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Registration Dossier
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EC number: 250-063-5 | CAS number: 30125-47-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening study: according to OECD 422, GLP, rat, oral, NOAEL for reproductive performance and fertility 1000 mg/kg bw/d (male and female)
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 2011 - June 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation:
- 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 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- - applied as a suspension in water
- 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 to GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- 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
- 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 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Positive control:
- no
- 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 - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - All male animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study, i.e. on study days 33 and 34. Also all female animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces at the end of the study.
- In one female animal of test group 1 (100 mg/kg bw/d) abdominal distension was observed on study days 49 and 50. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female animal of test group 1 (100 mg/kg bw/d) was sacrificed moribund on study day 50 (the animal was not pregnant). It revealed dilation of uterus and cervix with a cloudy fluid content. This was histopathologically diagnosed as a diffuse inflammation with dilation and was regarded to be the cause for the infertility of this mating pair and also the reason for sacrificing this animal. It was a single case and not regarded to be treatment-related but spontaneous in origin.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - During the first premating week as well as in the first gestation week the food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly increased. These findings were assessed as spontaneous in nature and not test substance-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - In males of test groups 1 and 2 (100 and 300 mg/kg bw/d), creatinine values were lower compared to controls, but the means were not dose-dependently decreased. Therefore, this alteration was regarded as incidental and not treatment-related.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test substance-treated groups and controls, were without a doseresponse
relationship or occurred in single rats only, these observations were considered to have been incidental.
- Motor activity measurement: Deviations to the control were only noted in test group 1 (100 mg/kg bw/d), i.e. increased value in interval 9 in male animals and increased value in interval 6 in female animals. As no significant deviations were noted with regard to the overall motor activity and no doseresponse relationship was observed, the findings were assessed as being incidental. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - 2 females of test group 3 (1000 mg/kg bw/d) revealed histopathological yellow crystalline particles within the alveoli, often close to or within macrophages, rarely inside multinucleated giant cells. The same finding was also observed for the female animal of test group 1 that revealed the macropscopic finding “discoloration” in the lung. These yellow particles were regarded to be test substance that was aspired subsequently to the gavage procedure. Therefore, these discolorations in lung and mediastinal lymph node were caused by the test substance but were not regarded to be a treatment-related adverse finding.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Histopathological examination of the stages of spermatogenesis did not reveal any adverse effects.
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - 2 control males, 2 male animals of test group 1, 3 male animals of test group 2 and 1 male animal of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 70% and 90%, what reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- 1 control female, 2 females of test group 1, 3 females test group 2 and 1 female of test group 3 were either sperm-negative or did not become pregnant
- 1 control female and 2 females of test group 2 (300 mg/kg bw/d) were pregnant after mating but had not delivered viable pups (implantation sites only). Thus, the female fertility index varied between 80% and 90%. These findings were assessed to be incidental and not related to treatment as no dose-response relationship was observed. - Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Remarks on result:
- other: no adverse effects observed up to and including the highest tested dose
- Clinical signs:
- no effects observed
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- - one pup of a female of test group 1 and one pup of a female of test group 3 (100 and 1000 mg/kg bw/d) were found dead. These findings were assessed to be incidental and not related to treatment.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Mean pup body weights were significantly lower on PND1 in test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
- One female runt was seen in test group 2 (300 mg/kg bw/d) and 2 male and 3 female runts in test group 3 (1000 mg/kg bw/d).
These values were within the range of the biological variation inherent in the strain of rats used for this study. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- not examined
- Nipple retention in male pups:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - A situs inversus of the heart was observed in 1 pup of test group 3 (1000 mg/kg bw/d). The finding was assessed as being spontaneous in nature and without biological relevance.
- Histopathological findings:
- not examined
- Other effects:
- no effects observed
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Remarks on result:
- other: no adverse effects observed up to and including the highest tested dose
- Reproductive effects observed:
- no
- Conclusions:
- Under the conditions of this Combined Repeated Dose Toxicity Study with the 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.
Reference
Table 1: Summary of female reproduction and delivery data
|
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
Females on Study |
N |
10 |
10 |
10 |
10 |
Females Mated |
N |
l0Fi |
10 |
8 |
10 |
Female Mating Index |
% |
100 |
100 |
80 |
100 |
Mating days until day 0 pc |
MEAN |
3.8 D |
1.9 |
2.3 |
4.2 |
|
S.D. |
3.39 |
1.66 |
1.49 |
3.33 |
|
N |
10 |
10 |
8 |
10 |
days 1 to 4 |
N |
9 |
9 |
8 |
9 |
|
% |
90 |
90 |
100 |
90 |
days 5 to 8 |
N |
0 |
1 |
0 |
0 |
|
% |
0.0 |
10 |
0.0 |
0.0 |
days 9 to 14 |
N |
1 |
0 |
0 |
1 |
|
% |
10 |
0.0 |
0.0 |
10 |
days 15 to 21 |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Females Pregnant |
N |
9Fi |
8 |
7 |
9 |
Female Fertility Index |
% |
90 |
80 |
88 |
90 |
Duration of Gestation (Days) |
MEAN |
22.1 D |
22.0 |
21.8 |
21.9 |
|
S.D. |
0.35 |
0.00 |
0.45 |
0.33 |
Implantation sites |
TOTAL |
101 |
90 |
71 |
123 |
|
MEAN |
11.2 D |
11.3 |
10.1 |
13.7 |
|
S.D. |
3.49 |
2.60 |
6.15 |
2.06 |
|
N |
9 |
8 |
7 |
9 |
Postimplantation Loss |
TOTAL |
5 |
1 |
3 |
2 |
|
MEAN |
0.6 D |
0.1 |
0.4 |
0.2 |
|
S.D. |
0.88 |
0.35 |
0.79 |
0.44 |
|
N |
9 |
8 |
7 |
9 |
% Postimplantation Loss |
MEAN |
13.8 D |
1.6 |
28.6 |
1.4 |
|
S.D. |
32.82 |
4.42 |
48.80 |
2.83 |
|
N |
9 |
8 |
7 |
9 |
Females withLiveborn |
N |
8Fi |
8 |
5 |
9 |
Gestation Index |
% |
89 |
100 |
71 |
100 |
with Stillborn Pups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
with all Stillborn |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
|
TOTAL |
96 |
89 |
68 |
121 |
Liveborn |
N |
96Fi |
88 |
68 |
121 |
Live Birth Index |
% |
100 |
99 |
100 |
100 |
Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Table 2: Summary of litter data
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
(Total Number of) Litters |
N |
8 |
8 |
5 |
9 |
Litters with LivebornPups |
N |
8Fi |
8 |
5 |
9 |
|
% |
100 |
100 |
100 |
100 |
Litters with StillbornPups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
Litters with all Stillborn Pups |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
TOTAL |
96 |
89 |
68 |
121 |
|
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
Pups Liveborn |
N |
96Fi |
88 |
68 |
121 |
|
% |
100 |
99 |
100 |
100 |
Pups Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Pups Died |
N |
0Fi |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Sacrificed Moribund |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Cannibalized |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Accidental Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Sacrificed, Maternal Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Pups dead day 0 |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
days 1 to 4 |
N |
0 |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Surviving days O to 4 |
N |
96Fi |
87 |
68 |
120 |
Viability Index |
% |
100 |
99 |
100 |
99 |
Table 3: Summary of necropsy observations
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Litters Evaluated |
N |
8 |
8 |
5 |
9 |
Pups Evaluated |
N |
95 |
89 |
68 |
120 |
Live |
N |
95 |
88 |
68 |
120 |
Stillborn |
N |
0 |
1 |
0 |
0 |
HEART, SITUS INVERSUS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
TOTAL PUP NECROPSY OBSERVATIONS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
Statistics: D=Dunnett-test (two-sided); Fi =Fisher's exact test (one-sided); Wi=Wilcoxon-test (one-sided)
* : p<=0.05 ** : p<=0.01
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
- Quality of whole database:
- According to OECD TG 422 and GLP, Klimisch 1.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
In a GLP-conform study according to OECD guideline 422, the test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/d. 0.5% Water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation period and 4 days of lactation and 2 weeks thereafter in females. The males were administered up to one-week post-mating. Regarding clinical examinations, no signs of general systemic toxicity were observed in male and female parental animals up to a dose level of 1000 mg/kg bw/d. Yellowish discolored feces were observed in all male and female animals towards the end of the administration period. The effects were related to the test substance but assessed as being non-adverse. Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, macroscopically yellow discoloration of the content of the digestive tract in numerous animals was observed. Beside the discoloration no signs of toxicity in the respective tissues were noted. Therefore, this finding was regarded to be a consequence to the incorporation of the yellow test substance and, therefore, treatment-related but not adverse in nature.
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance 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.
Effects on developmental toxicity
Description of key information
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening study: according to OECD 422, GLP, rat, oral, NOAEL for developmental toxicity was set at 1000 mg/kg bw/d
Prenatal Developmental Toxicity study: Read-across, according to OECD TG 414, GLP, rat, oral, NOAEL for developmental toxicity was set at 1000 mg/kg bw/d
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- August 2011 - June 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation:
- 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 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- - applied as a suspension in water
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - according to GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy - Details on mating procedure:
- - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy - 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
- 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 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization - Maternal 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 - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No - Fetal examinations:
- - External examinations: Yes, all pubs
- Soft tissue examinations: necropsy observations macroscopically
- Skeletal examinations: No
- Head examinations: No
- other: Sex ratio, Pup body weight data, Pup clinical observations - 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 - Indices:
- Pup viability/mortality; Live birth index
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - All female animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces at the end of the study.
- In one female animal of test group 1 (100 mg/kg bw/d) abdominal distension was observed on study days 49 and 50. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- - One female of the low dose group (100 mg/kg bw/d) was sacrificed moribund on study day 50 (the animal was not pregnant). It revealed dilation of uterus and cervix with a cloudy fluid content. This was histopathologically diagnosed as a diffuse inflammation with dilation and was regarded to be the cause for the infertility of this mating pair and also the reason for sacrificing this animal. It was a single case and not regarded to be treatment-related but spontaneous in origin.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - During the first premating week as well as in the first gestation week the food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly increased. These findings were assessed as spontaneous in nature and not test substance-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test substance-treated groups and controls, were without a dose-response
relationship or occurred in single rats only, these observations were considered to have been incidental.
- Motor activity measurement: Deviations to the control were only noted in test group 1 (100 mg/kg bw/d), i.e. increased value in interval 6 in female animals. As no significant deviations were noted with regard to the overall motor activity and no dose-response relationship was observed, the findings were assessed as being incidental. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The weight increase in absolute and relative spleen weight in females of test group 2 (300 mg/kg bw/d) was regarded to be incidental due to a missing dose-response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw/d).
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - 7 females of test group 3 (1000 mg/kg bw/d) revealed a yellow discoloration of the glandular stomach contents. 3 females of test group 3 (1000 mg/kg bw/d) showed the same discoloration of the contents of the jejunum.
- One female animal of test group 1 (100 mg/kg bw/d) revealed a yellow discoloration of the lung (regarded to be test substance that was aspired subsequently to the gavage procedure or due to a gavage error into the trachea) and the mediastinal lymph nodes (regarded to be the physiologic clearing route of the lung).
These discolorations were caused by the test substance but were not regarded to be a treatmentrelated adverse finding. - Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - 2 females of test group 3 (1000 mg/kg bw/d) revealed histopathological yellow crystalline particles within the alveoli, often close to or within macrophages, rarely inside multinucleated giant cells. The same finding was also observed for the female animal of test group 1 that revealed the macropscopic finding “discoloration” in the lung. These yellow particles were regarded to be test substance that was aspired subsequently to the gavage procedure. Therefore, these discolorations in lung and mediastinal lymph node were caused by the test substance but were not regarded to be a treatment-related adverse finding.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Number of abortions:
- not examined
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The mean postimplantation loss was highest in test group 2 (300 mg/kg bw/d), i.e. 28.6% compared to the control group (13.8%). As no dose-response relationship was observed, the finding was assessed as not being related to treatment.
- Total litter losses by resorption:
- not examined
- Early or late resorptions:
- not examined
- Dead fetuses:
- not examined
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 1 female of the control group, 2 females of test group 1 (100 mg/kg bw/d), 3 females of test group 2 (300 mg/kg bw/d) and 1 female of test group 3 (1000 mg/kg bw/d) were either sperm-negative or did not become pregnant. These findings were assessed to be incidental and not related to treatment as no dose-response relationship was observed.
- Other effects:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the limit dose
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- (instead of fetal body weight changes, the body weight of the pups was assessed)
Mean pup body weights were significantly lower on PND1 in test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
One female runt was seen in test group 2 (300 mg/kg bw/d) and 2 male and 3 female runts in test group 3 (1000 mg/kg bw/d).
These values were within the range of the biological variation inherent in the strain of rats used for this study. - Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- one stillborn pup in test group 1 (100 mg/kg bw/d)
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The viability index as indicator for pup mortality between PND 0 and 4 was 99% for test groups 1 and 3 (100 and 1000 mg/kg bw/d; 1 pup of 1 female in group 1 and 3 was found dead).
These findings were assessed to be incidental and not related to treatment. - External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A situs inversus of the heart was observed in one pup of test group 3 (1000 mg/kg bw/d). The finding was assessed as being spontaneous in nature and without biological relevance.
- Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects: no effects
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to and including the limit dose
- Abnormalities:
- not specified
- Developmental effects observed:
- no
- Conclusions:
- Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please see the attached justification.
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Abnormalities:
- not specified
- Description (incidence and severity):
- the study is currently ongoing
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May 2021 - 24 Feb 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 25 June 2018
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- TEST MATERIAL
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g
- Expiry date: 29 December 2027
- Appearance: Solid, orange
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: proven.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspensions of the test item, in 0.5 % CMC, was prepared using the following procedure:
1. The required amount of test item was weighed.
2. The required amount of vehicle was added.
3. The mixture was treated with a Silverson (medium head) for approximately 5 minutes.
4. The resulting suspension was left under magnetic stirring for at least 1 hour prior to dosing or analysis. The formulation was prepared daily or weekly, since the stability data supported the weekly preparation.
FORM AS APPLIED IN THE TEST: suspension in vehicle - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Hannover
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks (virgin females); 11 weeks (males)
- Weight at study initiation: 200-225 g (females); at least 350 g (males)
- Fasting period before study: not specified
- Housing: no more than 5 of one sex to a cage (before mating for all animals and after mating for males); one male with one female rat (during the mating period); individually (mated females)
- Diet (e.g. ad libitum): commercially available laboratory rodent diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 4 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 - 20
- Photoperiod (hrs dark / hrs light): 12 hours
IN-LIFE DATES: From: 2021-05-27 (arrival) To: 2021-07-19 (start of necropsy) - Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5% in water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
1. The required amount of test item was weighed.
2. The required amount of vehicle was added.
3. The mixture was treated with a Silverson (medium head) for approximately 5 minutes.
4. The resulting suspension was left under magnetic stirring for at least 1 hour prior to dosing or analysis. The formulation was prepared daily or weekly.
VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analysis were performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the homogeneity and concentration.
- Details on mating procedure:
- - Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy - Duration of treatment / exposure:
- From Day 6 through Day 19 post coitum.
- Frequency of treatment:
- Once daily
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 300 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- 25 mated female rats/ dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dose levels have been selected by the Sponsor.
- Rationale for animal assignment (if not random): On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified.
- Time of day for (rat) dam blood sampling: The blood sampling was performed on the morning of the day of necropsy. - Maternal examinations:
- CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked early in each working day and again in the afternoon for mortality. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Severely debilitated animals were observed carefully. Clinical signs were recorded daily starting from allocation until sacrifice.
BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 post coitum
- All animals, including those found dead, were euthanized by carbon dioxide inhalation and subjected to necropsy. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
- Organs examined: From all females completing the scheduled test period, the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin.
OTHER:
- Thyroid hormone determination (T3, T4 and TSH): all females, samples taken on day 20 post coitum, determination via immunoanalysis - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not obtained from animals found dead or killed during the study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); gross evaluation of placentae; Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation. - Blood sampling:
- - Plasma: Yes
- Serum: Yes
- Volume collected: approximately 1 mL
- Other: On Day 20 post coitum, blood samples for thyroid hormones determination were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. This procedure was performed within a short timeframe (e.g. two hours, if possible) on the morning of the day of necropsy. Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots (300µL in the aliquot A, the remaining in the aliquot B, if possible) and stored at -20°C, pending analysis.
- Immunoanalysis: Serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH)) - Fetal examinations:
- - External examinations: Yes: all live foetuses
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes: all live foetuses on Day 20 post coitum
Structural deviations were classified as follows:
- Malformations: major abnormalities that are rare and/or affect the survival or health of the species under investigation.
- Anomalies: minor abnormalities that are detected relatively frequently.
- Variants: a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development. - Statistics:
- For continuous variables the significance of the differences amongst group means were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables were carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test. - Indices:
- - Corrected maternal body weight (Body weight on Day 20 post coitum minus gravid uterus weight)
- Corrected maternal body weight gain (Body weight on Day 20 post coitum minus gravid uterus weight minus maternal weight on Day 6 post coitum)
- Pre-implantation loss: [(no. of corpora lutea - no. of implantations) x 100] / no. of corpora lutea
- Post-implantation loss: [(no. of implantations - no. of live foetuses) x 100] / no. of implantations
- Total implantation loss: [(no. of corpora lutea - no. of live foetuses) x 100] / no. of corpora lutea
- Sex ratios of the foetuses were calculated as the percentage of males per litter. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment related adverse clinical signs were described.
Hairloss was observed in one control female, in one mid- dose female and in 2 high dose females.
Considering the low incidence of hariloss and the presence of the sign also in a control animal the observation was deemed representative of normal background variability within the Wistar Han rat.
During the last days of gestation period, yellow faeces were observed in all high dose group females.
One low dose female showed a palpable mass on gestation Days 18-20. The presence of mass is considered spontaneous in origin. - Mortality:
- no mortality observed
- Description (incidence):
- No mortality occurred during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Maternal body weight and body weight gain were unaffected by treatment.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption was comparable between groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No changes were noted. Lower T4 levels were noted at 1000 mg/kg body weight, but the values were within the historical control data. Furthermore, no changes occurred in T3 and TSH values, thyroid gland weights and histopathological examination of the thyroid gland. Therefore, this difference was considered as incidental.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No changes in organ weights were seen between the controls and the treated females.
No changes in gravid uterus weight was observed. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Macroscopic examinations of thyroid
- No changes were seen between the controls and the treated females at macroscopic observations. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Microscopic examinations of thyroid
- No changes were seen between the controls and the treated females at microscopic observations. - Histopathological findings: neoplastic:
- not specified
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group.
- Changes in pregnancy duration:
- not examined
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female.
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Post-implantation losses and the mean number of fetuses per dam were not affected by treatment with the test item at all dose levels. In group 3, a statistically significant higher pre-implantation loss was noted. Since pre-implantation loss occurs before treatment start and in the absence of dose-dependency this difference was considered to be incidental.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- Sex ratios were comparable between the control and the treated groups.
- Changes in litter size and weights:
- not specified
- Anogenital distance of all rodent fetuses:
- no effects observed
- Description (incidence and severity):
- Anogenital distance was unaffected by treatment.
- Changes in postnatal survival:
- not examined
- External malformations:
- no effects observed
- Description (incidence and severity):
- No treatment related findings were observed.
- Skeletal malformations:
- no effects observed
- Description (incidence and severity):
- Observations noted at the skeletal examination were similar between the control and the treated groups.
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A minimally higher incidence of slight enlargement of brain ventricles (56% of the litters) was noted in high dose foetuses, compared to controls. These changeswere also observed in 32% of control litters, 44% of the low dose litters and 30% of the mid-dose litters. Therefore, the slightly higher incidences in the high dose group are considered to be incidental.
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- On the basis of the results, the dosage of 1000 mg/kg/day is considered the NOAEL for maternal and embryo-foetal development.
- Executive summary:
In a GLP-conform study according to OECD guideline 414, the effects of the test item on pregnant female Wistar Han rats and embryo-fetal development were assessed when administered orally by gavage once daily to mated female rats from Day 6 through to Day 19 post coitum, inclusive. Each group consisted of 25 mated female rats. Each group consisted of 25 mated female rats. The test material was administered once daily at dose levels of: 0 mg/kg bw/day (vehicle control); 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (0.5 % carboxy- methylcellulose (CMC)).
Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.
No mortality occurred during the study. All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female. The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group. No treatment related clinical signs were described. Yellow faeces were noted for all high dose females during the last days of gestation period. Maternal body weight and body weight gain were unaffected by treatment. Food consumption was comparable between groups. No changes in thyroid hormone levels were observed. No changes in terminal body weight, gravid uterus weight or absolute weight gain was observed. No changes in organ weights were seen between the controls and the treated females. Litter data and sex rations was comparable between the control and the treated groups. Anogenital distance was unaffected by treatment. No changes were seen between the controls and the treated females at macroscopic or microscopic observations. Regarding the external, visceral and skeletal examinations of foetuses, no treatment related abnormal findings were observed.
Referenceopen allclose all
Table 1: Summary of female reproduction and delivery data
|
ORAL ADMINISTRATION(GAVAGE) - F0 FEMALES (Fl LITTER) SUMMARY OF FEMALE REPRODUCTION AND DELIVERY DATA |
|
|
|||
|
|
TEST GROUP 0 0 MG/KG BW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Females on Study |
N |
10 |
10 |
10 |
10 |
|
Females Mated |
N |
10Fi |
10 |
8 |
10 |
|
Female Mating Index |
% |
100 |
100 |
80 |
100 |
|
Mating days until day 0 pc |
MEAN |
3.8D |
1.9 |
2.3 |
4.2 |
|
|
S.D. |
3.39 |
1.66 |
1.49 |
3.33 |
|
|
N |
10 |
10 |
8 |
10 |
|
days 1 to4 |
N |
9 |
9 |
8 |
9 |
|
|
% |
90 |
90 |
100 |
90 |
|
days 5 to8 |
N |
0 |
1 |
0 |
0 |
|
|
% |
0.0 |
10 |
0.0 |
0.0 |
|
days 9 to 14 |
N |
1 |
0 |
0 |
1 |
|
|
% |
10 |
0.0 |
0.0 |
10 |
|
days 15 to 21 |
N |
0 |
0 |
0 |
0 |
|
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
Females Pregnant |
N |
9Fi |
8 |
7 |
9 |
|
Female Fertility Index |
% |
90 |
80 |
88 |
90 |
|
Duration of Gestation (Days) |
MEAN |
22.1D |
22.0 |
21.8 |
21.9 |
|
|
S.D. |
0.35 |
0.00 |
0.45 |
0.33 |
|
Implantation sites |
TOTAL |
101 |
90 |
71 |
123 |
|
|
MEAN |
11.2 D |
11.3 |
10.1 |
13.7 |
|
|
S.D. |
3.49 |
2.60 |
6.15 |
2.06 |
|
|
N |
9 |
8 |
7 |
9 |
|
Postimplantation Loss |
TOTAL |
5 |
1 |
3 |
2 |
|
|
MEAN |
0.6 D |
0.1 |
0.4 |
0.2 |
|
|
S.D. |
0.88 |
0.35 |
0.79 |
0.44 |
|
|
N |
9 |
8 |
7 |
9 |
|
% Postimplantation Loss |
MEAN |
13.8 D |
1.6 |
28.6 |
1.4 |
|
|
S.D. |
32.82 |
4.42 |
48.80 |
2.83 |
|
|
TEST GROUP 0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Females with Liveborn |
N |
8Fi |
8 |
5 |
9 |
Gestation Index |
% |
89 |
100 |
71 |
100 |
with Stillborn Pups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
with all Stillborn |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
|
TOTAL |
96 |
89 |
68 |
121 |
Liveborn |
N |
96Fi |
88 |
68 |
121 |
Live Birth Index |
% |
100 |
99 |
100 |
100 |
Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Table 2: Summary of litter data
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
(Total Number of) Litters |
N |
8 |
8 |
5 |
9 |
Litters with LivebornPups |
N |
8Fi |
8 |
5 |
9 |
|
% |
100 |
100 |
100 |
100 |
Litters with StillbornPups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
Litters with all Stillborn Pups |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
TOTAL |
96 |
89 |
68 |
121 |
|
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
Pups Liveborn |
N |
96Fi |
88 |
68 |
121 |
|
% |
100 |
99 |
100 |
100 |
Pups Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Pups Died |
N |
0Fi |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Sacrificed Moribund |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Cannibalized |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Accidental Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Sacrificed, Maternal Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Pups dead day 0 |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
days 1 to 4 |
N |
0 |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Surviving days O to 4 |
N |
96Fi |
87 |
68 |
120 |
Viability Index |
% |
100 |
99 |
100 |
99 |
Table 3: Summary of necropsy observations
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Litters Evaluated |
N |
8 |
8 |
5 |
9 |
Pups Evaluated |
N |
95 |
89 |
68 |
120 |
Live |
N |
95 |
88 |
68 |
120 |
Stillborn |
N |
0 |
1 |
0 |
0 |
HEART, SITUS INVERSUS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
TOTAL PUP NECROPSY OBSERVATIONS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
Statistics: D=Dunnett-test (two-sided); Fi =Fisher's exact test (one-sided); Wi=Wilcoxon-test (one-sided)
* : p<=0.05 ** : p<=0.01
TAB. 1: SUMMARY OF REPRODUCTION DATA – GROUP DATA
Observations | Dose Levels [mg/kg/day] | |||
0 | 100 | 300 | 1000 | |
No. Dams Inseminated | 25 | 25 | 25 | 25 |
No. Dams That Conceived | 25 | 25 | 23 | 25 |
Percent Dams Conceived | 100.0 | 100.0 | 92.0 | 100.0 |
No. Dams Died During Study | 0 | 0 | 0 | 0 |
No. Dams with Resorptions only | 0 | 0 | 0 | 0 |
No. of Litters | 25 | 25 | 23 | 25 |
Total No. Corpora Lutea | 276 | 301 | 263 | 303 |
Mean No. Corpora Lutea/Pregnancy | 11.0 | 12.0 | 11.4 | 12.1 |
Total No. Implantation | 269 | 285 | 243 | 293 |
Mean No. Implants/Pregnancy | 10.8 | 11.4 | 10.6 | 11.7 |
Total No. Live Fetuses | 263 | 281 | 235 | 288 |
Mean No. Live Fetuses/Pregnancy | 10.5 | 11.2 | 10.2 | 11.5 |
Total No. Dead Fetuses | 0 | 0 | 0 | 0 |
Mean No. Dead Fetuses/Pregnancy | 0.0 | 0.0 | 0.0 | 0.0 |
No. Dams with Resorptions and/or Dead Fetuses | 6 | 2 | 5 | 4 |
Total No. Resorptions | 6 | 4 | 8 | 5 |
Mean No. Resorptions/Pregnancy | 0.2 | 0.2 | 0.3 | 0.2 |
Preimplantation Loss (%)(mean) | 2.1 | 4.8 | 7.6 | 2.9 |
Post Implantation Loss (%)(mean) | 3.6 | 1.7 | 3.3 | 1.6 |
Mean Foetal Wt. Live Fetuses | 4.13 | 4.09 | 4.18 | 4.11 |
TAB. 2: CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE
Dose Levels [mg/kg/day] | 0 | 100 | 300 | 1000 | |||||
Observations | a | b | a | b | a | b | a | b | |
APPEARANCE | |||||||||
Hairloss | 1 | 4.0 | 0 | 0.0 | 1 | 4.0 | 2 | 8.0 | |
Presence of palpable mass | 0 | 0.0 | 1 | 4.0 | 0 | 0.0 | 0 | 0.0 | |
Coloured faeces, yellow | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 25 | 100.0 | |
Key: () = Number of animals alive at start of interval
a = Number of animals affected
b = Percent of animals with observation during interval
TAB. 3: BODY WEIGHT (g) OF PREGNANT FEMALES - GROUP MEAN DATA
Dose Levels [mg/kg/day] | Day of Phase | ||||||||
| 0! | 3" | 6 | 9 | 12 | 15 | 18 | 20 | |
0 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 215.67 | 225.12 | 237.05 | 244.64 | 259.30 | 274.26 | 306.16 | 330.42 | |
SD | 11.96 | 13.19 | 13.16 | 13.29 | 15.88 | 16.92 | 21.13 | 24.61 | |
100 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 212.70 | 220.04 | 231.39 | 239.71 | 252.47 | 268.15 | 302.95 | 327.30 | |
SD | 14.19 | 17.38 | 16.13 | 17.66 | 17.02 | 19.15 | 20.56 | 25.22 | |
300 | (n) | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
Mean | 216.02 | 226.23 | 236.17 | 243.73 | 257.54 | 274.63 | 308.58 | 333.25 | |
SD | 12.82 | 12.60 | 12.98 | 13.41 | 13.22 | 18.42 | 18.59 | 21.16 | |
1000 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 215.65 | 6 | 5 | 4 | 7 | 3 | 6.98 | 3 | |
SD | 17.29 | 8.54 | 9.24 | 9.35 | 0.93 | 2.21 | 5.26 | 7.77 | |
Note: ! = Gestation phase; " = Dosing/Gestation phase
* = mean value of group is significantly different from control at p < 0.05
** = mean value of group is significantly different from control at p < 0.01
Statistical analysis: Dunnett`s test if group variances are homogeneous
Modified t test if group variances are inhomogeneous ($)
TAB. 4: BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES - GROUP MEAN DATA
Dose Levels [mg/kg/day] | Day of Phase | |||||||
| 3" | 6 | 9 | 12 | 15 | 18 | 20 | |
0 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 225.12 | 237.05 | 244.64 | 259.30 | 274.26 | 306.16 | 330.42 | |
SD | 13.19 | 13.16 | 13.29 | 15.88 | 16.92 | 21.13 | 24.61 | |
100 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 220.04 | 231.39 | 239.71 | 252.47 | 268.15 | 302.95 | 327.30 | |
SD | 17.38 | 16.13 | 17.66 | 17.02 | 19.15 | 20.56 | 25.22 | |
300 | (n) | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
M n | 226.23 | 236.17 | 243.73 | 257.54 | 274.63 | 308.58 | 333.25 | |
S | 12.60 | 12.98 | 13.41 | 13.22 | 18.42 | 18.59 | 21.16 | |
1000 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 6 | 5 | 4 | 7 | 3 | 6.98 | 3 | |
SD | 8.54 | 9.24 | 9.35 | 0.93 | 2.21 | 5.26 | 7.77 | |
Note: Data for Dosing/Gestation phase
* = mean value of group is significantly different from control at p < 0.05
** = mean value of group is significantly different from control at p < 0.01
Statistical analysis: Dunnett`s test if group variances are homogeneous
Modified t test if group variances are inhomogeneous ($)
° = mean daily body weight gain over the previous period starting from gestation day 0
TAB. 5: FOOD CONSUMPTION° (g/animal/day) OF PREGNANT FEMALES – GROUP MEAN DATA
Dose Levels [mg/kg/day] | Day of Phase | |||||||
| 3 | 6 | 9 | 12 | 15 | 18 | 20 | |
0 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 8.55 | 0.90 | 1.02 | 2.54 | 3.82 | 5.84 | 6.04 | |
SD | 3.66 | 2.02 | 2.67 | 2.69 | 1.75 | 2.60 | 2.03 | |
100 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 8.55 | 0.76 | 0.97 | 2.09 | 3.50 | 6.10 | 5.81 | |
SD | 3.40 | 2.51 | 3.31 | 2.07 | 2.24 | 2.85 | 1.93 | |
300 | (n) | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
M n | 8.56 | 0.89 | 1.40 | 2.77 | 5.07 | 7.08 | 6.41 | |
S | 2.74 | 2.32 | 2.27 | 2.50 | 3.57 | 3.15 | 2.31 | |
1000 | (n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Mean | 8.17 | 0.51 | 1.25 | 2.78 | 3.63 | 4.88 | 5.63 | |
SD | 3.58 | 2.50 | 2.71 | 2.84 | 2.32 | 2.64 | 2.75 | |
Note: Data for Dosing/Gestation phase
* = mean value of group is significantly different from control at p < 0.05
** = mean value of group is significantly different from control at p < 0.01
Statistical analysis: Dunnett`s test if group variances are homogeneous
Modified t test if group variances are inhomogeneous ($)
° = food consumed over the previous period starting from Day 0 post coitum
TAB. 6: TERMINAL BODY WEIGHT, GRAVID UTERUS WEIGHT, CORRECTED MATERNAL BODY WEIGHT AND CORRECTED MATERNAL BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA
Dose Levels [mg/kg/day] |
| Terminal Body weight | Gravid uterus weight | Body weight Day 6 | Corrected maternal body weight^ | Corrected body weight gain# |
0 | Mean | 325.81 | 64.98 | 237.96 | 260.83 | 23.77 |
SD | 25.81 | 15.16 | 13.15 | 17.78 | 8.33 | |
(n) | 25 | 25 | 25 | 25 | 25 | |
100 | Mean | 323.78 | 69.04 | 231.40 | 254.74 | 23.34 |
SD | 23.03 | 12.22 | 16.13 | 16.84 | 7.86 | |
(n) | 25 | 25 | 25 | 25 | 25 | |
300 | Mean | 329.40 | 64.38 | 236.17 | 265.02 | 28.84 |
SD | 21.11 | 13.17 | 12.97 | 18.18 | 11.89 | |
(n) | 23 | 23 | 23 | 23 | 23 | |
1000 | Mean | 327.70 | 66.29 | 235.85 | 261.41 | 25.56 |
SD | 26.98 | 15.89 | 19.24 | 32.34 | 22.56 | |
(n) | 25 | 25 | 25 | 25 | 25 |
^ = Corrected maternal body weight at necropsy minus gravid uterus weight
# = Corrected maternal body weight at necropsy minus gravid uterus weight, minus body weight at day 6 of pregnancy
* = mean value of group is significantly different from control
Statistical analysis: Kruskall Wallis test
William’s test if group means are different from control at p < 0.05
TAB. 7: THYROID HORMONE DETERMINATION ON DAY 20 POST COITUM – GROUP MEAN DATA
| Dose Levels [mg/kg/day] | 0 | 100 | 300 | 1000 |
Parameter/units |
|
|
|
|
|
Triiodothyronine nmol/L | Mean | 0.820 | 0.793 | 0.880 | 0.783 |
| SD | 0.134 | 0.103 | 0.127 | 0.113 |
| n | 25 | 25 | 25 | 25 |
Thyroxine nmol/L | Mean | 25.0 | 23.8 | 24.7 | 22.0+ |
| SD | 3.5 | 3.7 | 3.7 | 2.0 |
| n | 25 | 25 | 25 | 25 |
Thyroid stimulating hormone ng/mL | Mean | 7.24 | 7.51 | 7.86 | 7.02 |
| SD | 2.23 | 2.60 | 2.49 | 1.37 |
| n | 25 | 25 | 25 | 25 |
Controls from group(s): 1 Subgroup(s): 1
* = mean value of group is significantly different from control at p < 0.05
+ = mean value of group is significantly different from control at p < 0.01
TAB. 8: LITTER DATA AND SEX RATIOS OF FEMALES - GROUP MEAN DATA
Dose Levels [mg/kg/day] |
| Corpora Lutea | Implan-tations | Uterine Deaths | Viable young | % | Implantation loss (%) | Litter Weight (g) | Mean Foetal Weight (g) | ||||||||
Early | Late | Total | Total | Male | Female | Pre | Post | Total | Male | Female | combined | ||||||
0 | Mean | 11.04 | 10.76 | 0.24 | 0.00 | 0.24 | 10.52 | 4.60 | 6.17 | 45.05 | 2.11 | 3.55 | 5.64 | 43.31 | 4.23 | 4.07 | 4.13 |
SD | 2.75 | 2.57 | 0.44 | 0.00 | 0.44 | 2.57 | 1.68 | 1.83 | 17.98 | 4.67 | 10.18 | 10.68 | 11.63 | 0.73 | 0.68 | 0.69 | |
(n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 24 | 25 | 25 | 25 | 25 | 25 | 25 | 24 | 25 | |
100 | Mean | 12.04 | 11.40 | 0.16 | 0.00 | 0.16 | 11.24 | 5.64 | 5.60 | 50.64 | 4.78 | 1.69 | 6.40 | 45.52 | 4.19 | 4.00 | 4.09 |
SD | 2.26 | 1.96 | 0.55 | 0.00 | 0.55 | 2.19 | 1.96 | 2.22 | 15.65 | 6.96 | 5.85 | 8.82 | 8.87 | 0.59 | 0.57 | 0.56 | |
(n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | |
300 | Mean | 11.44 | 10.57 | 0.35 | 0.00 | 0.35 | 10.22 | 5.30 | 4.91 | 51.35 | 7.60* | 3.34 | 10.91 | 42.74 | 4.30 | 4.07 | 4.18 |
SD | 1.70 | 1.85 | 0.89 | 0.00 | 0.89 | 2.04 | 2.20 | 1.91 | 17.25 | 10.45 | 8.78 | 11.79 | 11.02 | 0.68 | 0.61 | 0.62 | |
(n) | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | |
1000 | Mean | 12.12 | 11.72 | 0.20 | 0.00 | 0.20 | 11.52 | 5.96 | 5.56 | 51.78 | 2.94 | 1.64 | 4.58 | 47.35 | 4.23 | 4.02 | 4.14 |
SD | 1.94 | 1.84 | 0.50 | 0.00 | 0.50 | 1.85 | 1.90 | 1.98 | 14.63 | 7.30 | 4.13 | 7.76 | 8.01 | 0.59 | 0.69 | 0.62 | |
(n) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | |
* = mean value of group is significantly different from control
Statistical analysis: Kruskall Wallis test
William’s test if group means are different from control at p < 0.05
TAB. 9: ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA, MALES
Parameter/units | Group | 0 mg/kg/day | 100 mg/kg/day | 300 mg/kg/day | 1000 mg/kg/day |
ANOGENITAL DISTANCE (mm) | Mean | 4.19 | 4.18 | 4.13 | 4.16 |
SD | 0.37 | 0.41 | 0.27 | 0.32 | |
(n) | 25 | 25 | 23 | 25 | |
Pup weight (g) | Mean | 4.23 | 4.19 | 4.30 | 4.23 |
SD | 0.73 | 0.59 | 0.68 | 0.59 | |
(n) | 25 | 25 | 23 | 25 | |
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g⅓ | Mean | 2.60 | 2.60 | 2.55 | 2.58 |
SD | 0.18 | 0.20 | 0.20 | 0.24 | |
(n) | 25 | 25 | 23 | 25 |
^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)
Statistical analysis: Kruskall Wallis test
T test if group mean differences are different from control at p < 0.05
* = mean value of group is significantly different from control
TAB. 10: ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA, FEMALES
Parameter/units | Group | 0 mg/kg/day | 100 mg/kg/day | 300 mg/kg/day | 1000 mg/kg/day |
ANOGENITAL DISTANCE (mm) | Mean | 2.50 | 2.31 | 2.46 | 2.48 |
SD | 0.44 | 0.43 | 0.39 | 0.35 | |
(n) | 24 | 25 | 23 | 25 | |
Pup weight (g) | Mean | 4.07 | 4.00 | 4.07 | 4.02 |
SD | 0.68 | 0.57 | 0.61 | 0.69 | |
(n) | 24 | 25 | 23 | 25 | |
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g⅓ | Mean | 1.57 | 1.46 | 1.55 | 1.57 |
SD | 0.25 | 0.25 | 0.27 | 0.24 | |
(n) | 24 | 25 | 23 | 25 |
^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)
Statistical analysis: Kruskall Wallis test
T test if group mean differences are different from control at p < 0.05
* = mean value of group is significantly different from control
TAB. 11: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA, FEMALES, BRAIN
Group | 0 mg/kg/day | 100 mg/kg/day | 300 mg/kg/day | 1000 mg/kg/day |
(n) | 25 | 25 | 25 | 25 |
Mean | 1.828 | 1.810 | 1.806 | 1.810 |
SD | 0.094 | 0.116 | 0.109 | 0.068 |
Group diff. at p < 0.05 |
| 0.067 | 0.067 | 0.067 |
Group diff. at p < 0.01 |
| 0.083 | 0.083 | 0.083 |
Data homogeneous by Bartlett's test (Dunnett's test)
Analysis of variance: F ratio = 0.26 Df = 3/ 96 F probability = 0.854
Note: a * indicates group mean is significantly different from control at level of significance shown.
TAB. 12: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA, FEMALES, THYROID
Group | 0 mg/kg/day | 100 mg/kg/day | 300 mg/kg/day | 1000 mg/kg/day |
(n) | 25 | 25 | 25 | 25 |
Mean | 0.0223 | 0.0224 | 0.0244 | 0.0233 |
SD | 0.0037 | 0.0041 | 0.0042 | 0.0039 |
Group diff. at p < 0.05 |
| 0.0027 | 0.0027 | 0.0027 |
Group diff. at p < 0.01 |
| 0.0034 | 0.0034 | 0.0034 |
Data homogeneous by Bartlett's test (Dunnett's test)
Analysis of variance: F ratio = 1.42 Df = 3/ 95 F probability = 0.240
Note: a * indicates group mean is significantly different from control at level of significance shown.
TAB. 13: ORGAN WEIGHTS° TO BRAIN WEIGHT - GROUP MEAN DATA, THYROID, FEMALES
Group | 0 mg/kg/day | 100 mg/kg/day | 300 mg/kg/day | 1000 mg/kg/day |
(n) | 25 | 25 | 25 | 25 |
Mean | 1.220 | 1.238 | 1.352 | 1.288 |
SD | 0.188 | 0.234 | 0.261 | 0.220 |
Group diff. at p < 0.05 |
| 0.153 | 0.155 | 0.153 |
Group diff. at p < 0.01 |
| 0.192 | 0.194 | 0.192 |
Data homogeneous by Bartlett's test (Dunnett's test)
Analysis of variance: F ratio = 1.65 Df = 3/ 95 F probability = 0.181
Note: a * indicates group mean is significantly different from control at level of significance shown.
° = expressed as % organ to brain weight ratio
TAB. 14: MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE - GROUP INCIDENCE, FEMALES
Group | 0 mg/kg/day | 100 mg/kg/day | 300 mg/kg/day | 1000 mg/kg/day |
(n) | 25 | 25 | 25 | 25 |
Whole animal |
|
|
|
|
Forelimbs |
|
|
|
|
Skin |
|
|
|
|
Uterus |
|
|
|
|
TAB. 15: EXTERNAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
Group | 0 mg/kg/day | 100 mg/kg/day | 300 mg/kg/day | 1000 mg/kg/day |
(n) | 25 | 25 | 25 | 25 |
Thyroid THYRO-GLOSSAL DUCT REMNANT | 1 | 1 | 1 | 1 |
Thyroid ECTOPIC THYMIC TISSUE | 1 | 2 | 0 | 1 |
TAB. 16: MICROSCOPIC OBSERVATIONS – FINAL SACRIFICE - GROUP INCIDENCE
Dose Levels [mg/kg/day] | Organ | Cat | Observations | No. Foetuses | No. Litters | ||||
Observed | Affected | % | Observed | Affected | % | ||||
0 | Forelimb | AN | Short | 263 | 1 | 0.38 | 25 | 1 | 4.00 |
Head | AN | Domed shape | 263 | 1 | 0.38 | 25 | 1 | 4.00 | |
Hindlimb | AN | Short | 263 | 1 | 0.38 | 25 | 1 | 4.00 | |
Mouth | AN | Abnormal shape | 263 | 1 | 0.38 | 25 | 1 | 4.00 | |
Whole Foetus |
| No abnormalities detected | 263 | 262 | 99.62 | 25 | 24 | 96.00 | |
100 | Whole Foetus |
| No abnormalities detected | 281 | 281 | 100.00 | 25 | 25 | 100.00 |
300 | Tail | AN | Short | 235 | 1 | 0.43 | 23 | 1 | 4.35 |
Whole Foetus |
| No abnormalities detected | 235 | 234 | 99.57 | 23 | 23 | 100.00 | |
1000 | Hindlimb | AN | Abnormal shape | 288 | 2 | 0.69 | 25 | 2 | 8.00 |
Whole Foetus |
| No abnormalities detected | 288 | 286 | 99.31 | 25 | 25 | 100.00 | |
TAB. 17: FIXED VISCERAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
Dose Levels [mg/kg/day] | Organ | Cat | Observation(s) | No. Foetuses | No. Litters | ||||
|
|
|
| Obs | Aff | % | Obs | Aff | % |
0 | Abdomen | VA | Umbilical vein left | 126 | 11 | 8.73 | 25 | 8 | 32.00 |
| Abdomen | VA | Haemorrhagic | 126 | 3 | 2.38 | 25 | 3 | 12.00 |
| Brain | AN | Ventricles enlarged moderate | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Brain | VA | Ventricles enlarged slight | 126 | 12 | 9.52 | 25 | 8 | 32.00 |
| Ear | MA | Malpositioned | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Forelimb | MA | Focomelia | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Head | MA | Ankyloglossia | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Heart | AN | Septum abnormal size thin | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Hindlimb | MA | Focomelia | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Kidneys | VA | Pelvic dilatation slight | 126 | 9 | 7.14 | 25 | 5 | 20.00 |
| Mouth | MA | Nasal conchae absent | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Tail | AN | Bent | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Testis | AN | Displaced | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Ureter | AN | Enlarged moderate | 126 | 1 | 0.79 | 25 | 1 | 4.00 |
| Ureter | VA | Enlarged slight | 126 | 5 | 3.97 | 25 | 3 | 12.00 |
100 | Abdomen | VA | Umbilical vein left | 134 | 8 | 5.97 | 25 | 5 | 20.00 |
| Abdomen | VA | Haemorrhagic | 134 | 2 | 1.49 | 25 | 2 | 8.00 |
| Brain | AN | Ventricles enlarged moderate | 134 | 1 | 0.75 | 25 | 1 | 4.00 |
| Brain | VA | Ventricles enlarged slight | 134 | 20 | 14.93 | 25 | 11 | 44.00 |
| Great vessels | VA | Innominate artery longer | 134 | 1 | 0.75 | 25 | 1 | 4.00 |
| Kidneys | VA | Pelvic dilatation slight | 134 | 11 | 8.21 | 25 | 7 | 28.00 |
| Testis | AN | Displaced | 134 | 4 | 2.99 | 25 | 3 | 12.00 |
| Ureter | VA | Enlarged slight | 134 | 7 | 5.22 | 25 | 4 | 16.00 |
300 | Abdomen | VA | Umbilical vein left | 111 | 5 | 4.50 | 23 | 5 | 21.74 |
| Brain | VA | Ventricles enlarged slight | 111 | 10 | 9.01 | 23 | 7 | 30.43 |
| Great vessels | VA | Innominate artery longer | 111 | 2 | 1.80 | 23 | 2 | 8.70 |
| Heart | AN | Septum abnormal size thin | 111 | 1 | 0.90 | 23 | 1 | 4.35 |
| Kidneys | VA | Pelvic dilatation slight | 111 | 8 | 7.21 | 23 | 6 | 26.09 |
| Testis | AN | Displaced | 111 | 1 | 0.90 | 23 | 1 | 4.35 |
| Ureter | VA | Enlarged slight | 111 | 4 | 3.60 | 23 | 4 | 17.39 |
1000 | Abdomen | VA | Haemorrhagic | 138 | 1 | 0.72 | 25 | 1 | 4.00 |
| Abdomen | VA | Umbilical vein left | 138 | 13 | 9.42 | 25 | 11 | 44.00 |
| Brain | AN | Ventricles enlarged moderate | 138 | 1 | 0.72 | 25 | 1 | 4.00 |
| Brain | VA | Ventricles enlarged slight | 138 | 23 | 16.67 | 25 | 14 | 56.00 |
| Great vessels | VA | Innominate artery longer | 138 | 1 | 0.72 | 25 | 1 | 4.00 |
| Heart | AN | Septum abnormal size thin | 138 | 1 | 0.72 | 25 | 1 | 4.00 |
| Kidneys | AN | Pelvic dilatation moderate | 138 | 1 | 0.72 | 25 | 1 | 4.00 |
| Kidneys | VA | Pelvic dilatation slight | 138 | 9 | 6.52 | 25 | 5 | 20.00 |
| Testis | AN | Displaced | 138 | 5 | 3.62 | 25 | 5 | 20.00 |
| Thoracic cavity | AN | Haemorrhage | 138 | 1 | 0.72 | 25 | 1 | 4.00 |
| Ureter | AN | Enlarged moderate | 138 | 1 | 0.72 | 25 | 1 | 4.00 |
| Ureter | VA | Enlarged slight | 138 | 8 | 5.80 | 25 | 5 | 20.00 |
TAB. 18: SKELETAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
Dose Levels [mg/kg/day] | Organ | Cat | Observation(s) | No. Foetuses | No. Litters | ||||
|
|
|
| Obs | Aff | % | Obs | Aff | % |
0 | Forepaw(s) | AN | Metacarpal(s) no ossification 4th | 137 | 17 | 12.41 | 24 | 7 | 29.17 |
| Ribs | AN | Wavy | 137 | 13 | 9.49 | 24 | 8 | 33.33 |
| Ribs | VA | 14 ribs | 137 | 16 | 11.68 | 24 | 8 | 33.33 |
| Ribs | VA | Short 14th | 137 | 60 | 43.80 | 24 | 21 | 87.50 |
| Ribs | VA | Rudimentary 14th | 137 | 17 | 12.41 | 24 | 13 | 54.17 |
| Skull | AN | Temporal incomplete ossification | 137 | 10 | 7.30 | 24 | 7 | 29.17 |
| Skull | VA | Parietal incomplete ossification | 137 | 24 | 17.52 | 24 | 12 | 50.00 |
| Skull | VA | Interparietal incomplete ossification | 137 | 47 | 34.31 | 24 | 17 | 70.83 |
| Skull | VA | Hyoid incomplete ossification | 137 | 2 | 1.46 | 24 | 2 | 8.33 |
| Skull | VA | Supraoccipital incomplete ossification | 137 | 47 | 34.31 | 24 | 18 | 75.00 |
| Sternebrae | AN | Asymmetrical ossification | 137 | 3 | 2.19 | 24 | 3 | 12.50 |
| Sternebrae | AN | Asymmetrical ossification 5th | 137 | 4 | 2.92 | 24 | 4 | 16.67 |
| Sternebrae | VA | Incomplete ossification 6th | 137 | 17 | 12.41 | 24 | 9 | 37.50 |
| Sternebrae | VA | No ossification 5th | 137 | 3 | 2.19 | 24 | 2 | 8.33 |
| Sternebrae | VA | Incomplete ossification 5th | 137 | 13 | 9.49 | 24 | 12 | 50.00 |
| Thoracic vertebrae | VA | Centrum incomplete ossification | 137 | 2 | 1.46 | 24 | 2 | 8.33 |
100 | Forepaw(s) | AN | Metacarpal(s) no ossification 4th | 147 | 7 | 4.76 | 25 | 6 | 24.00 |
| Ribs | AN | Wavy | 147 | 6 | 4.08 | 25 | 4 | 16.00 |
| Ribs | VA | Short 14th | 147 | 62 | 42.18 | 25 | 22 | 88.00 |
| Ribs | VA | Rudimentary 14th | 147 | 19 | 12.93 | 25 | 14 | 56.00 |
| Ribs | VA | 14 ribs | 147 | 12 | 8.16 | 25 | 8 | 32.00 |
| Skull | AN | Temporal incomplete ossification | 147 | 8 | 5.44 | 25 | 6 | 24.00 |
| Skull | VA | Supraoccipital incomplete ossification | 147 | 50 | 34.01 | 25 | 15 | 60.00 |
| Skull | VA | Parietal incomplete ossification | 147 | 18 | 12.24 | 25 | 10 | 40.00 |
| Skull | VA | Interparietal incomplete ossification | 147 | 35 | 23.81 | 25 | 14 | 56.00 |
| Skull | VA | Hyoid incomplete ossification | 147 | 5 | 3.40 | 25 | 4 | 16.00 |
| Sternebrae | AN | Asymmetrical ossification | 147 | 2 | 1.36 | 25 | 2 | 8.00 |
| Sternebrae | AN | Asymmetrical ossification 5th | 147 | 7 | 4.76 | 25 | 5 | 20.00 |
| Sternebrae | AN | Bipartite 5th | 147 | 2 | 1.36 | 25 | 1 | 4.00 |
| Sternebrae | VA | Incomplete ossification 6th | 147 | 5 | 3.40 | 25 | 4 | 16.00 |
| Sternebrae | VA | Incomplete ossification 5th | 147 | 10 | 6.80 | 25 | 7 | 28.00 |
| Sternebrae | VA | No ossification 5th | 147 | 3 | 2.04 | 25 | 2 | 8.00 |
| Thoracic vertebrae | VA | Centrum incomplete ossification | 147 | 3 | 2.04 | 25 | 2 | 8.00 |
300 | Cervical vertebrae | AN | Cervical rib(s) | 124 | 1 | 0.81 | 23 | 1 | 4.35 |
| Forepaw(s) | AN | Metacarpal(s) no ossification 4th | 124 | 7 | 5.65 | 23 | 5 | 21.74 |
| Ribs | AN | Wavy | 124 | 11 | 8.87 | 23 | 7 | 30.43 |
| Ribs | VA | Short 14th | 124 | 48 | 38.71 | 23 | 21 | 91.30 |
| Ribs | VA | 14 ribs | 124 | 8 | 6.45 | 23 | 5 | 21.74 |
| Ribs | VA | Rudimentary 14th | 124 | 17 | 13.71 | 23 | 12 | 52.17 |
| Skull | AN | Temporal incomplete ossification | 124 | 6 | 4.84 | 23 | 4 | 17.39 |
| Skull | VA | Supraoccipital incomplete ossification | 124 | 35 | 28.23 | 23 | 12 | 52.17 |
| Skull | VA | Interparietal incomplete ossification | 124 | 23 | 18.55 | 23 | 12 | 52.17 |
| Skull | VA | Parietal incomplete ossification | 124 | 17 | 13.71 | 23 | 9 | 39.13 |
| Sternebrae | AN | Asymmetrical ossification 5th | 124 | 3 | 2.42 | 23 | 3 | 13.04 |
| Sternebrae | AN | Asymmetrical ossification | 124 | 2 | 1.61 | 23 | 2 | 8.70 |
| Sternebrae | VA | Incomplete ossification 6th | 124 | 10 | 8.06 | 23 | 5 | 21.74 |
| Sternebrae | VA | Incomplete ossification 5th | 124 | 11 | 8.87 | 23 | 8 | 34.78 |
| Thoracic vertebrae | VA | Centrum incomplete ossification | 124 | 6 | 4.84 | 23 | 6 | 26.09 |
1000 | Forepaw(s) | AN | Metacarpal(s) no ossification 4th | 150 | 5 | 3.33 | 25 | 4 | 16.00 |
| Ribs | AN | Wavy | 150 | 10 | 6.67 | 25 | 6 | 24.00 |
| Ribs | VA | Rudimentary 14th | 150 | 19 | 12.67 | 25 | 15 | 60.00 |
| Ribs | VA | Short 14th | 150 | 44 | 29.33 | 25 | 21 | 84.00 |
| Ribs | VA | 14 ribs | 150 | 15 | 10.00 | 25 | 7 | 28.00 |
| Sacral vertebrae | AN | Arch(es) incomplete ossification | 150 | 1 | 0.67 | 25 | 1 | 4.00 |
| Skull | AN | Temporal incomplete ossification | 150 | 9 | 6.00 | 25 | 4 | 16.00 |
| Skull | AN | General incomplete ossification | 150 | 5 | 3.33 | 25 | 2 | 8.00 |
| Skull | VA | Interparietal incomplete ossification | 150 | 28 | 18.67 | 25 | 16 | 64.00 |
| Skull | VA | Supraoccipital incomplete ossification | 150 | 44 | 29.33 | 25 | 17 | 68.00 |
| Skull | VA | Parietal incomplete ossification | 150 | 14 | 9.33 | 25 | 10 | 40.00 |
| Skull | VA | Hyoid incomplete ossification | 150 | 3 | 2.00 | 25 | 3 | 12.00 |
| Sternebrae | AN | Asymmetrical ossification | 150 | 3 | 2.00 | 25 | 3 | 12.00 |
| Sternebrae | AN | Asymmetrical ossification 5th | 150 | 4 | 2.67 | 25 | 3 | 12.00 |
| Sternebrae | VA | Incomplete ossification 5th | 150 | 8 | 5.33 | 25 | 6 | 24.00 |
| Sternebrae | VA | Incomplete ossification 6th | 150 | 7 | 4.67 | 25 | 5 | 20.00 |
| Sternebrae | VA | No ossification 5th | 150 | 1 | 0.67 | 25 | 1 | 4.00 |
| Thoracic vertebrae | VA | Centrum asymmetrical dumb-bell shaped | 150 | 1 | 0.67 | 25 | 1 | 4.00 |
| Thoracic vertebrae | VA | Centrum incomplete ossification | 150 | 1 | 0.67 | 25 | 1 | 4.00 |
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
- Quality of whole database:
- - According to OECD TG 422 and GLP, Klimisch 1.
- Read-across to CAS 106276-80-6: according to OECD TG 414 and GLP, Klimisch 1.
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
In a GLP-conform study according to OECD guideline 422, the test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/d. 0.5% Water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation period and 4 days of lactation and 2 weeks thereafter in females. The males were administered up to one-week post-mating. As mentioned above, clinical examinations, reproductive performance, clinical pathology, histopathology and gross pathology did not reveal any finding in treated animals. Clinical examination and gross pathology did not reveal any treatment related adverse effect on pups.
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance 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.
Besides this screening study no data on the developmental toxicity of the actual test substance is available. However, a read-across to an OECD Guideline 414 study with a strucurally analogue substance (CAS 106276-80-6) was performed.
In a GLP-conform study according to OECD guideline 414, the effects of the test item on pregnant female Wistar Han rats and embryo-fetal development were assessed when administered orally by gavage once daily to mated female rats from Day 6 through to Day 19 post coitum, inclusive. Each group consisted of 25 mated female rats. Each group consisted of 25 mated female rats. The test material was administered once daily at dose levels of: 0 mg/kg bw/day (vehicle control); 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (0.5 % carboxy- methylcellulose (CMC)).
Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.
No mortality occurred during the study. All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female. The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group. No treatment related clinical signs were described. Yellow faeces were noted for all high dose females during the last days of gestation period. Maternal body weight and body weight gain were unaffected by treatment. Food consumption was comparable between groups. No changes in thyroid hormone levels were observed. No changes in terminal body weight, gravid uterus weight or absolute weight gain was observed. No changes in organ weights were seen between the controls and the treated females. Litter data and sex rations was comparable between the control and the treated groups. Anogenital distance was unaffected by treatment. No changes were seen between the controls and the treated females at macroscopic or microscopic observations. Regarding the external, visceral and skeletal examinations of foetuses, no treatment related abnormal findings were observed.
On the basis of the results, the dosage of 1000 mg/kg/day is considered the NOAEL for maternal and embryo-foetal development.
Based on the physico-chemical, structural as well as toxicological similarities of the two source substances and the target substance, the same outcome is assumed for the actual substance as for the source substance.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects on fertility or development were observed in a screening study in rats (OECD 422/421). As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.
Additional information
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