Reaction products of furan-2,5-dione and octadec-1-ene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

OECD-guideline and GLP-compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

EC Number:

272-221-2

EC Name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Cas Number:

68784-12-3

Molecular formula:

Due to a mixture of numerous isomers a specific molecular formular cannot be provided ( UVCB-substance) .

IUPAC Name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Details on test material:

Name of test material (as cited in study report): ASA
- Substance type: organic, uvcb
- Physical state: liquid
- Analytical purity:97.1%
- Purity test date: no data
- Lot/batch No.: 5500001727
- Expiration date of the lot/batch: 31 Oct 2013
- Stability under test conditions:guaranteed by the sponsor
- Storage condition of test material:room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (females) and Charles River, UK (males)
- Age at study initiation:10-12 weeks (males/females)
- Weight at study initiation: m (314.4-316.7); f (195.8-199.5)
- Fasting period before study: no data
- Housing:individually in type M III polycarbonate cages supplied by Becker & Co., Castrop Rauxel, Germany (floor area of about 800 cm2)
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24 °C
- Humidity (%):30-70%
- Air changes (per hr):10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (propylene glycol) was filled up to the desired volume, subsequently mixed using a magnetic stirrer.
The test-substance solutions were prepared in such intervals that the stability was guaranteed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle:

group dose conc. Male Female Male
0 0 0 5 10 10
1 100 2.0 5 10 10
2 300 6.0 5 10 10
3 1000 20.0 5 10 10

Details on mating procedure:

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 16.00 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 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:

Analyses of the test-substance preparations
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory (building A 030) of Experimental Toxicology and Ecology of BASF SE as a part of the study.
The stability of the test substance in propylene glycol stored deep frozen over a period of 7 days followed by 4 hours at room temperature was demonstrated before the start of the study (Project No.: 01Y0471/09Y004).
Concentration control and homogeneity analyses of the test-substance preparations were performed in samples of all concentrations at the start and at the end of the administration period.

Food analysis
The food used in the study was assayed for chemical and microbiological contaminants.

Drinking water analysis
The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for bacteria by a contract laboratory. The German “Trinkwasserverordnung” (Drinking Water Regulation) will serve as the guideline for maximum tolerable contaminants.

Bedding and enrichment analysis
The bedding and the enrichment are regularly assayed for specific contaminants (chlorinated hydrocarbons and heavy metals).

Duration of treatment / exposure:

females were treated for 49 days and males for 35

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 m/ 10 f

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previously performed 14-days test study (Project No.: 10C0471/09069) ASA was administered by gavage to groups of 3 male and 3 female Wistar rats at dose levels of 0, 100, 300, 1000 mg/kg body weight/day (mg/kg bw/d). Propylene glycol served as vehicle.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before and after treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:before and after treatment. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.The day of littering was considered to be a 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

BODY WEIGHT: Yes
- Time schedule for examinations:once a week. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Once a week (in a period of 7 days) 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 on PND 0 and 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).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:towards the end of the administration period (study days 35 (males) and 49 (females))
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals: 5 randomly selected
- Parameters c Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:towards the end of the administration period (study days 35 (males) and 49 (females))
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume were examined.

NEUROBEHAVIOURAL EXAMINATION: YES (Functional Observation Battery with Home cage observations, Open field observations and Sensorimotor Tests/Reflexes)

Oestrous cyclicity (parental animals):

no

Sperm parameters (parental animals):

no

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.

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 finally confirmed at necropsy.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.


Pup body weight data
The pups were weighed one day after birth (PND 1) and on day 4 after birth. Pups' body weight change was calculated from these results.
Furthermore the body weights on PND 1 post partum were used for the calculation of "runts" (pups, which weighed more than 25% less than the mean weight of the respective control pups).
The individual weights were always determined at about the same time of the day (in the morning).

Postmortem examinations (parental animals):

Organ weights
Selected organ weights were determined in all animals sacrificed on schedule and gross pathology was done

1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles with coagulation glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands (with parathyroid glands)
17. Uterus

Organ/tissue fixation
The following organs or tissues of parental animals were fixed in 4% formaldehyde solution or in modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulation glands
9. Colon
10. Duodenum
11. Epididymides (modified Davidson`s solution)
12. Esophagus
13. Eyes with optic nerve
14. Female mammary gland
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 (mesenteric and axillary lymph nodes)
24. Nose (nasal cavity)
25. Ovaries
26. Oviducts
27. Pancreas
28. Parathyroid glands
29. Pharynx
30. Pituitary gland
31. Prostate
32. Rectum
33. Salivary glands (mandibular and sublingual glands)
34. Seminal vesicle
35. Sciatic nerve
36. Skeletal muscle
37. Spinal cord (cervical, thoracic and lumbar cords)
38. Spleen
39. Sternum with marrow
40. Stomach (forestomach and glandular stomach)
41. Testes (modified Davidson’s solution)
42. Thymus
43. Thyroid glands
44. Trachea
45. Urinary bladder
46. Uterus
47. Vagina

Histopathology
Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings of following organs
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulation gland
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Peyer’s patches
20. Prostate
21. Rectum
22. Sciatic nerve
23. Seminal vesicle
24. Spleen
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Stomach (forestomach and glandular stomach)
27. Testes
28. Thymus
29. Thyroid glands (with parathyroid glands)
30. Trachea
31. Urinary bladder
32. Uterus
33. Vagina

Postmortem examinations (offspring):

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) and in the morning on Saturdays, Sundays or public holidays.

The number and percentage of dead pups on the day of birth (PND 0) and pups dying between PND 1-4 were determined; however, pups which died accidentally and pups which were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the PND 0 and on PND 4.
Pup necropsy observations
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups were discarded after their evaluation.

Statistics:

Statistics of the clinical examinations
DUNNETT-test , FISHER'S EXACT test, WILCOXON-test, KRUSKAL-WALLIS test

Statistics of clinical pathology
FISHER'S EXACT test, KRUSKAL-WALLIS test

Statistics of pathology
KRUSKAL-WALLIS test, WILCOXON-test

Reproductive indices:

Male mating index (%) = (number of males with confirmed mating*/ number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (numbers of maels proving fertility* / number of males placed with females) x 100
*defined by a female with implants in utero

Female mating index (%) = (number of females mated*/number of males placed with females) x 100
*defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of pregnant females* / number of females mated**) x 100
*defined as the number of females with implants in utero
**defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pubs at day of birth / number of females pregnant*) x 100
*defined as the number of females with implants in utero

Offspring viability indices:

Live birth index (%) = (number of liveborn pubs at birth / total number of pubs born) x 100

Post implanatation loss (%) = ((number of implantations - number of pubs delivered) / number of implantations) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection, poor general state in female animals during lactation, insufficient maternal care

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Diffuse squamous cell hyperplasia in the forestomach of all males and females. Multifocal atypical bile duct hyperplasia in the liver of males and females.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No animal died prematurely during the study period.
In test group 3 (1000 mg/kg bw/d), salivation after treatment was observed in 6 males from study week 1 onwards and in 2 males in study weeks 3 and 4. Three male animals in test group 2 (300 mg/kg bw/d) showed salivation after treatment in study weeks 3 and 4. In females no salivation was observed during the premating period. One male of test group 0 (control) showed an eye injury in study week 4.
Salivation after treatment was observed in 3 to 4 female animals of test group 3 (1000 mg/kg bw/d) from GD 3 onwards. Salivation after treatment was also observed in 1 female animal of test group 2 (300 mg/kg bw/d) from GD 8 onwards.
Five female animals of test group 3 (1000 mg/kg bw/d), i.e. animal Nos. 132, 134, 137, 138 and 139, showed insufficient maternal care of pups. In two litters no more pups were alive on PND 1 (animal No. 139) and 2 (animal No. 138). Four of these animals also showed piloerection, i.e. animal Nos. 132, 134, 138 and 139. In addition urine smeared anogenital region was observed in female No. 139. Three dams of test group 3 (1000 mg/kg bw/d), i.e. animal Nos. 135, 136 and 139, showed salivation after treatment.
Animal No. 124 of test group 2 (300 mg/kg bw/d) showed insufficient maternal care, piloerection and salivation after treatment.
Poor general state, piloerection and insufficient maternal care of pups were observed in animal No. 111 of test group 1 (100 mg/kg bw/d).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No significant changes in food consumption values for male and female animals were observed during the entire study period. Although not significantly decreased, food consumption in female animals of test group 3 (1000 mg/kg bw/d) was only 78% compared to test group 0 (control) during lactation.
No significant changes in body weight and body weight change values for male and female animals were observed during the entire study period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) no data

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
males: For all F0 parental males, which were placed with F0 females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in all test groups.
Fertility was proven for most of the F0 parental males of test groups within the scheduled mating interval for the F1 litter. One control male (animal No. 5 mated with female No. 105) did not generate F1 pups. Thus, the male fertility index ranged between 90% 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: The female mating index calculated after the mating period for F1 litter was 100% for test groups 0, 1 and 2 (0, 100 and 300 mg/kg bw/d, respectively). The female mating index calculated after the mating period for F1 litter of test group 3 (1000 mg/kg bw/d) was 90%.
The mean duration until sperm was detected (GD 0) amounted to 3.1, 1.9, 2.4, and 2.0 days (0, 100, 300 and 1000 mg/kg bw/d, respectively). Although the value of test group 1 (100 mg/kg bw/d) was significantly lower a relation to treatment was excluded as no dose-response relationship was observed. Consequently, the differences between the test groups were assessed as being spontaneous in nature and without biological relevance.

All sperm positive rats delivered pups or had implantations in utero with the exception of female No 105 (control), which was mated with male No. 5 but did not become pregnant. The fertility index varied between 90% and 100%.

The mean duration of gestation was between 21.9 and 22.4 days and did not show significant differences. The gestation index was 100% in all test groups.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (13.8, 12.0, 13.9, and 13.1 implants per dam in test groups 0-3). Furthermore, there were no indications for test substance-induced intrauterine embryo /fetolethality since the postimplantation loss did not show any significant differences between the test groups, and the mean number of F1 pups delivered per dam remained unaffected (13.7, 12.0, 13.5, and 12.2 pups per dam at 0, 100, 300 and 1000 mg/kg bw/d, respectively).

The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 96% (test group 0 and 3), 92% (test groups 1) and 94% (test group 2). Moreover, the number of stillborn pups was comparable between the test groups and a dose-response relationship was not observed.


ORGAN WEIGHTS
All mean absolute weight parameters in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) did not show significant differences when compared to test group 0 (control).
When compared to test group 0 (control; set to 100%), the mean relative weights of following organs were significantly increased: kidney, testes (male); liver (female)
All other mean relative weight parameters did not show significant differences when compared to test group 0 (control).

The increase of the relative kidney weight in males of test group 1 (100 mg/kg bw/d) was regarded to be incidental as no dose-response relationship occurred. The increased relative liver weight in females of test group 2 (300 mg/kg bw/d) was also considered to be incidental because the mean absolute weight was not significantly increased and no histopathological correlate was present.
For the increase of the relative testes weight in males of test group 3 (1000 mg/kg bw/d) there were no histopathological correlates. Furthermore, the absolute weight of testes did not show significant differences. Therefore, the increased testes weight was related to the slightly but not significantly decreased terminal body weight (-5%) in these males.

GROSS PATHOLOGY
Forestomach: Three males of test group 2 (300 mg/kg bw/d) and 6 males of test group 3 (1000 mg/kg bw/d) showed a yellow-white deposition on the forestomach epithelium. A thickening of the forestomach wall was observed in all males of test group 3 (1000 mg/kg bw/d).
Duodenum: The duodenal wall was slightly thickened in 7 males of test group 3 (1000 mg/kg bw/d).
All other gross lesions occurred singly and were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Forestomach: A diffuse squamous cell hyperplasia was observed in the forestomach of 1 control female and of 1 female in test group 1 (100 mg/kg bw/d). In test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d), incidence and severity of diffuse squamous cell hyperplasia were increased dose-related. The squamous cell hyperplasia was characterized by an increased number of epithelial cells showing small exophytic structures into the lumen or small endophytic finger-like projections. The hyperplasia was accompanied by an infiltration of some lymphoid cells and granulocytes. The hyperplasia correlated with the thickening of the forestomach wall that was grossly diagnosed in all males of test group 3 (1000 mg/kg bw/d). The increased occurrence of squamous cell hyperplasia was considered to be treatment-related.
The squamous cell hyperplasia occurred mostly in combination with hyperkeratosis. The macroscopically diagnosed yellow-white deposition on the forestomach epithelium corresponded histopathologically with a diffuse orthokeratotic hyperkeratosis, characterized by an increased thickening of the superficial non-nucleated keratin layer. The incidence of hyperkeratosis was increased treatment-related in males of test group 2 (300 mg/kg bw/d) as well as in males and females of test group 3 (1000 mg/kg bw/d).
The occurrence of the minimal squamous cell hyperplasia in the forestomach of 1 control female and 1 female of test group 1 (100 mg/kg bw/d) as well as the occurrence of hyperkeratosis in 1 control female, in 2 females of test group 1 (100 mg/kg bw/d) and in 2 females in test group 2 (300 mg/kg bw/d) were considered to be incidental.

Liver: A multifocal atypical bile duct hyperplasia was observed in 2 males of test group 2 (300 mg/kg bw/d) as well as in 8 males and all females of test group 3 (1000 mg/kg bw/d). This finding was characterized by a multifocal proliferation of bile ducts in the portal region. The bile duct epithelium seemed slightly atypical. It was single layered, often cuboidal and slightly basophilic. Some mitotic figures and necrotic cells were present within the epithelium. The hyperplasia was associated with periductular fibrosis and periductular lymphohistiocytic infiltrates.
Furthermore, a minimal centrilobular, hepatocellular hypertrophy was observed in 8 males and 7 females of test group 3 (1000 mg/kg bw/d).

The occurrence of multifocal atypical bile duct hyperplasia and of centrilobular, hepatocellular hypertrophy was considered to be treatment-related.

Thyroid glands: A minimal or slight follicular hypertrophy/hyperplasia was observed in 1 control male, in 2 males of test group 1 (100 mg/kg bw/d), in one male of test group 2 (300 mg/kg bw/d), and in 4 males of test group 3 (1000 mg/kg bw/d). In affected animals the number of small follicles was slightly increased or the follicular epithelium was higher, varying in size from cuboidal cells to columnar cells.
For the slightly increased incidence of hypertrophy/hyperplasia in males of test group 3 (1000 mg/kg bw/d), a treatment related effect could not be ruled out.

Duodenum: For the macroscopically described thickening of duodenal wall in 7 males of test group 3 (1000 mg/kg bw/d), there was no histopathological correlate.

All other findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Diffuse squamous cell hyperplasia in the forestomach of males and females. Multifocal atypical bile duct hyperplasia in the liver of males and of females.

2

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: live birth index was not affected

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
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.
No pup of test group 0 (control) died ahead of schedule. However, the number of pups that died was significantly increased in test group 1-3 (100, 300 and 1000 mg/kg bw/d) showing a relation to dosing. In addition several pups were cannabilized between PND 1 and 4. The viability index as indicator for pup mortality between PND 0-4 was 96% for test group 0 (control), 89% for test group 1 (100 mg/kg bw/d), 83% for test group 2 (300 mg/kg bw/d) and 70% for test group 3 (1000 mg/kg bw/d).

CLINICAL SIGNS (OFFSPRING)
The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.
Two runts of each gender were seen in test group 0 (control) and 2 female runts were seen in test group 2 (300 mg/kg bw/d). Both values were within the range of the biological variation inherent in the strain of rats used for this study.

Pup necropsy observations
Post mortem autolysis was observed for several pups in all test groups including the control. In 13 pups of test group 3 (1000 mg/kg bw/d) and in eight pups of test group 2 (300 mg/kg bw/d) the stomach was found empty. The same was true for 5 pups of test group 1 (100 mg/kg bw/d) and 1 pub of test group 0 (control).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 100 - <= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity (F1)

Key result

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Any other information on results incl. tables

Test group 3: 1000 mg/kg bw/d

 

F0 parental animals

• Clinical Examinations:
• Piloerection in up to 4 of 10 female animals during lactation
• Poor general state in 2 of 10 female animals during lactation
• Insufficient maternal care (pups were found scattered, cold, not cleaned and with empty stomach) in 5 out of 9 dams, two of these dams had a complete and one of these dams had a partial litter loss
• Fertility:
• No test-substance related, relevant findings
• Clinical Pathology:
• Increased platelet counts in rats of both sexes
• Increased ALT activities and bilirubin values in rats of both sexes
• Increased SGGT activities in males
• Increased WBC counts, absolute neutrophil, relative and absolute monocyte as well as relative and absolute LUC counts in males
• Reduced prothrombin time in males
• Decreased total protein and albumin values in males
• Increased cholesterol values in males
• Pathology:
• Diffuse squamous cell hyperplasia in the forestomach of all males and females.
• Multifocal atypical bile duct hyperplasia in the liver of 8 males and of all females.

 

F1 pups

• Increased pup mortality (30% dead/cannibalized), almost exclusively due to insufficient maternal care in 3 dams

 

 

Test group 2: 300 mg/kg bw/d

 

F0 parental animals

• Clinical Examinations:
• Piloerection in up to 1 of 10 female animal during lactation
• Insufficient maternal care in 1 of 10 dams, this dam had a complete litter loss
• Fertility:
• No test-substancerelated, relevant findings
• Clinical Pathology:
• No test-substancerelated, relevant findings
• Pathology:
• Diffuse squamous cell hyperplasia in the forestomach of 9 males and 5 females.
• Multifocal atypical bile duct hyperplasia in the liver of 2 males.

 

F1 pups

• Increased pup mortality (17% dead/cannibalized), for a big part due to insufficient maternal care in 1 dam

 

 

Test group 1: 100 mg/kg bw/d

 

F0 parental animals

• Clinical Examinations:
• Poor general state and piloerection in 1 of 10 female animal during lactation
• Insufficient maternal care in 1 of 10 dams, this dam had a complete litter loss
• Fertility:
• No test-substancerelated, relevant findings
• Clinical Pathology:
• No test-substancerelated, relevant findings
• Pathology:
• No test-substancerelated, relevant findings

 

F1 pups

• Increased pup mortality (10% dead/cannibalized), for the most part due to insufficient maternal care in 1 dam

 

Functional observational battery (P)

Home cage observations:

No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

  

Open field observations:

One female animal of test group 2 (300 mg/kg bw/d;animal No. 128) showed piloerection. All other male and female animals of all test groups did not reveal any test substance-related findings during home cage observation.

 

Sensorimotor tests/reflexes:

There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore,these observations were considered as being incidental.

 

Quantitative Parameters:

No test substance-related impaired parameters were observed in male and female animals of all test group

 Motor activity measurement (P)

There were no significant deviations concerning the overall motor activity (summation of all intervals) in the male and female animals of all test groups in comparison to the concurrent control group.

Regarding single intervals, in males of test group 1 (100 mg/kg bw/d) one isolated significantly increased value was measured at interval 10. This finding was considered as being incidental since the overall motor activity was not changed and no findings were observed for female animals.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats the no observed adverse effect level (NOAEL) for systemic toxicity in F0 males was 100 mg/kg bw/d due to pathological findings at 300 mg/kg bw/d and above.
The NOAEL for systemic toxicity in F0 females could not be established. The dose of 100 mg/kg bw/d was without pathological findings. However, insufficent maternal care at all dose levels correlates with increased pup mortality. The cause for the insufficent maternal care is presently undecided as the effect may be due to systemic maternal toxicity or due to an effect on the pups via lactation. The NOAEL for fertility in male and female Wistar rats was 1000 mg/kg bw/d.

Executive summary:

C15 -20 ASA was administered by gavage to Wistar rats at 0, 100, 300 and 1000 mg/kg bw/d. Systemic toxicity was observed in female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the lactation period. The most severe effect was

a poor condition and insufficient maternal care of pups, mainly in the high dose, but present in all test groups. As consequence, in two litters no pups were alive on PND 1 and 2.

Fertility indices for male and female animals were not impaired by test-substance administration and live birth index of pups in all test groups were not influenced. In contrast, the viability index as indicator for pup mortality was dose-dependently increased in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) and clearly outside the expected range.