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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 5 AUG 2010 to 24 APR 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 5 AUG 2010 to 24 APR 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 422). Although this study is usually not designed to cover this endpoints requirements additional information related to developmental toxicity is reported here.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
not applicable
GLP compliance:
yes
Remarks:
according to Swiss Ordinance [SR 813.115.1]
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: OP 1
- Expiration date of the lot/batch: 16 Oktober 2014
- Certificate of analysis. N° 1873, 15/04/2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 +/- 5 °C)
- Stability of the test substance in the solvent/vehicle: 7 days
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- strain: RccHan: Wistar
- Source: Harlan Laboratories, B.V., Horst, The Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: group means males: 291-319 g; group means females: 185-218 g
- Fasting period before study: no
- Housing:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed.
Afterwards animals were housed individually in Macrolon cages (Type III).
- Diet: pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, Kaiserazgst, Switzerland, batch no. 22/10), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-75.8
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
highly purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- formulations (w/v) were prepared weekly
- storage at room temperature (20 +/-5 °C)
- homogeneity of the test item in the vehicle was maintained during daily administration period using a magnetic stirrer

VEHICLE
- vehicle: highly purified water
- dose volume: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical study was performed to check accuracy of preparation and to determine stability and homogeneity of test substance preparation (HPLC coupled to an ELSD detector).
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: copulation plug observed or daily vaginal smear was sperm positive
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
F0-males:
- at least 28 days (14 days pre-pairing, 14 days maximum during mating, until the day before sacrifice)
F0-females:
- approx. 7 weeks (2 weeks prior to mating, throughout mating (14 days maximum), and approx. 21 days gestation and at least up to day 4 post partum, and including the day before sacrifice)
Frequency of treatment:
once daily
Duration of test:
approx. 7 weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Maternal examinations:
OTHER: Detailed description of examinations performed and observations made on parental animals are reported in chapter 7.5.1 or 7.8.1 respectively.
Fetal examinations:
- External examinations: Yes: [all per litter] macroscopically
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Details on maternal toxic effects:
Maternal toxic effects: no effects. Remark: for detailled description of observations made see results presented in chapter 7.5.1 and 7.8.1.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects were observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
Viability (offspring): no effects
Clinical signs (offspring): no effects
Body weight (offspring): no effects
Sexual maturation (offspring): not examined
Organ weights (offspring): not examined
Gross pathology (offspring): no effects
Histopathology (offspring): not examined

Details on results (offspring)
VIABILITY (OFFSPRING)
Litter size at first litter check: In group 4, one female (no. 78) had only one female pup. Taking this into consideration, birth index and litter size were not considered to be affected.
In order of ascending dose level, birth index was 96.3, 89.6, 93.8 and 95.5. and mean litter size at first litter check was 13.2, 12.4, 12.0 and 11.9.

Postnatal Loss Days 0 - 4 Post Partum: In group 4, one female (no. 78) lost the only one pup on day 2 post partum. The other postnatal losses were: one male pup in another female (no. 79) in group 4 and one male pup in one female (no. 62) in group 3. Excluding the female which lost the only pup on day 2 post partum, the viability index (number of living pups on day 4 p.partum / number of pups born alive) was 100.0%, 100.0%, 99.2% and 99.1%, in groups 1, 2, 3, and 4, respectively.

CLINICAL SIGNS (OFFSPRING)
No test item-related abnormal findings were observed at first litter check or during the lactation until day 4 post partum. The only clinical signs noted were wound on lower jaw in one female pup in group 2 and wound on snout in one female pup in group 4.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 1 post partum were unaffected by treatment with the test item. On day 1 post partum mean pup weights were 5.9, 6.0, 6.0 and 5.9 g in order of ascending dose level. During the lactation period (to day 4 post partum), no test item-related effects were observed in body weight development. Mean pup weights on day 4 post partum were 8.7, 9.2, 9.3 and 9.0 g in order of ascending dose level.

GROSS PATHOLOGY (OFFSPRING)
No abnormal macroscopical findings were observed at macroscopic examination of F1 pups.

OTHER FINDINGS (OFFSPRING)
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.
The proportion of males on day 4 post partum was 41%, 53%, 42% and 48%, in order of ascending dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse toxic effects at higest dose group
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
There were no effects on parental animals or any changes seen at macroscopic examination of the offspring that were considered to be an effect of treatment.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
not applicable
GLP compliance:
yes
Remarks:
according to Swiss Ordinance [SR 813.115.1]
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: OP1
- Expiration date of the lot/batch: 16 October, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 +/- 5 °C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- strain: RccHan:Wistar
- Source: Harlan Laboratories, B.V., Horst, The Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: group means males: 291-319 g; group means females: 185-218 g
- Fasting period before study: no
- Housing:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed.
Afterwards animals were housed individually in Macrolon cages (Type III).
- Diet: pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, Kaiserazgst, Switzerland, batch no. 22/10), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-75.8
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(highly purified)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- formulations (w/v) were prepared weekly
- storage at room temperature (20 +/-5 °C)
- homogeneity of the test item in the vehicle was maintained during daily administration period using a magnetic stirrer

VEHICLE
- vehicle: highly purified water
- dose volume: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: copulation plug observed or daily vaginal smear was sperm positive
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical study was performed to check accuracy of preparation and to determine stability and homogeneity of test substance preparation (HPLC coupled to an ELSD detector).
Duration of treatment / exposure:
F0-males:
- at least 28 days (14 days pre-pairing, 14 days maximum during mating, until the day before sacrifice)
F0-females:
- approx. 7 weeks (2 weeks prior to mating, throughout mating (14 days maximum), and approx. 21 days gestation and at least up to day 4 post partum, and including the day before sacrifice)
Frequency of treatment:
once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a dose range finding study with the test item (in Han Wistar rats, Harlan Laboratories Study, using dose levels of 100, 300 and 1000 mg/kg/day).
Positive control:
none

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- clinical signs time schedule: daily cage-side observation (during acclimatisation up to necropsy), females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
-detailed clinical signs time schedule: Once prior to the first administration of the test item and weekly thereafter, observations were performed outside the home cage.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0-generation (parental animals): daily from treatment start to day of necropsy
F1-generation (pups): weighed on day 0 (if possible), day 1 and 4 post partum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly, but not during the mating period
(in detail: Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

OTHER:
Observations on haematology, clinical chemistry and neurobehavioural examination are reported in section 7.5.
Sperm parameters (parental animals):
Parameters examined in males: Epididymides, Prostate Glands, Seminal Vesicles and Coagulating Glands, microscopic examination of the integrity of the spermatogenietic cycle


Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The litters were examined for
- litter size,
- live births,
- still births and
- any gross anomalies.
- The sex ratio of the pups was recorded.
- Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: at least until the minimum total dosing period of 28 days
- Maternal animals: On day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

GROSS PATHOLOGY: Yes (with special attention to the reproductive organs; The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites)

HISTOPATHOLOGY: Yes
- from all males: prostate, testes, seminal vesicles with coagulating gland, epididymides
- from all females: ovaries
- from 5 animals/sex/group: any gross lesions, adrenal glands, brain, heart, kidneys, liver, spleen, thymus, spinal chord, small and large intestines, stomach, thyroids, trachea and lungs (inflation and immersion), uterus with vagina, urinary bladder, lymph nodes (mandibular, mesenterical), peripheral nerv (sciatic), bone marrow

Other examinations
ORGAN WEIGHTS
- from all males: testes, epididymides
- from 5 animals/sex/group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic).

GROSS NECROPSY
- Gross necropsy and external examinations were performed

HISTOPATHOLOGY / ORGAN WEIGTHS: No
Statistics:
- means and standard deviations were calculated
- Dunnett-test
- Steel-test
- exact Fisher-test



Reproductive indices:
Percentage mating = (Females mated / Females paired) * 100
Fertility index = (Females achieving a pregnancy / Females paired) * 100
Conception rate = (Females achieving a pregnancy / Females mated) * 100
Gestation index = (Number of females with living pups / Number of females pregnant) * 100
Offspring viability indices:
Birth index = (number of pups born alive / number of implantations) * 100
Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100
- percentage live males at first litter check: (number of live male pups)/(number of live pups) * 100
- percentage live females at first litter check: (number of live female pups)/(number of live pups) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Description (incidence and severity):
Test substance intake: not applicable for a gavage study

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related clinical signs were observed (see section 7.5.1).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls (see section 7.5.1).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no differences in food consumption (absolute and relative) between treated and control animals (see section 7.5.1).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
The histopathological evaluation of the reproductive organs did not reveal any test-item related changes in animals of the high-dose group, even in the evaluation with special emphasis on stages of spermatogenesis and histopathology of interstitial cell morphology.
Higher degree of oligospermia in epididymides was recorded in animal nos. 6 and 8, which was considered to be associated with the tubular atrophy of testes. Nothing special related to the infertility was observed in prostate glands, seminal vesicles and coagulating glands of these infertile animals.
Nothing special related to the infertility was observed in the other infertile animals (nos. 1 and 4 of group 1, no. 16 of group 2, and no. 36 of group 4).
Overall the assessment of the integrity of the spermatogenesis in testes did not provide any evidence of impairment related to the treatment of test item.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Six females (4 females on vehicle control group and one in each of group 2 and 4 (los and high dose)) were non-pregnant.

One female in group 2 (no. 52) mated during the second additional pairing period. All the other females mated during the first pairing period. In groups 1, 2, 3 and 4, mean precoital time was 4.4, 1.7, 1.9 and 2.2 days and median precoital time was 3, 2, 2 and 2 days, respectively. For the
female which mated during the second period the precoital time was 4 days.
Four females in group 1, one female each in groups 2 and 4 were not pregnant. All the pregnant females delivered pups. Therefore, the gestation index was 100% in all groups.

The mean duration of gestation was unaffected by the treatment with the test item. Mean duration of gestation was 21.2, 21.3, 21.4 and 21.2 days, in order of ascending dose level.

Mean number of corpora lutea per dam (determined at necropsy) was 14.7, 15.9, 14.7 and 14.1 in order of ascending dose level and gave no indication of a test item-related effect.

The mean number of implantations per dam did not give any indication of a test item-related effect.
The higher incidence of post-implantation loss which occurred in groups 2 and 3 (10.4% and 6.3% compared to 3.7% in the control group) was considered to be incidental since it did not follow a dose-dependent pattern. The mean numbers of implantations per dam were 13.7, 13.9, 12.8 and 12.4 in order of ascending dose level.
The mean incidence of post-implantation loss as a percentage of total implantations was 3.7%, 10.4%, 6.3% and 4.5%, in order of ascending dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weights and organ:body weight of treated animals were considered to be similar to those of control animals (see section 7.5.1).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations of the remaining animals at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment (see section 7.5.1).

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no microscopic findings recorded which could be attributed to treatment with the test substance (see section 7.5.1).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

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 examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Litter size at first litter check: In group 4, one female (no. 78) had only one female pup. Taking this into consideration, birth index and litter size were not considered to be affected.
In order of ascending dose level, birth index was 96.3, 89.6, 93.8 and 95.5. and mean litter size at first litter check was 13.2, 12.4, 12.0 and 11.9.

Postnatal Loss Days 0 - 4 Post Partum: In group 4, one female (no. 78) lost the only one pup on day 2 post partum. The other postnatal losses were: one male pup in another female (no. 79) in group 4 and one male pup in one female (no. 62) in group 3. Excluding the female which lost the only pup on day 2 post partum, the viability index (number of living pups on day 4 p.partum / number of pups born alive) was 100.0%, 100.0%, 99.2% and 99.1%, in groups 1, 2, 3, and 4, respectively.

CLINICAL SIGNS (OFFSPRING)
No test item-related abnormal findings were observed at first litter check or during the lactation until day 4 post partum. The only clinical signs noted were wound on lower jaw in one female pup in group 2 and wound on snout in one female pup in group 4.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 1 post partum were unaffected by treatment with the test item. On day 1 post partum mean pup weights were 5.9, 6.0, 6.0 and 5.9 g in order of ascending dose level. During the lactation period (to day 4 post partum), no test item-related effects were observed in body weight development. Mean pup weights on day 4 post partum were 8.7, 9.2, 9.3 and 9.0 g in order of ascending dose level.

GROSS PATHOLOGY (OFFSPRING)
No abnormal macroscopical findings were observed at macroscopic examination of F1 pups.

OTHER FINDINGS (OFFSPRING)
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.
The proportion of males on day 4 post partum was 41%, 53%, 42% and 48%, in order of ascending dose level.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no changes in litter observations that were considered to be an effect of treatmemt

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, reproduction, litter observation, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the test item administered by oral gavage in Wistar rats (10/sex/dose) was performed according to OECD guideline 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day. Oral dosing of male and female Wistar rats with the test item for at least 28 days (males; females were treated up to approx. 7 weeks), revealed no treatment-related findings on parental animals, on fertility, on embryo-foetal development, or on pup development.From the results presented (absence of adverse effects) in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.