Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

BASF SE, 2013, according to OECD 422, GLP compliant,no signs of systemic toxicity or reproductions toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 11-12 weeks (upon receipt)
- Weight at study initiation: the animals were of comparable size and weight
- Housing: individually 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
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
Route of administration:
oral: gavage
Vehicle:
other: water containing 5 mg/100 mL Cremophor EL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Korantin MAT was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 5 mg/100 mL Cremophor EL was filled up to the desired volume, subsequently released with a magnetic stirrer. 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 and were stored at room temperature. The administration volume was 10 mL/kg body weight.


VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was suitable for this type of test
- Concentration in vehicle: 1, 3 and 10 g/100 mL
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio 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.

The animals were paired by placing the female in the cage of the male mating partner from about 15.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
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 stability of the test substance in drinking water containing 5 mg/100 mL Cremophor EL for a period of 7 days at room temperature was proven during the study.
Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period.
Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study.
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.
Frequency of treatment:
once daily
Details on study schedule:
N/A
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: on request of the sponsor
- Rationale for animal assignment (if not random): random, the list of randomization instructions was compiled with a computer.
Positive control:
N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
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.

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 (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 when handled
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
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 fe¬males 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. These body weight data were solely used for the calculations of the dose volume.


FOOD CONSUMPTION: Yes
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, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
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).

WATER CONSUMPTION: Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.
Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
Special attention was given on stages of spermatogenesis in the male gonads upon Histopathology.
Litter observations:
LITTER DATA
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) 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. At the same time, the pups were also being examined for macroscopically evident changes. 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).
Pup body weights and pup body weight change are listed for males, females and males + females.
“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.
Postmortem examinations (parental animals):
GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
Weight assessment was carried out on all animals. The following weights were determined:

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 / 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
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. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

Paraffin embedding, sectioning and staining
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed, staining with Hematoxylin-eosin (H&E):
all gross lesions (all affected animals per group),
control and high dose animals only:
Adrenal glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Bone marrow (femur) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Brain (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cecum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cervix (all animals per group)
Coagulation glands (all animals per group)
Colon (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Duodenum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Epididymides (all animals per group)
Heart (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ileum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Jejunum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Kidneys (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Liver (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lung (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lymph nodes (mesenteric and axillary lymph nodes) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ovaries (all animals per group)
Oviducts (all animals per group)
Peyer’s patches (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Prostate (all animals per group)
Rectum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Sciatic nerve (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Seminal vesicles (all animals per group)
Spinal cord (cervical, thoracic and lumbar cords) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Spleen (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Stomach (forestomach and glandular stomach) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Testes (all animals per group)
Thymus (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Thyroid glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Trachea (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Urinary bladder (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Uterus (all animals per group)
Vagina (all animals per group)

Special attention was given on stages of spermatogenesis in the male gonads.

A correlation between gross lesions and histopathological findings was attempted.
Postmortem examinations (offspring):
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-by-case basis, depending on the type of finding noted.

Statistics:
Detailed statistical analyses were conducted
Reproductive indices:
For the males, mating and fertility indices were calculated. For the females, mating, fertility and gestation indices were calculated. The live birth index was calculated for F1 litters and the postimplantation loss (in %) was calculated.
Offspring viability indices:
The viability index and the sex ratio were calculated
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No animal died prematurely in the present study.

All animals of both sexes in test group 3 (1000 mg/kg bw/d) showed salivation within 2 hours after the administration on several days of the study. In test group 2 (300 mg/kg bw/d) male animal Nos. 25, 27 and 30 showed salivation within 2 hours after the administration on several days during the pre-mating period as well on mating day 0 (male animal No. 27, only). Female animal Nos. 122, 123, 126, 128 and 130 of test group 2 (300 mg/kg bw/d) showed salivation within 2 hours after the administration on several days during the pre-mating period. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.

During the mating phase male animal No. 6 of test group 0 (control group) showed respirations sounds on mating days 5-6. This finding was assessed as being incidental and not related to treatment.

The Detailed Clinical Observations on study days 0, 7, 14, 21, 28 in male animals and on study days 0, 7, 14, 21, 28, 35, 42, 49, 56 in female animals of test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) did not reveal any abnormalities.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test substance-related changes in mean body weights or body weight change values were observed in any test group.
On lactation day 4, mean body weights of female animals in test groups 2 and 3 (300 and 1000 mg/kg bw/d) were significantly lower. In addition, mean body weight change value during lactation was significantly lower in female animals of test group 2 (300 mg/kg bw/d) between study days 0-4. However, no significant changes were observed in on the other time points during the lactation period.

In female animals of test group 3 (1000 mg/kg bw/d) food consumption during gestation was significantly lower between study days 7-14 and 14-20. This finding was assumed to be incidental because the mean value was still within the normal range for pregnant animals at the end of gestation. In addition, no significant impacts on body weight data were observed.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Stages of spermatogenesis in the male gonads was comparable to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male reproduction data

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 (0, 100, 300, 1000 mg/kg bw/d).

Fertility was proven for nearly all F0 parental males within the scheduled mating interval to produce F1 litter. Male animal No. 38 of test group 3 (1000 mg/kg bw/d) did not generate pups with female animal No. 138. 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.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% for all test groups (0, 100, 300 and 1000 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 1.6, 2.2, 1.4 and 1.8 days in test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d, respectively).

All sperm-positive rats delivered pups with the exception of female animal No. 138 (test group 3), which was mated with male animal No. 38 but did not become pregnant. The female fertility index varied between 90% and 100%. Female animal No. 138, which delivered no pups had no implantation sites.
The mean duration of gestation was between 22.0 and 22.2 days and did not show significant differences.

The gestation index reached 100% in all test groups including the control group.

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99.1% (control group) and 100% (test groups 1, 2 and 3). One single stillborn pup was only seen in control group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
When compared with control group 0 (set to 100%), the mean relative weights of livers (males only) and kidneys (both sexes) were significantly increased in test group 3 (1000 mg/kg bw/d).

The increase in absolute kidney weights of males and females of test group 3 (1000 mg/kg bw/d) was most likely treatment-related due to a dose response-relationship and also altered relative kidney weights. However, as no histopathologic correlate was present the finding was regarded to be not adverse.
The increase of relative liver weights in males of test group 3 (1000 mg/kg bw/d) was also regarded to be most likely treatment-related although no histopathologic finding could explain the weight increase. But as no other parameter with regard to liver cell metabolism was altered it was regarded to be not adverse in nature.

All other mean weight parameters (absolute and relative) did not show significant differences when compared to the control groups

GROSS PATHOLOGY (PARENTAL ANIMALS)
All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

Fertility:
The female that was not pregnant and its male mating partner did not show gross lesions in reproduction relevant organs which could explain the lack of offspring.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:

The investigated not pregnant female as well as its male mating partner of test group 3 (1000 mg/kg bw/d) did not show relevant histopathological findings that could explain infertility.
Dose descriptor:
NOAEL
Remarks:
systemic, reproductive performance and fertility, development
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Generation: P and F1 (migrated information)
LITTER DATA

Pup number and status at delivery
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study.

Pup viability/mortality
The viability index as indicator for pup mortality between PND 0 and 4 was 100% for test groups 0 and 1 (control and 100 mg/kg bw/d). In control group one cannibalized pup was found. In test group 2 (300 mg/kg bw/d) the viability index was 99% because of 1 pup found dead and 1 cannibalized pup in different litters. In test group 3 (1000 mg/kg bw/d) the viability index was 98% because of 2 pups found dead in different litters. The remaining pups which belonged to these animals did not show any findings until sacrifice.

The viability index was still within the historical control data and reflected the normal range of biological variation inherent in the strain used in this study.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

Pup clinical observations
One pup of female animal No. 134 (test group 3, 1000 mg/kg bw/d) showed a thread-like tail on PND 0-3 and a short tail on PND 4.
One pup of control group (female No. 108) was not assessed because it died during interval on PND 0. The surviving F1 pups of any test group did not show clinical signs up to scheduled sacrifice on PND 4.

Pup body weight data
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.
One female runt was seen in test group 0 as well as test group 2 (control group, 300 mg/kg bw/d) on PND 1. One male runt was seen in test group 3 (1000 mg/kg bw/d) on PND1.
All values were within the range of the biological variation inherent in the strain of rats used for this study.

Pup necropsy observations
In 1 pup of test group 3 (1000 mg/kg bw/d; female No. 134) a small tail was observed. This finding was assessed as being spontaneous in nature and without biological relevance as it can also be found in the historical control data. One pup of test group 0 (control group; female No. 107) was not assessed because it was cannibalized on PND4.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
not specified

Korantin MATwas administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2)and 1000 mg/kg bw/d (test group 3).

 

Regardingclinical examinations,signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire study period.

Salivation after treatment was seen in all animals of test group 3 (1000 mg/kg bw/d) and sporadically in a few animals of test group 2 (300 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

 

Fertility indicesfor male and female animals were not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. In addition,live birth indicesof pups in all test groups were not influenced.

 

Theviability indexas indicator for pup mortality was not altered dose-dependently.

 

Concerningclinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

 

Regardingpathology, the treatment of male and female Wistar rats with up to 1000 mg/kg bw/d of the test substance led to treatment-related increase in male and female absolute and relative kidney weights in test group 3 (1000 mg/kg bw/d). In addition, the relative liver weight in males of test group 3 (1000 mg/kg bw/d) was also significantly and treatment-related increased. As there were no histopathologic findings in both organs these weight increases were not regarded to be adverse in nature but an adaptive effect.

No findings on the reproduction tract were observed.

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.


CONCLUSION

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of KORANTIN MAT to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000mg/kg bw/d in male and in female Wistar rats.

The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d in male and female Wistar rats.

TheNOAEL for developmental toxicity was effective 1000 mg/kg bw/d.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Drinking watercontaining 5 mg/100 mL Cremophor ELserved as vehicle. The study was conducted according to OECD 422 guideline and GLP (BASF SE, 2013) 

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.

 

Observations

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.

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, after 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 CO2and examined macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinalyses 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 the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) 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.

Results

Analytics

The various analyses confirmed

-     the stability of the test substance in drinking water at room temperature over a period of 7 days,

-     the homogeneous distribution of the test substancein drinking water,

-     the correctness of the prepared concentrations of the test-substance preparations in drinking water.

 

Findings

The following test substance-related, relevant findings were noted:

 

Test group 3: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

- No testsubstance-related, adverse findings were noted.

 

F1 PUPS

 

Clinical Examinations/ Gross Findings

- Notestsubstance-related, adverse findings were noted.

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

- Notestsubstance-related, adverse findings were noted.

 

F1 PUPS

 

Clinical Examinations/ Gross Findings

- Notestsubstance-related, adverse findings were noted.


Test group 1: 100 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

- No testsubstance-related, adverse findings were noted.

 

F1 PUPS

 

Clinical Examinations/ Gross Findings

- Notestsubstance-related, adverse findings were noted.

 

 Conclusion

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus,the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000mg/kg bw/d in male and in female Wistar rats.

The NOAEL for reproductive performance and fertility was set to 1000mg/kg bw/d in male and female Wistar rats.

The NOAEL for developmental toxicity was effective 1000 mg/kg bw/d.


Short description of key information:
NOAEL (systemic, reproductive performance and development) = 1000 mg/kg bw/day (BASF SE, 2013)

Effects on developmental toxicity

Description of key information

BASF SE, 2019, according to OECD 414, GLP compliant, no test substance-related adverse effects on dams, gestational parameters or fetuses in any test group (100, 300, 1000 mg/kg bw/day), NOAEL (developmental/teratogenicity) = 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-25 until 2019-03-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Limit test:
no
Specific details on test material used for the study:
Please note: This kind of substance was in the past handle under the CAS nr 85204-21-3
After analytical characterisation the substance was renamed and received a new CAS nr. Now the substance is characterised by the CAS 1471311-93-9 EC Number: 939-488-3

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and 14671736W0
- Expiration date of the lot/batch: 01 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the vehicle: stability in drinking water with 5 mg/100 mL Cremophor EL over a period of 7 days at room temperature verified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: prepared at the beginning of the administration period and thereafter at intervals, specific amount of test substance was weighed, topped up with drinking water or deionized water
- Final dilution of a dissolved liquid: suspension in drinking water or deionized water with 5 mg/100 mL Cremophor EL, respectively

FORM AS APPLIED IN THE TEST: suspension; preparations were kept homogeneous with a magnetic stirrer

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 146-193 g (day of arrival = gestation day 0 (GD 0))
- Housing: housed individually in Polycarbonate cages type III, floor area about 800 cm² (TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany)
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland
- Water: ad libitum, tap water in water bottles
- Acclimation period: 6 days (GD 0 - GD 6)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 h to 18.00 h / 18.00 h to 6.00 h)

IN-LIFE DATES: From: 25 Jan 2018 To: 15 Feb 2018
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water, from day 8 onwards deionized water (with 5 mg/100 mL Cremophor EL, respectively
Details on exposure:
APPLICATION OF DOSING SOLUTION:
- volume administered: 10 mL/kg body weight

PREPARATION OF DOSING SOLUTIONS:
- specific amount of test substance was weighed, topped up with drinking water or deionized water
- prepared in a calibrated beaker and intensely mixed with a magnetic stirrer
- Rate of preparation: at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: water with 5 mg/100 mL Cremophor EL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- samples of the test substance preparations were analyzed (at the beginning of administration and due to a change in the vehicle) for verification of the concentrations
- samples taken for the concentrations control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (100 and 1000 mg/kg bw/d)
- three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD 0)
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19)
Frequency of treatment:
once a day, always at approximately the same time in the morning
Duration of test:
21 days (GD 0 until GD 20)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
1 g/ 100 mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
3 g/ 100 mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
10 g/ 100 mL
No. of animals per sex per dose:
25 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: as requested by the sponsor, based on available studies e.g. 85R0586/11S125
- Rationale for animal assignment: each cohort was evenly distributed among the dose groups
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day, once on Saturdays, Sundays or on public holidays (GD 0-20)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment period (GD 0-5 and 20); during GD 6-19 all rats were checked daily before administration as well as within 2 hours and within 5 hours after administration
- Parameters: any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: apart from uterus, not further specifications
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
- Simultaneous comparison of all dose groups with the control group, DUNNETT-test (two-sided) for the hypothesis of equal means: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

- Pairwise comparison of each dose group with the control group, FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

- Pairwise comparison of each dose group with the control group, WILCOXONtest (one-sided) for the hypothesis of equal medians: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Indices:
The calculations of the indices are given below in "Any other information on materials and methods".
- conception rate, preimplantation loss, postimplantation loss
Historical control data:
Historical control data are available for animals of this strain and age for parameters of conception rate, the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses.
Clinical signs:
no effects observed
Description (incidence and severity):
- occasional salivation: all high-dose group (1000 mg/kg bw/d) females and 3/25 of the middose group (300 mg/kg bw/d) females
- salivation occurred only shortly, i.e. within 0-2h, after treatment and was observed during GD 8-19 (test group 3) or only on GD 19 (test group 2)
The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic toxicity.

- no clinical signs or changes of general behavior (attributed to the test substance), were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period
Mortality:
no mortality observed
Description (incidence):
No test substance-related or spontaneous mortalities in any females of all test groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- mean body weights, average body weight gain of all dams (all dose groups) generally comparable to the concurrent control group throughout the entire study period
- indicental statistically significantly increased body weight gain value in test group 3 during GD 1-3
- corrected body weight gain of all test groups revealed no difference of any biological relevance; mean carcass weights of all test groups remained unaffected
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- mean food consumption of all test groups was generally comparable to the concurrent control group throughout the entire study period
- incidental statistically significantly increased food consumption value in test group 3 during GD 1-3
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- mean gravid uterus weights of the animals of all test groups not influenced
- any differences between test groups and the control group revealed no dose-dependency and were assessed to be without biological
relevance
- mean placental weights of test groups 1-3 were comparable to the concurrent control group
Gross pathological findings:
no effects observed
Description (incidence and severity):
- no necropsy findings observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups for the pre- and post-implantation losses
Early or late resorptions:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups for the number of resorptions
Dead fetuses:
no effects observed
Description (incidence and severity):
- no test substance-related and/or biologically relevant differences between the different test groups for the number of viable fetuses
- two dead fetuses were found at cesarean section of one mid-dose dam (300 mg/kg bw/d); this rare finding may occur spontaneously in this rat strain
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
- conception rate was 92% in the control group and 100% in the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d)
- no test substance-related and/or biologically relevant differences between the different test groups in conception rate,in the mean number of corpora lutea and implantation sites
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: no differences of toxicological relevance between the control and the treated groups for any reproductive parameters observed
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
- mean fetal weights of all test groups were not influenced by the test substance
- no biologically relevant differences in comparison to the control group
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- sex distribution of the fetuses in all test group animals was comparable to the control fetuses
External malformations:
no effects observed
Description (incidence and severity):
- no external malformations recorded
- one external variation in one single fetus was recorded in the highest test group: limb hyperflexion; details given in table 1
- incidence not statistically significantly different from control and the finding was within the range of the historical control data; not considered as treatment-related and adverse
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- malformations noted in two fetuses, in test groups 1 and 3 (100 and 1000 mg/kg bw/d), individual details given in table 3 and total skeletal malformations given in table 4
- one female fetus of the high-dose group had multiple skeletal malformations concerning the skull, the vertebral column and ribs
- mean value of affected fetuses per litter was within the historical control range
- ‘fused mandible’ in one further individual fetus in the high-dose group; found in the historical control data in comparable incidences
- isolated findings not assessed as treatment-related and adverse
The total incidences of skeletal malformations in treated animals did not differ significantly from the control group and were comparable to the historical control data.

- skeletal variations of different bone structures were observed in all dose groups, with or without effects on corresponding cartilages, details for total fetal skeletal variations are given in table 5 and an overview of statistically significantly increased variations are given in table 6
- skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose
- overall incidences of skeletal variations were comparable to the historical control data
- ‘incomplete ossification of skull’, ‘incomplete ossification of nasal’ and ‘supernumerary rib (14th); cartilage present’: not related to dose and the mean values were clearly inside the historical control ranges
- findings were not assessed as treatment related
- ‘supernumerary rib (14th); cartilage not present’: statistically significantly increased in test group 3 (affected fetuses/litter: 65.6%)
- value was marginally outside of the range of the historical control data
The rudimentary rib finding is very frequent in this rat strain and is not adverse per se. Since the finding was quite near the range of the historical control data, it was not assessed as treatment-related and adverse.
Visceral malformations:
no effects observed
Description (incidence and severity):
- no soft tissue malformations recorded
- four soft tissue variations were detected: short innominate in test groups 0 and 2, malpositioned subclavian origin in test group 1, dilated renal pelvis and dilated ureter in all test groups; details given in table 2
- dilated renal pelvis: mean values for affected fetuses per litter were outside the historical control range in the concurrent control and in the high-dose group alike
- values did, however, not significantly differ from each other; the finding was neither assessed as treatment-related nor as adverse
- dilated ureter: within the historical control range for all test groups
Overall, the incidences of all these variations were neither statistically significantly nor dosedependently increased in the treated groups.
Details on embryotoxic / teratogenic effects:
External and soft tissue malformations did not occur in any of the fetuses in this study. There were noted skeletal malformations in test groups 1 and 3 (100 and 1000 mg/kg bw/d).
One fetus of test group 3 carried multiple skeletal malformations (such as misshapen basisphenoid, cervical hemivertebra, thoracic hemivertebra with detached rib, misshapen thoracic vertebrae). Another fetus of test group 3 showed a fused mandible. Further malformations, i.e. absent lumbar vertebra and malpositioned and bipartite sternebra, were observed in individual fetuses, unrelated to dose.
All these findings were single cases, most of them can be found in the historical control data, except for ‘absent lumbar vertebra’. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study.
There is no evidence for any association of these scattered findings with the treatment.
The total incidences of malformations are summarized in table 7.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose
Abnormalities:
effects observed, non-treatment-related
Developmental effects observed:
no

Table 1: Total external variations in one single fetus (no malformations) observed

 

 

Test group 0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N N

23

232

25

250

25

228

25

255

 

Fetal incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (0.4)

 

Litter incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (4.0)

Affected fetuses/litter

 

Mean %

 

0.0

 

0.0

 

0.0

 

0.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 2: Total soft tissue variations in the fetuses (no malformations) observed

 

 

Test group 0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N N

23

111

25

119

25

108

25

121

Fetal incidence

 

N (%)

 

13 (12)

 

9 (7.6)

 

5 (4.6)

 

16 (13)

Litter incidence

 

N (%)

 

6 (26)

 

8 (32)

 

5 (20)

 

10 (40)

Affected fetuses/litter

 

Mean %

 

12.6

 

7.1

 

4.6

 

12.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 3: Individual fetal skeletal malformations observed

Test group

Dam No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

28-08 M

absent lumbar vertebra

47-05 F

malpositioned and bipartite sternebra

2 (300 mg/kg bw/d)

none

 

3 (1000 mg/kg bw/d)

91-07 F

multiple skeletal malformations

97-09 M

fused mandible

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Table 4: Total fetal skeletal malformations observed

 

 

Test group 0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N N

23

121

25

131

25

120

25

134

Fetal incidence

 

N (%)

 

0.0

 

2 (1.5)

 

0.0

 

2 (1.5)

Litter incidence

 

N (%)

 

0.0

 

2 (8.0)

 

0.0

 

2 (8.0)

Affected fetuses/litter

 

Mean %

 

0.0

 

1.5

 

0.0

 

1.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 5: Total fetal skeletal variations observed

 

 

Test group 0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N N

23

121

25

131

25

120

25

134

Fetal incidence

 

N (%)

 

116 (96)

 

122 (93)

 

114 (95)

 

125 (93)

Litter incidence

 

N (%)

 

23 (100)

 

25 (100)

 

25 (100)

 

25 (100)

Affected fetuses/litter

 

Mean %

 

95.6

 

93.1

 

94.4

 

93.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 6: Statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

 

Finding

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

HCD

Mean % (range)

 

Incomplete ossification of skull; unchanged cartilage

 

4.1

 

10.7

 

12.6*

 

10.0

 

7.8

(2.1 - 15.9)

 

Incomplete ossification of nasal; unchanged cartilage

 

0.0

 

2.3*

 

0.0

 

0.8

 

0.8

(0.0 - 5.5)

 

Supernumerary rib (14th); cartilage present

 

2.4

 

3.5

 

8.7*

 

8.2*

 

7.8

(1.9 - 14.7)

 

Supernumerary rib (14th); cartilage not present

 

45.6

 

40.1

 

57.2

 

65.6*

 

54.0

(42.1 - 65.2)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p<=0.05 (Wilcoxon-test [one-sided]) ** = p<=0.01 (Wilcoxon-test [one-sided])

Table 7: Summary of the total fetal malformations observed

 

 

Test group 0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

NN

23

232

25

250

25

228

25

255

Fetal incidence

 

N (%)

 

0.0

 

2 (0.8)

 

0.0

 

2 (0.8)

Litter incidence

 

N (%)

 

0.0

 

2 (8.0)

 

0.0

 

2 (8.0)

Affected fetuses/litter

 

Mean%

 

0.0

 

0.8

 

0.0

 

0.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Conclusions:
The oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.
In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.
Executive summary:

The test substance (UVCB) was tested for its prenatal developmental toxicity in Wistar rats according to OECD 414.

The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water; from day 8 onwards deionized water, with 5 mg/100 mL Cremophor EL) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 23-25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

The stability of the test substance in drinking water with 5 mg/100 mL Cremophor EL over a period of 7 days at room temperature was demonstrated and a homogeneous distribution of the test substance in the vehicle together with the correctness of the prepared concentrations was shown.

There were no test substance-related adverse effects of toxicological relevance on dams, gestational parameters or fetuses observed in any test group.

Based on these findings and under the conditions of the prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day

of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

Prenatal development, according to OECD 414, BASF SE, 2019

The Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine was tested for its prenatal developmental toxicity in Wistar rats according to OECD 414.

The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water; from day 8 onwards deionized water, with 5 mg/100 mL Cremophor EL) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 23-25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

The stability of the test substance in drinking water with 5 mg/100 mL Cremophor EL over a period of 7 days at room temperature was demonstrated and a homogeneous distribution of the test substance in the vehicle together with the correctness of the prepared concentrations was shown.

There were no test substance-related adverse effects of toxicological relevance on dams, gestational parameters or fetuses observed in any test group.

Based on these findings and under the conditions of the prenatal developmental toxicity study, the oral administration of Korantin MAT to pregnant Wistar rats from implantation to one day prior to the expected day

of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.

Justification for classification or non-classification

Based on the available data, the test substance is not classified with regard to toxicity to reproduction according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP), respectively.