one group of rats received a single oral dose of 0.4 mg β-estradiol/kg bw/day for 3 consecutive days. The vehicle used for the test substance and β-estradiol was 0.5 % aqueous carboxymethylcellulose.

Bodyweights and detailed clinical observations were recorded and uterus was removed from each animal and trimmed of any fat and adhering non-uterine tissue at the end of the study (approximately 24 hours after administration of the final dose).

The uterus was then opened with a small incision, squeezed and blotted onto filter paper to remove any excess fluid and the uterine wet weight was recorded.

Clinical signs of toxicity related to the test substance did not occur during this study. Although, two animals died soon after dosing and one animal required euthanasia during the study, post-mortem examination revealed dosing accidents to be the cause of death. One animal showed some adverse clinical signs; on termination, examination revealed a dosing accident had occurred. There was no effect of the test item administration on body weight. There was no effect of test item administration on blotted uterine weight. Administration of 0.4 mg β-estradiol/kg bw/day resulted in increased blotted uterine weights. In conclusion, no changes to clinical condition, body weight or uterine weight was observed following oral gavage administration to the immature rat at doses up to 1000 mg/kg bw/day for three consecutive days. There was no evidence of an uterotrophic response to non-micronized ETH50.

Justification for classification or non-classification

The present data on reproductive toxicity do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.

Additional information