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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid/clear, colorless
Details on test material:
Name of test substance: Laurylacrylate 1214
Batch No. 110014P040
CAS No.: 84238-60-8
Purity: 93.9 % (corr. area % as a sum of three main compounds)
Date of production: July 2011
Expiry date: 18 aug Jan 2012
Physical state/ appearance: liquid/clear, colorless
Storage conditions: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks (males/females)
- Fasting period before study: no
- Housing:
During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material (the present supplier is documented in the raw data).
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Dust-free wooden bedding was used in this study. Wooden gnawing blocks (Type NGM E-022) supplied by Abed® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (ad libitum): ground Kliba maintenance diet mouse-rat “GLP”
- Water (ad libitum): from water bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod: 12 hours light from 06.00-18.00h, 12 hours dark from 18.00-06.00h
- Acclimation period: 5-6 days before the beginning of the treatment period

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired weight, subsequently released with a magnetic stirrer. The test substance preparations were produced at least once a week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice.
The stability of the test substance in corn oil for a period of 7 days at room temperature was confirmed.
Concentration control Analysis of the test substance preparations were performed in samples of all concentrations at the start of the administration period.
Duration of treatment / exposure:
After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals, daily. The treatment lasted up to one day prior to sacrifice.
Frequency of treatment:
daily in the morning
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 male and 10 female rats per dose group
Control animals:
yes, concurrent vehicle
Positive control:
No positive control.

Examinations

Observations and examinations performed and frequency:
Parental animals

- Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The littering 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 public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering 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
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). 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

- Food consumption
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.

- Body weight data
Body weight was determined 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). The body weight change of the animals was calculated from these results.
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 1) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

- Functional observational battery
A functional observational battery was performed in the first five male animals per test group, the first 5 female animals with litter of all test groups at the end of the administration period starting at about 10.00 h. The FOB started 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.

- Home cage observations:
The animals were observed in their closed home cages; during this period 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. Impairment of gait
Open field observations:

The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior when removed from cage
2. Fur
3. Skin
4. Salivation
5. Nose discharge
6. Lacrimation
7. Eyes/ pupilsize
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Impairment of gait
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes

- Sensory motor tests/ reflexes:
The animals were then 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 (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

- Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and females (with litter) per group. Motor activity was measured on the same day as 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 numbers of beam interrupts was counted over 12 intervals of 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 rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and 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.


Clinical pathology

In the morning blood was taken from the retrobulbar venous plexus from fasted animals.
The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group:

- Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and
Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

Parameters: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular
hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes, Prothrombin time

- Clinical chemistry
Parameters: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; γ-Glutamyltransferase
(GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase; Sodium (Na+), Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total bilirubin, Total proteins, Albumin,Total cholesterol, Triglycerides , Biles acids

- Urinalysis (parameters)
pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment Color, turbidity, Volume



Litter observations

-. Pup number and status at delivery
All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

- Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. 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. The viability index was calculated.

- Sex ratio
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; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally
confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

- Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

- Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results.
The individual weights were always determined at about the same time of the day (in the morning). “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.







Sacrifice and pathology:
Parental animals

Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

Weight parameters
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 per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological 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 4% buffered formaldehyde solution or 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 gland
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
48. Vagina

- Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings from the following organs of all animals/test group, all animals affected/test group or 5 animals per sex/test group, females with litters only, same animals as used for
clinical pathological examinations.
Special attention was given to stages of spermatogenesis in the male gonads.
A correlation between gross lesions and histopathological findings was attempted.

Organs:
Adrenal glands, All gross lesions, Bone marrow (femur) , Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart,
Ileum, Jejunum, Kidneys, Live, Lungs, Lymph nodes (axillary and mesenteric, Ovaries , Oviducts, Prostate gland, Peyer’s patches , Rectum,
Sciatic nerve , Seminal vesicles, Spinal cord (cervical, thoracic, lumbar) , Spleen, Stomach (forestomach and glandular stomach), Testes , Thymus , Thyroid glands , Trachea , Urinary bladder , Uterus , Vagina



Offsprings

- Pup necropsy observations
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.


Other examinations:
Reproductive indices
The following indeces were determined: mating and fertility index for both males and females, gestation index, live birth index

Offspring viability index
The following indices were determined: sex ratio and viability index
Statistics:
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.

Results and discussion

Results of examinations

Description (incidence and severity):
PARENTAL ANIMALS
- One female animal of test group 3 (No.138) showed a swelling of the left mouth region on study days 21 and 28. No other abnormal clinical signs in all test groups were observed.
- Summary clinical observations for males and females: Slight to moderate salivation in 3 male animals of test groups 2 and all male animals of test group 3 was observed after treatment in the pre-mating period. Slight to moderate salivation in 4 male animals of test groups 2 and all male animals of test group 3 was observed after treatment in the mating period. Slight to moderate salivation in 5 male animals of test groups 2 and all male animals of test group 3 was observed after treatment in the post-mating period. Slight to moderate salivation in 1 female animal of test groups 2 and in 7 female animals of test group 3 was observed after treatment in the pre-mating period. Slight to moderate salivation in 1 female animal of test groups 2 and in 7 female animals of test group 3 was observed after treatment in the mating period. Animal No. 126 (mated with Animal No. 26) did not show sperm in vaginal smear.
- Summary clinical observations for females during gestation: Animal No. 138 of test group 3 showed a swelling of the left mouth region from gestation day 1 to 17. Because of a single incidence this finding was assessed as being incidental.
Animal Nos. 123, 125, 127 and 128 of test group 2 showed slight salivation after treatment. All animals of test group 3 showed slight to moderate salivation after treatment. These findings were substance-related because of the smelling of the test substance, but no adverse effects.
Animal Nos. 123 and 127 of test group 2 and animal No. 131 and 134 of test group 3 did not deliver any pups. These findings were assessed as being spontaneous in nature and without biological relevance.
Animal No. 109 of control group showed apathy and reduced nutritional condition on gestation day 23.
- No other clinical signs were observed.
- Clinical observations for females during lactation: Animal Nos. 125 and 128 of test group 2 showed slight salivation after treatment on different study days. Animal Nos. 132, 135, 136, 137, 138, 139 and 140 of test group 3 showed slight to moderate salivation after treatment on different lactation days. These findings were substance-related because of the smelling of the test substance, but no adverse effects. Animal No. 109 of the control group showed a complete litter loss after insufficient after-birth and maternal care. Due to the reduced nutritional condition and the apathy, this animal was sacrificed in a moribund state on lactation day 1.

OFFSPRING
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
Description (incidence):
PARENTAL ANIMALS
Animal No. 109 was sacrificed in a moribund state on lactation day 1.
No other animal died prematurely in the present study.

OFFSPRING
The viability index indicating pup mortality during lactation (PND 0 - 4) was 90% in test group 0 and 100 % in test groups 1 - 3.
The value of test group 0 was in the normal range of biological variation inherent in the strain of rats used for this study.
Description (incidence and severity):
PARENTAL ANIMALS
Body weight of females of test group 2 (300 mg/kg bw/d) was significantly decreased on gestation day 20 (-5.4%).
Body weight change was significantly decreased in females of test group 3 (1000 mg/kg bw/d) when regarding the premating period from day 0 to day 13 (-6.1%).
Due to the lack of dose response relationship these findings were assessed as being incidental.

OFFSPRING
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. In test group 1 (100 mg/kg bw/d) one female runt was seen (Animal No. 118).
Description (incidence and severity):
PARENTAL ANIMALS
The food consumption was significantly increased (+10.7%) on day 7 in males of test group 3 (1000 mg/kg bw/d) during premating period.
Description (incidence and severity):
PARENTAL ANIMALS
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 as incidental.
The following examinations were performed during FOB and are assessed individually:
Home cage observations
No test substance-related effects were observed.
Open field observations
No test substance-related effects were observed.
Sensorimotor tests/reflexes
No test substance-related effects were observed.
Quantitative Parameters
No test substance-related effects were observed.

Motor activity measurement
Comparing the single intervals with the control groups, no significant deviations were measured with the exception of slightly decreased values in all interval in males of test group 3 (1000 mg/kg bw/d). As a consequence, the overall motor activity measurement in male animals of test group 3 was significantly decreased. As no FOB parameter was changed and no findings occurred during DCO, the change was assessed as being spontaneous and not related to treatment.
No changes were noted for male animals of test groups 1 and 2 (100 and 300 mg/kg bw/d) as well as female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.
Description (incidence and severity):
PARENTAL ANIMALS
- Absolute weights: When compared to the control group 0 (set to 100%), the mean absolute kidney and liver weights were significantly increased in male animals. All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group 0.
- Relative weights: When compared to the control group 0 (set to 100%), the mean relative kidney and liver weights were significantly increased in male animals. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
As there was no dose response or a histopathological correlate, the increased kidney weights were regarded as incidental.
The increase in absolute (test group 3, 1000 ppm) and relative liver weights in test group 2 and 3 (300 and 1000 mg/kg bw/day) in male animals was considered to be treatment - related, as they followed a dose response although a histopathological correlate was missing.
Description (incidence and severity):
PARENTAL ANIMALS
Female animal No. 109 that died showed light red foci on the kidney, and an effusion in the thoracic cavity, and a retained fetus in the uterus.
Two male animals (Animal Nos. 23 and 26) of dose group 2 (300 ppm) showed a focus on the liver.
All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Description (incidence and severity):
PARENTAL ANIMALS
Animal No. 109 showed multifocal necroses in the kidney correlating to red foci in the macroscopic diagnosis. The uterus was necrotic with inflammation, fetal tissue was no longer recognizable histologically. Both findings are likely to have contributed to the moribund state of this animal.
Male animal Nos.23 and 26 showed macroscopically a focus on the liver which correlated histologically with focal necrosis. This was not considered to be treatment – related as it was focal and not present in test group 3.
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.
Details on results:
PARENTAL ANIMALS
- Male mating index: For F0 parental males, which were placed with females to generate F1 pups, mating was confirmed, except for animal No. 26. The male mating index was 90% in test group 2 (300 mg/kg bw/d) and 100% in all other test groups. This finding 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.
- Male fertility index: Fertility was proven for all of the F0 parental males within the scheduled mating interval to produce F1 litter. Two males of test group 3 (No. 31 mated with 131 and No. 34 mated with No. 134), three males of test group 2 (No. 23 mated with female No. 123, No. 26 mated with female No. 126 and No. 27 mated with No. 127) did not generate F1 pups. The male fertility index was between the range of 70% and 100%.
This finding 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.
- Female mating index: The female mating index calculated after the mating period for F1 litter was between 90% in test group 2 (300 mg/kg bw/d) and 100% for all other test groups. The mean duration until sperm was detected (GD 0) was 2.7, 2.0, 1.6 and 2.0 days in test groups 0 - 3.
- Female fertility index: All sperm positive rats delivered pups with the exception of female Nos. 131 and 134 (test group 3; 1000 mg/kg bw/d) and Nos.123, 126 and 127 (test group 2; 300 mg/kg bw/d), which were mated with male Nos. 31, 34 and 23, 26 and 27, did not become pregnant. The female fertility index was 80% in the high dose group, 77.8% in the mid dose group and 100% in the low and control group. Female animals Nos. 131, 134, and 127 which delivered no pups, showed no implantation sites. These data reflect 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.
- Gestation index: The gestation index was 100% in all test groups.
- Live birth indices: The live birth index was 100% in all test groups. Three females (Nos. 133, 135 and 140) in test group 3 (1000 mg/kg bw/d) and two females (Nos. 104 and 109) in test group 0 (0 mg/kg bw/d) with single stillborn and found dead pups were observed.
- Postimplantation loss: The postimplantation loss was between 2.96% (test group 0), 3.41% (test group 1), 5.56% (test group 2) and 2.53% (test group 3). The values were in the range of the historical control data.
- Fertility (pathology): Three mating pairs of test group 2 ([300 mg/kg bw/day] Nos. 23/123, 26/126, 27/127) and two pairs of test group 3 ([1000 mg/kg bw/day] 31/131, 34/134) were recorded as no offspring/ not pregnant. None of the examined animals showed relevant gross findings.
- Fertility (histopathology): Three mating pairs of test group 2 ([300 mg/kg bw/day] Nos. 23/123, 26/126, 27/127) and two pairs of test group 3 ([1000 mg/kg bw/day] 31/131, 34/134) were recorded as no offspring/ not pregnant. None of the examined animals showed relevant histopathological findings (134 [stomach erosion/ulcus], 23 [liver necrosis], 26 [liver necrosis], 34 [no lesions], 31 [thyroid hypertrophy/hyperplasia, grade 1]. These findings were not considered to be treatment - related and spontaneous in origin.

OFFSPRING
- Pup number and status at delivery: The mean number of delivered F1 pups was 13.9 (test group 0), 11.9 (test group 1), 10.7 (test group 2) and 10.9 (test group 3). The decreased values of test group 2 and test group 3 were consequent to the increase of the mean number of delivered pups in the control group, which were higher than the historical control data. The three stillborn pups in test group 3 and six stillborn pups in test group 0 were incidental and in the normal range of biological variation inherent in the strain of rats used for this study.
- Sex ratio: TThe 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.
- Pup necropsy observations: Two stillborn pups of test group 0 showed post mortem autolysis (Animal no. 109). Two found dead pups of test group 0 (animal No. 109) showed an empty stomach. These findings were assessed as being spontaneous in nature and without biological relevance, because of the insufficient after-birth and maternal care of the female no. 109.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed
Dose descriptor:
NOAEL
Remarks:
toxic effect, reproductive performance and on developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL (no observed adverse effect level) for general, systemic toxicity was 1000 mg/kg bw/day.
The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/day for the F0 parental rats.
The NOAEL for developmental toxicity in the F1 progeny was found to be 1000 mg/kg bw/day.
Executive summary:

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/day (test group 0), 100 mg/kg bw/day (test group 1), 300 mg/kg bw/day (test group 2) and 1000 mg/kg bw/day (test group 3).

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. 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 and two weeks thereafter in females.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

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 was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in 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 and mating. 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 under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinanalysis were performed in 5 animals per sex and group towards the end of the administration period.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test 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 confirmed the stability of the test substance in corn oil at room temperature over a period of 7 days and the correctness of the prepared concentrations of the test substance preparations in corn oil.

The following test substance-related adverse findings were noted:

- Test group 3: 1000 mg/kg bw/day

No test substance-related, adverse findings were noted in neither F0 parental animals nor in F1 pups.

- Test group 2: 300 mg/kg bw/day

No test substance-related, adverse findings were noted in neither F0 parental animals nor in F1 pups.

- Test group 1: 100 mg/kg bw/day

No test substance-related, adverse findings were noted in neither F0 parental animals nor in F1 pups.

The NOAEL (no adverse observed effect level) for general, systemic toxicity was 1000 mg/kg bw/day.

The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/day for the F0 parental rats.

The NOAEL for developmental toxicity in the F1 progeny was found to be 1000 mg/kg bw/day.