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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:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 01256 BJ7
- Expiration date of the lot/batch: 24 june 2017

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 11 weeks
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 9th, 2011 To:October 6th, 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): in intervals, which took into account the analytical results of the stability verification
- Mixing appropriate amounts with: drinking water 1% CMC
- Storage temperature of food: RT
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and litter together
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- according GLP
- Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study. Samples of the test substance preparations were sent to the analytical laboratory twice (at the beginning and towards the end of the administration period) for verification of the concentrations.
- method stability of test item in drinking water: UV/VIS spectroscopy
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13-14 weeks

Doses / concentrationsopen allclose all
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: range finder test at 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization
Positive control:
no

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Sperm parameters (parental animals):
stages of spermatogenesis were examined in histopathology
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 up to day 4, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

HISTOPATHOLOGY
ORGAN WEIGHTS: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups delivered from the F0 parents were examined as soon as possible on the day of birth

GROSS NECROPSY
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
Statistics:
Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test
Reproductive indices:
Male reproduction data:
- Male mating index
- Male fertility index

Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index
- Live birth index
- Post implantation loss
Offspring viability indices:
Pup number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data

Results and discussion

Results: P0 (first parental animals)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

All male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) had orange discolored feces from study day 1 onwards until terminal sacrifice. This feces discoloration mirrors the presence of the test substance (or its metabolites) in the gastrointestinal tract. It was not considered to be an adverse toxic effect.

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: Orange discolored feces, no adverse toxic effect observed

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
Histopathological findings:
not examined

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 adverse effect observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Thus, under the conditions of this Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.