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REACH

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

EC number: 701-338-8 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No changes in fertility at doses up to 1000 mg/kg bw/d.

Link to relevant study records

Reference 1

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

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

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

Dose / conc.:

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

Dose / conc.:

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

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

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):

Sperm parameters (parental animals):

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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.

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

Key result

Critical effects observed:

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

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).

Key result

Dose descriptor:

NOAEL

Generation:

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

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

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.

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was conducted on 2,5-furandione, dihydro-, mono-C15- 20-alkenyl derivatives (CAS 68784-12-3), an analogue substance used as the source of information for the assessment of the target substance through read-across. Therefore, this study is informative for evaluation of the environmental fate and toxicity of the target substance, Reaction products of furan-2,5-dione and octadec-1-ene (known here as n-ODSA EC 701-338-8; no CASRN available), and it is adequate for classification and risk assessment.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Details on mating procedure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

females were treated for 49 days and males for 35

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

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:

Positive control:

none

Parental animals: Observations and examinations:

Oestrous cyclicity (parental animals):

Sperm parameters (parental animals):

Litter observations:

Postmortem examinations (parental animals):

Postmortem examinations (offspring):

Statistics:

Reproductive indices:

Offspring viability indices:

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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

Key result

Critical effects observed:

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

Key result

Dose descriptor:

NOAEL

Generation:

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

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

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:

Sensorimotor tests/reflexes:

Quantitative Parameters:

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

Motor activity measurement (P)

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of the analogue test substance, C15-20 ASA, 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. This study is informative for evaluation of the toxicity of the registered substance, n-ODSA EC 701-338-8, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.

Executive summary:

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.

This study is informative for evaluation of the toxicity of the registered substance, n-ODSA, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.

Reference 3

Endpoint:: fertility, other
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Justification for type of information:: Guideline 90-day repeated dose toxicity study with information on reproductive organs
Reason / purpose for cross-reference:: reference to same study
Qualifier:: according to guideline
Guideline:: other: OECD 408, 90-day Repeated Dose Toxicity Study in Rats
Principles of method if other than guideline:: OECD 408 Guideline, 90-day repeated dose toxicity study with information on reproductive organs
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Details on species / strain selection:: Male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK.
Sex:: male/female
Route of administration:: oral: gavage
Vehicle:: arachis oil
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 90 day
Frequency of treatment:: daily
Dose / conc.:: 10 mg/kg bw/day (actual dose received)
Dose / conc.:: 100 mg/kg bw/day (actual dose received)
Dose / conc.:: 300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:: 10
Control animals:: yes, concurrent vehicle
Postmortem examinations (parental animals):: Pathology
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Brain, Spleen, Heart, Thymus, Kidneys, Liver, and including these reproductive organs: OVARIES, EPIDIDYMIDES, TESTES, UTERUS.
Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:

Adrenals, OVARIES, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint)•, PITUITARY, Bone & bone marrow (sternum), PROSTATE, Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, SEMINAL VESICLES, EPIDIDYMIDES (Preserved in Modified Davidson’s fluid), Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes (Eyes fixed in Davidson’s fluid), Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), TESTES (Preserved in Modified Davidson’s fluid), Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Tongue (Retained only and not processed), Lungs (with bronchi, lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, MAMMARY GLANDS, UTERUS (with cervix), Muscle (skeletal, Retained only and not processed): VAGINA.

All tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). All tissues from control and 300 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Microscopic examination was conducted by the Study Pathologist (Peter Millar at Peter Millar Associates Ltd., 3 Queen Charlotte Lane, Edinburgh, EH6 6AY).
Statistics:: Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Histopathological findings: non-neoplastic:: no effects observed
: Key result
Dose descriptor:: NOAEL
Effect level:: > 300 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable due to absence of adverse toxic effects
: Key result
Critical effects observed:: no
: Key result
Reproductive effects observed:: no
Conclusions:: After 90 days of repeated oral exposure to doses up to 300 mg/kg bw/d of C15-20 ASA, rats showed no evidene of pathologic, histopathologic or toxic effects to organs, and in particular, the reproductive organs. There is no reason to expect that reproductive toxicity based on impaired organ function would result from exposure to this test material. The NOAEL is > 300 mg/kg bw/d for any adverse organ effect.

Reference 4

Endpoint:: fertility, other
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study
Justification for type of information:: This study was conducted on 2,5-furandione, dihydro-, mono-C15- 20-alkenyl derivatives (CAS 68784-12-3), an analogue substance used as the source of information for the assessment of the target substance through read-across. Therefore, this study is informative for evaluation of the environmental fate and toxicity of the target substance, Reaction products of furan-2,5-dione and octadec-1-ene (known here as n-ODSA EC 701-338-8; no CASRN available), and it is adequate for classification and risk assessment.
Reason / purpose for cross-reference:: read-across source
Qualifier:: according to guideline
Guideline:: other: OECD 408, 90-day Repeated Dose Toxicity Study in Rats
Principles of method if other than guideline:: OECD 408 Guideline, 90-day repeated dose toxicity study with information on reproductive organs
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Details on species / strain selection:: Male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK.
Sex:: male/female
Route of administration:: oral: gavage
Vehicle:: arachis oil
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 90 day
Frequency of treatment:: daily
Dose / conc.:: 10 mg/kg bw/day (actual dose received)
Dose / conc.:: 100 mg/kg bw/day (actual dose received)
Dose / conc.:: 300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:: 10
Control animals:: yes, concurrent vehicle
Postmortem examinations (parental animals):: Pathology
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Brain, Spleen, Heart, Thymus, Kidneys, Liver, and including these reproductive organs: OVARIES, EPIDIDYMIDES, TESTES, UTERUS.
Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:

Adrenals, OVARIES, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint)•, PITUITARY, Bone & bone marrow (sternum), PROSTATE, Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, SEMINAL VESICLES, EPIDIDYMIDES (Preserved in Modified Davidson’s fluid), Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes (Eyes fixed in Davidson’s fluid), Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), TESTES (Preserved in Modified Davidson’s fluid), Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Tongue (Retained only and not processed), Lungs (with bronchi, lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, MAMMARY GLANDS, UTERUS (with cervix), Muscle (skeletal, Retained only and not processed): VAGINA.

All tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). All tissues from control and 300 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Microscopic examination was conducted by the Study Pathologist (Peter Millar at Peter Millar Associates Ltd., 3 Queen Charlotte Lane, Edinburgh, EH6 6AY).
Statistics:: Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Histopathological findings: non-neoplastic:: no effects observed
: Key result
Dose descriptor:: NOAEL
Effect level:: > 300 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable due to absence of adverse toxic effects
: Key result
Critical effects observed:: no
: Key result
Reproductive effects observed:: no
Conclusions:: After 90 days of repeated oral exposure to doses up to 300 mg/kg bw/d of the structural analogue, C15-20 ASA, rats showed no evidene of pathologic, histopathologic or toxic effects to organs, and in particular, the reproductive organs. There is no reason to expect that reproductive toxicity based on impaired organ function would result from exposure to this test material. The NOAEL is > 300 mg/kg bw/d for any adverse organ effect. This study is informative for evaluation of the toxicity of the registered substance, n-ODSA EC 701-338-8, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.

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
Quality of whole database:: adequate

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

This information is from the substance 2,5-furandione, dihydro-,mono-C15-20-alkenylderivatives (CAS 68784-12-3, a mixture of a hexadecenyl- and octadecenyl succinic anhydrides), an analogue used for the assessment of several endpoints through read-across. The hypothesis for read-across between the substance being registered (Reaction products of furan-2,5-dione and octadec-1-ene; known here as n-ODSA EC 701-338-8; no CASRN available), and the analogue substance is a common functional group: a 2,5-furandione, dihydro- structure, also known as a succinic anhydride, to which is attached a long-chain monounsaturated olefin. In the environment, the anhydride moiety is quickly hydrolysed to form a dioic acid. When the substance to be registered and the analogue substance are compared, changes in the purity of the starting olefin stock, or small differences in the length (between sixteen and twenty) or arrangement (linear or branched) of the carbon chain are not anticipated to significantly affect the environmental fate properties or the toxicity of the substances. For each endpoint study based upon read-across, the analogue approach is substantiated by an evaluation provided in the Analogue Approach Report Format (AARF) attached to the endpoint study summary file. The AARF allows the read-across information to fulfil the information requirements of the REACH Annexes VII-X, to be the basis for classification and labelling decisions, and for risk assessment.

Regarding reproductive toxicity, in a guideline GLP combined repeated dose toxicity and reproductive toxicity study (OECD 422) with Wistar rats given C15 -20 ASA at doses of 100, 300 and 1000 mg/kg bw/day by oral gavage, there was no evidence of a decrease in fertility in the highest dose. Maternal toxicity was observed at all doses, especially during the lactation period, as seen as piloerection upon dosing, partial or complete loss of litters at post-natal day 2, and inadequate maternal care given to offspring (pups left alone, pups cold, absence of milk in stomachs of pups). In this study, Wistar rats at all doses showed symptoms of severe irritation of the forestomach, which may have resulted in pain and distress causing lactating mothers to be inattentive to pups. These effects in the stomach were not duplicated in the 90 -day repeated dose toxicity study (in Wistars) nor in the teratology study (OECD 414) with CD rats. Therefore, this effect is not considered reproducible. However neither the fertility index nor the live birth index was altered in males or females. The NOAEL was 1000 mg/kg bw/day.

Short description of key information:
No adverse effects on fertility were noted for an analogue substance, C15-20 ASA.

Justification for selection of Effect on fertility via oral route:
experimental data on analogue substance

Effects on developmental toxicity

Description of key information

No adverse effects on fetal development was observed for a structural analogue, C15-20 ASA.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

OECD 414

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 March 2014 - 10 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP guideline study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)

Deviations:

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Monitoring authority, Dept. of Health UK, 2014

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 196 to 269g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 14. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Test item formulation and experimental preparation
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Arachis Oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services as part of Harlan Laboratories Ltd. Study Number: 41304264 and results showed the formulations to be stable for twenty-one days. Formulations were therefore prepared once and stored at approximately +4 °C in the dark.

Samples were taken of each test item formulation and were analyzed for concentration of AS1100 at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by gas chromatography using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.
The formulations investigated during the study were found t ocomprise test item in the range of 101 to 110 % and thus ther required content limit with reference to the nominal concentration was met.
The test item was found to be stable in the formulations when kept for 21 days in the refrigerator ( 4°C).
In conclusion, the restults indicated the accurate use of the test item and vehicle during this study, The formulations were found t obe homogenously prepared and sufficient formulation stability under storage conditions was proven.

Details on mating procedure:

Not specified

Duration of treatment / exposure:

The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage.

Frequency of treatment:

Daily

Duration of test:

The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 females / group

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen based on previous toxicity data.

Maternal examinations:

Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

Ovaries and uterine content:

The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position

Fetal examinations:

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative (excluding fetuses in the litter of Female No. 83). Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, an alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths.

Clinical Observations

Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Eighteen females treated with 1000 mg/kg bw/day showed episodes of increased salivation from Day 12 onwards. One female treated with 300 mg/kg bw/day had increased salivation from Day 18 onwards. Observations of this nature are commonly experienced following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.

No such effects were detected in females treated with 100 mg/kg bw/day.

Body Weight
No treatment-related effects in body weight development were detected.
Statistical analysis of the data did not reveal any significant intergroup differences.

Food Consumption
No treatment-related effects were detected in food consumption.
Statistical analysis of the data did not reveal any significant intergroup differences.

Water Consumption
Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies
No macroscopic abnormalities were detected.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Data and Litter Placental and Fetal Weights
There were no obvious adverse effects of maternal treatment on litter data as assessed by the number of implantations, early and late embryonic/fetal deaths and live fetuses or sex ratio, as assessed by percentage male.

Females treated with 300 mg/kg bw/day had a statistically significant increase in the number of corpora lutea. In the absence of a true dose related response, this intergroup difference was considered to represent normal biological variation rather than an effect of treatment.

Fetal Examination
For all dose groups, there were no significant treatment-related trends in the proportion of fetuses (or litters) with evidence of visceral or skeletal anomalies. The type of visceral and skeletal anomalies were those commonly observed for this type of study.

Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in the number of fetuses with dumb-bell shaped thoracic centrum. The lower incidence of this parameter was considered to indicate a higher number of fetuses showing normal development of the thoracic centrum. Therefore, in isolation and in the absence of any differences in a number of variants or a syndrome of variance, the intergroup difference was considered not to be toxicologically significant.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in the number of fetuses showing incomplete ossification of the thoracic centrum. In the absence of a true dose relationship and in isolation the intergroup difference was considered to be of no toxicological importance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Conclusions:

The oral administration of AS1100 (C15-20 ASA) to pregnant rats by gavage during gestation at dose levels of 100, 300, 1000 mg/kg/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.

Executive summary:

Introduction

The study was performed according to the study plan presented in Appendix13 and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

· US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

· Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

· OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

· Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods…….

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis Oil BP) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results…….

Mortality

There were no unscheduled deaths.

Clinical Observations

Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Body Weight

No treatment-related effects in body weight development were detected.

Food Consumption

No treatment-related effects were detected in food consumption.

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies

No macroscopic abnormalities were detected.

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Fetal Examination

No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

Conclusion

The oral administration of AS1100 to pregnant rats by gavage during gestation at dose levels of 100, 300, 1000 mg/kg/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: adequate

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

The oral administration of the analogue material, AS1100 (C15-20 ASA), to rats in an OECD 414 guideline teratology study at dose levels of 100, 300, 1000 mg/kg/day did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 1000 mg/kg/day.

This information is from the substance 2,5-furandione, dihydro-,mono-C15-20-alkenylderivatives (CAS 68784-12-3, a mixture of a hexadecenyl- and octadecenyl succinic anhydrides), an analogue used for the assessment of several endpoints through read-across. The hypothesis for read-across between the substance being registered (Reaction products of furan-2,5-dione and octadec-1-ene; known here asn-ODSA EC 701-338-8; no CASRN available), and the analogue substance is a common functional group: a 2,5-furandione, dihydro- structure, also known as a succinic anhydride, to which is attached a long-chain monounsaturated olefin. In the environment, the anhydride moiety is quickly hydrolysed to form a dioic acid. When the substance to be registered and the analogue substance are compared, changes in the purity of the starting olefin stock, or small differences in the length (between sixteen and twenty) or arrangement (linear or branched) of the carbon chain are not anticipated to significantly affect the environmental fate properties or the toxicity of the substances. For each endpoint study based upon read-across, the analogue approach is substantiated by an evaluation provided in the Analogue Approach Report Format (AARF) attached to the endpoint study summary file. The AARF allows the read-across information to fulfil the information requirements of the REACH Annexes VII-X, to be the basis for classification and labelling decisions, and for risk assessment.

Justification for selection of Effect on developmental toxicity: via oral route:
experimental result on an analogue substance

Justification for classification or non-classification

The read-across substance (C15 -20 ASA) displayed no developmental toxicity effects in a guideline 414 study. This analogue substance is used as the source of information for the assessment of the target substance through read-across, and the target registered substance is expected to behave similarly. This study is informative for evaluation of the toxicity of n-ODSA EC 701-338-8, and is adequate for classification and risk assessment. Classification under Regulation EC No. 1272/2008 is not indicated based on the absence of adverse developmental effects in this study and in reproductive toxicity studies of the analogue substance..

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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Functional observational battery (P)

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Functional observational battery (P)

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Reference 4

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Reference

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

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