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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD 422 compliant study with well characterized material
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: See chapter "Repeated dose toxicity: oral" describing results on repeated dose effects in more details.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Substance type: blue solid
- Physical state: solid
- Purity test date: 2012
- Lot/batch No.: 110075
- Expiration date of the lot/batch: April 12, 2014
- Stability under test conditions: stable
- Storage condition of test material: at room temperature
- Other: The test substance appeared to be homogeneous.

Test animals

Species:
rat
Strain:
other:
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks
- Weight at study initiation: males: ca 200 g (females) and 320 g (males)
- Housing: The rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). Pregnant animals and their litters were housed together until day of parturition (PND) 4 (end of lactation).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

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: 2012-03-19 to: 2012-05-14

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.

- The administration volume was 10 mL/kg bw.
Details on mating procedure:
- 14 days after the beginning of treatment, males and females from the same test group were mated for a maximum of 2 weeks.
Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy and the following day gestation day (GD) 1
- After successful mating each pregnant female was caged (how): Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- The females were allowed to litter and rear their pups until day 4 after parturition.
On PND 4, all pups were sacrificed under Isoflurane anesthesia with CO₂ and examined.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 95-101% of the nominal concentrations.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.
Females: 52 days
Males: 31 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 or 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 14-day range-finding study

Examinations

Parental animals: Observations and examinations:
see also repeated dose toxicity: rat.OECD422
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity; a check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. Thereby, the following parameters were examined:
1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals; during the administration period on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• 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.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Pup number and status at delivery
• Total number of pups and the number of liveborn and stillborn pups in each litter on the day of birth.
• Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
- Pup viability/mortality
• Check for dead or moribund pups twice daily on workdays and once on Saturdays, Sundays or public holidays.
• Number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period).
• Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations.
• The number of live pups/litter was calculated on the day after birth, and on lactation day 4.
- Pup clinical observations
• The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.
- Pup body weight data
• The pups were weighed on the day after birth (PND 1) and on PND 4.
• “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
• Sex ratio at day 0 and day 4 after birth = number of live male or female pups on day 0 and 4/ number of live male and female pups on day 0 and 4 x 100
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. The sex of the pups was finally confirmed at necropsy.

GROSS EXAMINATION OF DEAD PUPS:
• 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.
All pups were discarded after their evaluation.
Postmortem examinations (parental animals):
SACRIFICE
- All animals were sacrificed by decapitation under Isoflurane anesthesia.

GROSS NECROPSY
- The exsanguinated animals were necropsied and assessed by gross pathology.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight parameters:
Weight assessment was carried out on all animals.
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

- Organ / Tissue preservation
The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
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 (modified Davidson’s solution)
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

From the liver, each one slices of the lobus dexter medialis and the lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast.

HISTOPATHOLOGY: Yes
Organ samples / Methods-Scope of examinations / Test group
1. All gross lesions: Hematoxylin-eosin (H&E), all affected animals per group, all treatment groups
2. Adrenal glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
3. Bone marrow (femur): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
4. Brain: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
5. Cecum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
6. Cervix: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
7. Coagulation glands: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
8. Colon: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
9. Duodenum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
10. Epididymides: Hematoxylin-eosin (H&E), all affected animals per group
11. Heart: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
12. Ileum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
13. Jejunum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
14. Kidneys: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
15. Liver: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group; low and mid dose group: embedded in paraplast all animals per group.
16. Lung: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
17. Lymph nodes (mesenteric and axillary lymph nodes): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
18. Ovaries: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
19. Oviducts: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
20. Peyer’s patches Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
21. Prostate: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
22. Rectum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
23. Sciatic nerve: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
24. Seminal vesicles: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
25. Spinal cord (cervical, thoracic and lumbar cords): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
26. Spleen: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
27. Stomach (forestomach and glandular stomach): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
28. Testes: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
29. Thymus: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
30. Thyroid glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
31. Trachea: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
32. Urinary bladder: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
33. Uterus: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
34. Vagina: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
Postmortem examinations (offspring):
SACRIFICE
- All surviving pups were sacrificed on PND 4 under isoflurane anesthesia with CO2.

GROSS NECROPSY
- All surviving pups, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups were discarded after their evaluation.
Statistics:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: DUNNETT-test (two-sided);
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions;
- Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, clinical pathology parameters (except for urine color and turbidity), weight parameters: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). 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 (two-sided) for the equal medians.
Reproductive indices:
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
- Male mating index (%) = number of males with confirmed mating*/number of males placed with females x 100
*defined by a female with vaginal sperm or with implants in utero
- Male fertility index (%) = number of males proving their fertility*/ number of males placed with females x 100
* defined by a female with implants in utero
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
- Female mating index (%) = number of females mated*/ number of females placed with males x 100
* defined as the number of females with vaginal sperm or with implants in utero
- Female fertility index (%) = number of females pregnant*/ number of females mated** x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero
- Gestation index (%) = number of females with live pups on the day of birth/number of females pregnant* x 100
* defined as the number of females with implants in utero
- Live birth index (%) = number of liveborn pups at birth/total number of pups born x 100
- Post implantation loss (%) = number of implantations number of pups delivered / number of implantations x 100
Offspring viability indices:
Viability index (%) = number of live pups on day 4 after birth/number of live pups on the day of birth x 100

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

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in all test groups.
Fertility was proven for the majority of the F0 parental males within the scheduled mating interval to produce F1 litter. However, one male (animal no. 26 mated with female no. 126) of test group 2 did not generate F1 pups. Thus, the male fertility index ranged between 90% and 100%. These values 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

The mean duration of gestation was 22.3 days in test group 0, 22.2 days in test group 1, 22.0 days in test group 2 and 22.1 days in test group 3 and did not show any significant difference.
The female mating index calculated after the mating period for F1 litter was 100% for control group, test group 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d) and 90% for test group 2 (300 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.7, 1.7 and 1.8 days in test groups 0, 2 and 3, respectively. In contrast, mean duration until sperm detection was significantly prolonged by +0.4 days versus control in test group 1 and amounted to 3.1 days. However, this finding was not dose-dependent and therefore regarded as incidental and spontaneous in nature.

All sperm positive rats delivered pups or had implantation sites with the exception of female animal no. 126 of test group 2, which was mated with male animal no. 26. This female was sperm-positive but did not become pregnant. Thus, the female fertility index varied between 90% and 100% .


The gestation index was 100% in all test groups.

The live birth index was 91.7%, 97.2, 100% and 95.7% in test groups 0-3, respectively. The female no. 101 of the control delivered 9 stillborn pups, female no. 115 of the low dose group delivered 3 stillborn pups and animal no. 136 of the high dose group delivered 4 stillborn pups.

The post implantation loss amounted to 0.91%, 9.87%, 8.27% and 6.48% in test groups 0, 1,2 and 3, respectively.

For all test groups the rate of liveborn and stillborn pups, as well as, post implantation loss reflected the normal range of biological variation inherent in the strain used in this study.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

The mean number of delivered pups per dam amounted to 10.8, 10.9, 9.4 and 9.4 in test groups 0-3, respectively. One female of the control delivered 9 stillborn pups, female no. 115 of the low dose group delivered 3 stillborn pups and animal no. 136 of the high dose group delivered 4 stillborn pups. For all test groups the rate of liveborn and stillborn pups reflected the normal range of biological variation inherent in the strain used in this study.

The viability index, an indicator for pup mortality between PND 0 and 4, amounted to 100% for the control, 98.5% for test group 1 (one pup of animal no. 116 was found dead / one pup was cannibalized), 99.0% for test group 2 (one pup was found dead) and 98% for test group 3 (one pup was found dead). All findings were regarded incidental and not related to treatment.

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show any biologically relevant differences between test groups.
On postnatal day 0 gender (males/females) within the test groups was distributed as follows: 51.5%/ 48.5% in the control group, 49.1%/ 50.9% in test group 1, 57.6%/ 42.4% in test group 2 and 50.0%/ 50.0% in test group 3.

Body weight development was normal. No treatment-related changes were noted during pup necropsy.

Effect levels (F1)

Dose descriptor:
NOEL
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: Observation until postnatal day 4

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

The NOEL for fertility and developmental toxicity was 1000 mg/kg bw/day based on the parameters examined in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.