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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on an OECD 422 compliant study, the NOAEL for oral repeated dose toxicity in rats was determined to be 10 mg/kg bw/d based on increased white blood cell count and thyroid hyperplasia findings in female and male animals, respectively.

The NOAEL for fertility and reproductive performance was 100 mg/kg bw/d for the F0 parental animals.

A NOAEL for developmental toxicity in the offspring was determined to be 30 mg/kg bw/d based on cleft palates and their subsequent adverse effects on pup survival and growth.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-19 to 2017-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0008924462
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 11-13 weeks
- Weight at study initiation: males: 318.3 g - 356.9 g; females 181.3 g - 213.5 g
- Females nulliparous and non-pregnant: yes
- Housing: individually, except during overnight matings and dams with their litters
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: ca. 5 days

Male and female Wistar rats, strain Crl:WI(Han), supplied by Charles River Laboratories Research Models and Services, Germany GmbH, which were free from any clinical signs of disease, were used for the investigations. The females were nulliparous and non-pregnant at the beginning of the study. The receipt of males (11-12 weeks old) and females (10 weeks old) at different age warrants that no sibling males and females will be paired during the study. These animals were used as F0 generation parental animals. All other animals used in this study (F1 generation pups) were derived from the supplier-provided animals.

DETAILS OF FOOD AND WATER QUALITY:
The supplier assayed the food used in the study for chemical and microbiological contaminants. The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 15 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose suspension in drinking water with 5 mg/100 mL Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance suspensions in 0.5% Carboxymethylcellulose suspension in drinking water with 5 mg/100 mL Tween 80 were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% Carboxymethylcellulose suspension in drinking water with 5 mg/ 100 mL Tween 80 and intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 1% Carboxymethylcellulose suspension in drinking water + 5 mg/100 ml Tween 80 for a period of 7 days in a refrigerator were carried out prior to the start of the study. Samples of the test substance preparations were sent to the analytical laboratory once during the study period for verification of the concentration. The samples, which were taken for the concentration control analysis at the beginning of the administration period, were also used to verify the homogeneity of the samples of the low- and high-concentration. From these concentrations three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running. Of each sample, one additional reserve sample (described by the suffix “R”) was retained. Details of the sampling schedule were recorded with the raw data.
Duration of treatment / exposure:
males: 14 days pre-mating; 14 days during mating; 2 days post-mating => 29 days
females: 14 days pre-mating; 14 days during mating; approx. 22 days gestation; 13 days lactation period => 63 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on availble acute toxicity data
- Rationale for animal assignment: random
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily before administration as well as within 2 hours and within 5 hours after the administration .
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week
Exceptions:
- 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 positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION: Yes
- once a week for parental animals with the exceptions:
- Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20.
- Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: Yes with isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: A functional observational battery (FOB) was performed in the first five male and the first five female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / home cage observations / open field observations

Home cage observations:
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observations:
The animals were transferred to a standard arena (50 * 50 * 25 cm) and observed. The following parameters were examined:
1. Behavior on removal from cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypy
14. Gait
15. Activity/arousal level
16. Feces (consistency/color) within 2 minutes
17. Urine (amount/color) within 2 minutes
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/Reflexes:
The animals were removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity measurement:
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
state of spermatogenesis, testis weight, epididymis weight, sperm morphology
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, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. The following animals died intercurrently (animal No. 36) or were sacrificed moribund (animal No. 138) and were necropsied and assessed by gross pathology as soon as possible after their death.

Organ weights:
The following weights were determined in all animals sacrificed on schedule:
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 fixation:
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson's solution:
1. All gross lesions
2. Adrenal glands
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 glands
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. Testes (modified Davidson's solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus (uteri of all cohabited female F0 parental animals were stained according to Salewski's method: see chapter: Female reproduction and delivery data)
48. Vagina

The testes, epididymides and ovaries of animals that died/were sacrificed intercurrently were fixed in 4% neutral-buffered formaldehyde solution.

HISTOPATHOLOGY: Yes
Please refer to table 7.
Animals that died/were sacrificed in a moribund state were processed histotechnically and assessed with all organs listed above like control animals.
Special attention was given on the stages of spermatogenesis in the testes. The organs were trimmed according to the "Revised guides for organ sampling and trimming in rats and mice" (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004). A correlation between gross lesions and histopathological findings was attempted.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at PND 4 days of age.
- These animals were subjected to postmortem examinations

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Please refer to any other information on materials and methods
Reproductive indices:
Please refer to any other information on materials and methods
Offspring viability indices:
Please refer to any other information on materials and methods
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Several male and female animals of test group 3 (100 mg/kg bw/d) showed soft feces and/or discolored feces (light brown) during premating.
Several male animals of test groups 3 and 2 (100 mg/kg bw/d and 30 mg/kg bw/d) showed salivation after treatment (grade: slight to severe) during premating (test group 3), mating and post-mating (test group 3 and 2).
Several female animals of test groups 3 and 2 showed salivation after treatment (grade: slight to severe) during premating and mating (test group 3) and gestation and lactation (test groups 3 and 2).
One mid-dose male (No. 21) showed labored respiration (slight to moderate) on mating days 2 - 6 and encrusted eyes (red) on mating days 3 - 6. These isolated findings in one animal were considered as possibly treatment related but not substance related as no dose-response relationship occurred.
Two high-dose females (Nos. 133 and 140) showed encrusted eye (both, red) on GD 22 and GD 22 - 23 and on PND 0 - 1 and PND 0, respectively. One high-dose (female No. 140) also showed piloerection on PND 3 - 4.
No clinical signs or changes of general behaviour, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals of test group 1 (10 mg/kg bw/d) during the entire study including gestation and lactation periods.
Four high-dose females did not properly nurse their pups (Nos. 131 [PND 1], 133 [PND 4], 136 [PND 1] and 137 [PND 1]).
Three high-dose females had all pups stillborn (Nos. 132, 138 and 140). One high-dose female had a complete litter loss (No. 136 on PND 2).
One sperm positive low-dose female (10 mg/kg bw/d - No. 115) and one sperm negative and one sperm positive control female (0 mg/kg bw/d - No. 102 and 103, respectively) did not deliver F1 pups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One parental male of test group 3 (No. 36 - 100 mg/kg bw/d) was found dead on mating day 7. One parental high-dose female (No. 138) was sacrificed moribund on PND 5 because it showed semiclosed eyelid (both), piloerection, reduced nutritional condition (severe), encrusted eye (both, red), closed eyelid (both), hypothermia and smeared fur (anogenital region, light brown). There were no other test substance-related mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of the high-dose parental females were statistically significantly below the concurrent control values on PND 4 (about 12%).
The mean body weights of all test-substance treated parental males and of the mid- and low-dose parental females during the entire study period as well as of the high-dose parental females during premating and gestation period were comparable to the concurrent control group.
The mean body weight change of the high-dose parental females was statistically significantly below the concurrent control values during GD 14 - 20 and 0 - 20 (about 25% and 19%, respectively) and during PND 0 - 4 (about 12%).
The mean body weight change of all test-substance treated parental males and of the mid- and low-dose parental females during the entire study period as well as of the high-dose parental females during premating period was comparable to the concurrent control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 females (100 mg/kg bw/d) was statistically significantly below the concurrent control values during premating days 0 - 7 and 0 - 13 (about 17% and 9%, respectively), during GD 14 - 20 (about 12%) and during PND 1 - 4 (about 60%).
The food consumption of all test substance-treated F0 male animals (100, 30 and 10 mg/kg bw/d) and the mid- and low-dose F0 females was comparable to the concurrent control values throughout the entire study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In female rats of test group 3 (100 mg/kg bw/d) hemoglobin and hematocrit values were lower and relative reticulocyte counts were higher compared to controls.
In females of test groups 2 and 3 (30 and 100 mg/kg bw/d) total white blood (WBC) counts were increased which was due to higher absolute neutrophil cell counts (in females of test group 2 not statistically significantly increased). Absolute and relative large unstained cell (LUC) counts were slightly increased in females of test group 3 (100 mg/kg bw/d). Absolute lymphocyte cell counts were also higher in females of test groups 2 and 3 (30 and 100 mg/kg bw/d; in test group 2 not statistically significant), but the means were within the historical control range (absolute lymphocytes 1.99-2.97 Giga/L). Relative basophil cell counts were decreased in females of test group 3 (100 mg/kg bw/d). No pathophysiological correlate to decreased basophil cell counts is known. Therefore, the mentioned changes of lymphocyte and basophil cell counts were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In rats of both sexes of test group 3 (100 mg/kg bw/d) aspartate aminotransferase (AST) activities were increased and additionally in females of the same test group alanine aminotransferase (ALT) activities and triglyceride values were higher compared to controls. In males the AST activity was within the historical control range (AST 1.32-2.00 ukat/L). Therefore, the AST alteration in males of test group 3 (100 mg/kg bw/d) was regarded as incidental and not treatment-related.
In females of test groups 1 and 2 (10 and 30 mg/kg bw/d) total bilirubin levels were higher compared to controls, but the values were not dose-dependently changed and therefore, this alteration was regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Detailed clinical observations (DCO)
One female animal of dose group 3 (No. 138 - 100 mg/kg bw/d) showed reduced nutritional condition (severe), piloerection and semiclosed eyelid (both) on DCO day 42 (This animal was sacrificed moribund on PND 5 see 4.2.1.1).
All male and all other female animals of all dose groups (10, 30 and 100 mg/kg bw/d) did not show any abnormalities.

Functional observational battery (FOB)
Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. One male animal of dose group 3 (No. 33 - 100 mg/kg bw/d) showed slight salivation (area around the mouth was moist).

Sensorimotor tests/reflexes: In the pupillary reflex test 4 out of 5 examined female animals of dose group 3 showed a retarded adaption of the pupil to light. This was assessed as substance related adverse finding. There were no other test substance-related findings in male and female animals of all test groups.

Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly decreased values of the landing foot splay test in females of dose group 1 was considered as spontaneous in nature and not treatment related as no dose-response relationship occurred.

Motor activity measurement (MA):
Motor activity measurement was statistically significantly below the concurrent control values in females of test group 3 (100 mg/kg bw/d) during early exploring phase (intervals 1 and 3, about 37% and 55%, respectively). Even though it did not reach statistical significance a decrease was also seen in males and therefore assessed as treatment related finding. The statistically significantly increased motor activity measurement in females of dose group 1 and 2 (10 and 30 mg/kg bw/d) during interval 9 and the statistically significantly decreased values in females of test group 1 (10 mg/kg bw/d) during interval 11 (about 83%) was considered as spontaneous in nature and not treatment related as no dose-response relationship occurred. No other statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the heart, there was necrosis of muscle fibers and infiltration of inflammatory cells, characterized by mainly macrophages and less granulocytes. The finding was regarded to be treatment-related. In the prostate an increase in numbers of nuclei and the height of the epithelium was noted. This finding was diagnosed as minimal hyperplasia and was regarded to be treatment-related. In the skeletal muscle of females only of test group 3 (100 mg/kg bw/day) slight to moderate necrosis of muscle fibers were observed. In addition infiltration of inflammatory cells, mainly macrophages were noted. This finding was regarded to be treatment-related. The same finding as described for the skeletal muscle was also noted in the area of the spine. This was also regarded to be treatment-related. In the testes there was an increase of abnormal residual bodies. The abnormal residual bodies were much larger and of irregular shape when compared to control males. This finding was regarded to be treatment-related. In the thyroid glands a minimal hypertrophy/hyperplasia, in combination with an increase in altered colloid was found in males of test group 2 and 3 (30 and 100 mg/kg bw/day) and females of test group 3 (100 mg/kg bw/day). The finding in males of test group 1 (10 mg/kg bw/day) was regarded to be incidental, as the same incidence was seen in a control male.
In males of test group 3 (100 mg/kg bw/day) four animals revealed a sperm granuloma in the epididymides, whereas only one male in the control showed this finding. This was still regarded to be incidental, as sperm granuloma can occur in relatively high incidences as spontaneous finding (Creasy et al., 2012).
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the high dose (100 mg/kg bw/day) were comparable to those of the controls.

Decedents: There were no histopathologic findings in the male that died (No. 36). The female (No. 138) that was sacrificed in a moribund state revealed findings in the heart and skeletal muscle. A test substance-related effect cannot be excluded but could also be not proven. Furthermore, this animal revealed inflammation in the intestinal tract. This could also be the cause of the moribund state of this animal.

Fertility: The female animals (Nos. 102 and 103), which were not pregnant as well as the male mating partners (Nos. 2 and 3) did not show relevant histopathological findings. Animal Nos. 15 and 115 were not investigated histopathologically.

For more details please also refer to table 8.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Male reproduction data
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for control male No. 2 paired with control female No. 102. Thus, the male mating index was 90% in the control and 100% in the low-, mid- and high-dose groups.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One low-dose male (10 mg/kg bw/d - No. 15) and two control males (0 mg/kg bw/d - Nos. 2 and 3) did not generate pregnancy.
Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show relevant gross lesions.

Female reproduction data:
The female mating index calculated after the mating period for F1 litter was 90% in test group 0 and 100% in test groups 1 - 3. The mean duration until sperm was detected (GD 0) varied between 2.1 and 2.9 days without any relation to dosing.
All female rats delivered pups or had implants in utero with the following exceptions:

• Low-dose female No. 115 (mated with male No. 15) did not become pregnant.
• Control female No. 102 (mated with male No. 2) did not become pregnant.
• Control female No. 103 (mated with male No. 3) did not become pregnant.

The fertility index varied between 88.9% in test group 0, 90% in test group 1 and 100% in test groups 2 and 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. None of the non-pregnant females had any relevant gross lesions.
The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.6 days). The gestation index was 100% in test groups 0 - 2 and 60% in test group 3. Although there was no statically significance the decreased gestation index in test group 3 was assessed as substance related finding.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (10.8 / 12.0 / 12.9 and 11.1 implants/dam in test groups 0 - 3, respectively).
The mean number of F1 pups delivered (10.5 / 11.6 / 11.7 and 9.2 pups/dam in test groups 0 - 3, respectively) was in test group 3 slightly lower, but achieved no statistical significance.
The post-implantation loss was statistically significantly above the concurrent control values in test group 3 (2.6 / 3.1 / 9.1 / 28.5 mean %* [*=p<=0.05] test groups 0 - 3, respectively). Also the perinatal loss (mean%/litter) was statistically significantly above the concurrent control values in test group 3 (45.8 [*=p<=0.05] vs. 0 in control).
The number of litters with stillborn pups was statistically significantly above the concurrent control values in test group 3 (6** [**=p<=0.01] vs. 0 in control).
The number of litters with all pups stillborn was significant above the concurrent control values in test group 3 (3 vs. 0 in control).
The above mentioned statistically significantly changes in post-implantation loss and delivery parameters where assessed as substance related adverse findings.

F1 generation pups/litter
The mean number of delivered F1 pups per dam was evenly distributed among the test groups. The mean number of liveborn pups/litter was statistically significantly below the concurrent control values in test group 3 (10.5 / 11.6 / 11.6 / 5.2* [*=p<=0.05] test groups 0 - 3, respectively). The absolute number of liveborn pups in this test group was also decreased (47 vs. 84 in control).
The mean number of stillborn pups/litter was statistically significantly above the concurrent control values in test group 3 (4.0* [**=p<=0.05] vs. 0 in control).
The number of found dead pups was increased in test group 3 (13 vs. 1 in control), as well as the number of stillborn pups (36 vs. 0 in control) and the number of cannibalized pups (19 vs. 0).
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 98.2%/ 100% /100% and 20.9%** [**=p<=0.01] in test groups 0 - 3, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
100 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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
musculoskeletal system
Organ:
myofibres
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Four male and four female pups of test group 3 showed reduced nutritional condition this was assessed as substance related. There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of test groups 1 and 2.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 98.2%, 100%, 100%, and 20.9%** [**=p<=0.01] in test groups 0 - 3, respectively.
The number of found dead pups was increased in test group 3 (13 vs. 1 in control), as well as the number of stillborn pups (36 vs. 0 in control) and the number of cannibalized pups (19 vs. 0).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose F1 male, female pups and both sexes combined (100 mg/kg bw/d) were statistically significantly below the concurrent control values on PND 1 (about 20%, 24% and 20%, respectively) and on PND 4 for female pups and both sexes combined (about 44% and 43%, respectively).
Due to the fact, that only 2 litters with male pups remained on PND 4, no statistical evaluation could be done on PND 4 for mean body weights and body weight change (PND 1 - 4).
The mean pup body weight change of the high-dose F1 female pups and both sexes combined was statistically significantly below the concurrent control values (about 95% and 92%, respectively).
No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1 - 2 (10 and 30 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Four male and four female pups of test group 3 showed reduced nutritional condition this was assessed as substance related.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One male runt was seen in the control, one female runt was seen in test group 1 and three male and four female runts were seen in test group 3. The increased number of runts in test group 3 was assessed as substance related.
A few pups showed spontaneous findings at gross necropsy, such as anasarca, partly cannibalized and post mortem autolysis.
In test group 3 (100 mg/kg bw/d) 16 male pups and 9 female pups in 8 litters showed cleft palates and 7 male and 8 female pups in 5 litters showed an empty stomach at necropsy. This was assessed as substance related and adverse.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
musculoskeletal system
Organ:
hard palate
Treatment related:
yes
Dose response relationship:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no

Table 7 Absolute and relative organ weights when compared to control in percent

 

Female animals

Test Group (mg/kg bw/d)

1 (10)

2 (30)

3 (100)

Absolute organ weights

Kidneys

103

106

113**

Liver

101

105

119**

Thymus

77

87

46**

Relative organ weights

Heart

106

104

118*

Kidneys

107

108

120*

Liver

105

107*

126*

Thymus

81

89

49*

 *p <= 0.05; **p <= 0.01

Table 8 Histopathological findings

 

Male

Female

Test group (mg/kg bw/d)

0 (0)

1 (10)

2 (30)

3 (100)

0 (0)

1 (10)

2 (30)

3 (100)

Heart

Necrosis, myocardial

0

1

2

6

0

0

0

5

Grade 1

 

 

2

3

 

 

 

3

Grade 2

 

1

 

3

 

 

 

2

Prostate

Hyperplasia, diffuse

0

0

1

5

-

-

-

-

Grade 1

 

 

1

5

 

 

 

 

Skeletal muscle

Necrosis, musclefiber

-

-

-

-

0

0

0

6

Grade 2

 

 

 

 

 

 

 

2

Grade 3

 

 

 

 

 

 

 

4

Skeletal muscles at spine

Cervical cord

Necrosis, musclefiber

0

0

0

1

0

0

0

0

Grade 1

 

 

 

1

 

 

 

 

Thoracic cord

Necrosis, musclefiber

0

0

0

0

0

0

0

3

Grade 1

 

 

 

 

 

 

 

2

Grade 3

 

 

 

 

 

 

 

1

Lumbar cord

Necrosis musclefiber

0

0

0

4

0

0

0

5

Grade 1

 

 

 

4

 

 

 

4

Grade 2

 

 

 

 

 

 

 

1

Testes

abnormal residual bodies, (multifocal)

0

4

8

7

-

-

-

-

Grade 1

 

4

7

5

 

 

 

 

Grade 2

 

 

1

2

 

 

 

 

Thyroid glands

Hypertrophy/hyperplasia

1

1

3

5

0

0

0

5

Grade 1

1

1

3

5

 

 

 

5

Altered colloid

0

1

1

1

0

0

0

1

Grade 1

 

1

1

1

 

 

 

1

 

 

 

Table 9 Reproductive parameter indices

Test group (mg/kg bw/d)

0 (0)

1 (10)

2 (30)

3 (100)

P0 Males

Mating index

90

100

100

100

Fertility index

80

90

100

100

P0 Females

Mating index

90

100

100

100

Fertility index

88.9

90

100

100

Gestation index

100

100

100

60

F1 Pups

Viability index

98.2

100

100

20.9**

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD 422 study, according to GLP and guideline.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 422 study in rats

The test item was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 10, 30 and 100 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100 mL Tween 80). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 3 days post-mating in males, and the entire gestation period as well as approximately 17 days of the lactation period (or in sperm negative females 25 days post-mating).

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 4.The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Clinico-chemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

 

The various analyses:

-Demonstrated the stability of the test substance in 1% Carboxymethylcellulose suspension in drinking water + 5 mg/100 mL TWEEN 80 over a period of 7 days in a refrigerator

-Confirmed overall the homogeneous distribution of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100 mL TWEEN 80

-Verified correct concentrations of the test substance preparations.

 

The following adverse treatment-related findings were noted:

 

100 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY

-One parental male animal was found dead on mating day 7 and one parental female animal was sacrificed moribund on PND 5

-Increased number of females which not properly nursed their pups (4 versus [vs.] 0 in control)

-Increased number of litters with all pups stillborn (3 vs. 0 in control) and one female with complete litter loss

- Decreased food consumption in the females during premating days 0 - 13 (up to 17% below control), during GD 14 - 20 (about 12% below control) and during PND 1 - 4 (about 60% below control)

-Decreased body weights in the females on PND 4 (about 12% below control)

-Decreased body weight change in the females during GD 0 - 20 (up to 25%) and during PND 0 - 4 (about 12%)

-Retarded adaption of the pupil to light during the pupillary reflex test in the functional observational battery in 4 out of 5 examined females

-Decreased motor activity values particularly during early exploring phase in males and females

-Decreased hemoglobin and hematocrit values in females

-Increased relative reticulocyte counts in females

-Increased total white blood (WBC) cell, absolute neutrophil and absolute and relative large unstained cell (LUC) counts in females

-Increased aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities and triglyceride levels in females

-Increased absolute (+19%) and relative (+26%) liver weight in females

-Minimal to slight myocardial necrosis in the heart of 6 males and 5 females

-Skeletal muscle fiber necrosis in 6 females Skeletal muscle fiber necrosis in the spine region in 5 males and 6 females (combined incidence)
-
Hypertrophy/hyperplasia (minimal) in the thyroid glands of 5 males

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

-Increased number of litters with stillborn pups (6 vs. 0 in control)

-Increased postimplantation loss (28.5 mean% vs. 2.6 mean% in control)

-Decreased gestation index (60% vs. 100% in control)

-Decreased number of liveborn pups (47 vs. 84 in control) resp. mean number of liveborn pups/litter (5.2 vs 10.5 in control)

-Increased number of stillborn pups (36 vs. 0 in control) resp. mean number of stillborn pups/litter (4.0 vs. 0 in control)

-Increased perinatal loss (45.8 mean% vs. 0 mean% in control)

-Increased number of found dead pups (13 vs. 1 in control)

-Increased number of cannibalized pups (19 vs. 0 in control)

-Decreased viability index (20.9% vs. 98.2% in control)

-Reduced nutritional condition in male and female pups

-Increased number of runts on PND1 (7 vs. 1 in control).

-Decreased pup body weights in male and female pups during the whole lactation period (up to 20% [males], 44% [females] and 43% [both sexes combined] below control)

-Decreased pup body weight change in females and both sexes combined (about 95% and -92% below control, respectively)

-25 pups with cleft palate and 15 pups with empty stomach at pup necropsy

 

30 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

-Increased total white blood (WBC) cell and absolute neutrophil counts in females

-Hypertrophy/hyperplasia (minimal) in the thyroid glands of 3 males

 

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

-No test substance-related adverse findings

10 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY

-No test substance-related adverse findings

 

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

-No test substance-related adverse findings

In conclusion, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of the test item was 10 mg/kg bw/d for male and female rats based on hypertrophy/hyperplasia in the thyroid glands of male rats and an increase of white blood cell counts in females at 30 mg/kg bw/d. The NOAEL for fertility and reproductive performance was 100 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the offspring was 30 mg/kg bw/d based on cleft palates and their subsequent adverse effects on pup survival and growth at 100 mg/kg bw/d. The high dose level (100 mg/kg bw/d) resulted in two mortalities and therefore was beyond the maximum tolerated dose. Therefore, the effects found with respect to developmental toxicity are not considered to be selective.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD 422 study, according to GLP and guideline.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is classified for toxicity to reproduction / development cat 2 (H361d "Suspected of damaging fertility or the unborn child") under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

Repeated oral administration of the test substance resulted in severe clinical symptoms, mortality and effects on several organs (skeletal and heart muscle tissue, liver, thyroid gland and white blood cells) at the dose level where the developmental/teratogenic effects were observed. Therefore, the effects found with respect to developmental toxicity/teratogenicity are not considered to be selective.

The test substance is classified for Specific Target Organ Toxicity (STOT RE cat.2) based on the effects observed at 30 and 100 mg/kg bw/day. In conclusion, the developmental toxicity/teratogenicity is not an isolated effect and was accompanied by systemic toxicity and mortality which requires classification for Specific Target Organ Toxicity. According to chapter 3.7.2.2.1. of Regulation EC 1272/2008 (CLP), classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on development. Therefore, classification with category 2 for developmental toxicity (H361d) is considered the most appropriate in line with the criteria laid down in Regulation EC 1272/2008 (CLP).

Additional information