N,N''-naphthalene-1,5-diylbis[N'-[3-[(2-ethylhexyl)oxy]propyl]urea]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD 422 compliant study with well characterized material

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Rat, Crl:WI(Han)

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
- Age at study initiation: 10-11 weeks
- Weight at study initiation: males: ca 205 g (females) and 320 g (males)
- Housing: The rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). Pregnant animals and their litters were housed together until day of parturition (PND) 4 (end of lactation).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2014-12-22 to: 2015-02-16

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.

- The administration volume was 4 mL/kg bw.

Details on mating procedure:

- Fourteen days after the beginning of treatment, males and females from the same test group were mated 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.
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy and the following day gestation day (GD) 1
- After successful mating each pregnant female was caged (how): Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- The females were allowed to litter and rear their pups until day 4 after parturition.
On PND 4, all pups were sacrificed under Isoflurane anesthesia with CO₂ and examined.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 95-101% of the nominal concentrations.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.
Females: 52 days
Males: 31 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 14-day range-finder study

Parental animals: Observations and examinations:

see also repeated dose toxicity: rat.OECD422
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity; a check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. Thereby, the following parameters were examined:
1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals; during the administration period on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The 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.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Pup number and status at delivery
• Total number of pups and the number of liveborn and stillborn pups in each litter on the day of birth.
• Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
- Pup viability/mortality
• Check for dead or moribund pups twice daily on workdays and once on Saturdays, Sundays or public holidays.
• Number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period).
• Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations.
• The number of live pups/litter was calculated on the day after birth, and on lactation day 4.
- 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 on the day after birth (PND 1) and on PND 4.
• “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
• Sex ratio at day 0 and day 4 after birth = number of live male or female pups on day 0 and 4/ number of live male and female pups on day 0 and 4 x 100
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. The sex of the pups was finally confirmed at necropsy.

GROSS EXAMINATION OF DEAD PUPS:
• 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.

Postmortem examinations (parental animals):

see also repeated dose toxicity: keyBASF85R0060/11C032, 2012.Repeated dose toxicity, oral, rat.OECD422
SACRIFICE
- All animals were sacrificed by decapitation under Isoflurane anesthesia.

GROSS NECROPSY
- The exsanguinated animals were necropsied and assessed by gross pathology.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight parameters:
Weight assessment was carried out on all animals.
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

- Organ / Tissue preservation
The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

From the liver, each one slices of the lobus dexter medialis and the lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast.

HISTOPATHOLOGY: Yes
Organ samples / Methods-Scope of examinations / Test group
1. All gross lesions: Hematoxylin-eosin (H&E), all affected animals per group, all treatment groups
2. Adrenal glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
3. Bone marrow (femur): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
4. Brain: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
5. Cecum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
6. Cervix: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
7. Coagulation glands: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
8. Colon: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
9. Duodenum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
10. Epididymides: Hematoxylin-eosin (H&E), all affected animals per group
11. Heart: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
12. Ileum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
13. Jejunum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
14. Kidneys: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
15. Liver: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group; low and mid dose group: embedded in paraplast all animals per group.
16. Lung: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
17. Lymph nodes (mesenteric and axillary lymph nodes): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
18. Ovaries: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
19. Oviducts: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
20. Peyer’s patches Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
21. Prostate: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
22. Rectum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
23. Sciatic nerve: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
24. Seminal vesicles: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
25. Spinal cord (cervical, thoracic and lumbar cords): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
26. Spleen: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
27. Stomach (forestomach and glandular stomach): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
28. Testes: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
29. Thymus: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
30. Thyroid glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
31. Trachea: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
32. Urinary bladder: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
33. Uterus: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
34. Vagina: Hematoxylin-eosin (H&E), control and high dose group, all animals per group

Postmortem examinations (offspring):

SACRIFICE
- All surviving pups were sacrificed on PND 4 under isoflurane anesthesia with CO2.

GROSS NECROPSY
- All surviving pups, 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:

- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: DUNNETT-test (two-sided);
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions;
- Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, clinical pathology parameters (except for urine color and turbidity), weight parameters: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Reproductive indices:

For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
- 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 (%) = number of males proving their fertility*/ number of males placed with females x 100
* defined by a female with implants in utero
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
- Female mating index (%) = number of females mated*/ number of females placed with males x 100
* defined as the number of females with vaginal sperm or with implants in utero
- Female fertility index (%) = number of females pregnant*/ 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 pups on the day of birth/number of females pregnant* x 100
* defined as the number of females with implants in utero
- Live birth index (%) = number of liveborn pups at birth/total number of pups born x 100
- Post implantation loss (%) = number of implantations number of pups delivered / number of implantations x 100

Offspring viability indices:

Viability index (%) = number of live pups on day 4 after birth/number of live pups on the day of birth x 100

Clinical signs:

no effects observed

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:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

The male mating index was 100% in all test groups.
One male animal of the control group (No. 5 mated with female No. 105), one male of test group 1 (No. 11 mated with female No. 111) and two males of test group 2 (Nos. 25 and 29 mated with female Nos. 125 and 129) did not generate F1 pups. The male fertility index was 100% in test 3, 90% in test groups 1 and 2, and 88.9% in control. This reflects the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility.
All stages of spermatogenesis were detected at histopathology.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 3.8, 2.1, 3.3 and 3.2 days in test groups 0 – 3. 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.

All sperm positive rats delivered pups or had implants in utero with the following exceptions:
􀁸 Control group female No. 105 (mated with male No. 5) did not become pregnant.
􀁸 Low dose group female No. 111 (mated with male No. 11) did not become pregnant.
􀁸 Mid dose group female No. 125 (mated with male Nos. 25) did not become pregnant.
􀁸 Mid dose group female No. 129 (mated with male No. 29) did get sperm in vaginal smear and showed implants but delivered no pups.
The female fertility index was 90% in test groups 0, 1 and 2, and 100% in test group 3.

The mean duration of gestation was similar in all test groups (22.1 day in test groups 0 and 1; 22.3 days in test group 2 and 22.2 days in test group 3). These findings 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.

The gestation index was 100% in test group 0, 1 and 3 and 89% in test group 2. The latter represents 8 females with live pups on the day of birth out of 9 females pregnant.

The rate live birth indices were 99.0% in test group 0, 99.1% in test group 1, 98.0% in test group 2 and 96.4% in test group 3. The values of test group 1, 2 and 3 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.

The postimplantation loss was 5.0 % in test group 0, 6.7 % in test group 1, 16.5 in test group 2 and 13.5 % in test group 3. These findings 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 (0.7% - 18.3%) of the historical control data.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

In test group 3 (1000 mg/kg bw/d; female No. 135) 4 pups were stillborn. In test group 2 (300 mg/kg bw/d; female Nos. 123, 130) each 1 pup was stillborn. In test group 1 (100 mg/kg bw/d; female No. 118) 1 pup was stillborn. In the control group (0 mg/kg bw/d;vfemale No. 103) 1 pup was stillborn. The mean number of delivered F1 pups per dam was evenly distributed about the groups 0, 1, 2 and 3 and in the range of the historical control data.
The viability index indicating pup mortality during lactation (PND 0 - 4) was 98.6% (test group 0), 100.0% (test group 1 and 2) and 99.2% (test group 3) based on one pup of test group 3 (1000 mg/kg bw/d) was missing (cannibalized) and 1 pup of the control group (0 mg/kg bw/d) was found dead. These findings 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.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups.
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. One female runt was recorded for test group 0 (0 mg/kg bw/d). Furthermore, four female runts were determined in test group 1 (100mg/kg bw/d) and one male runt was seen in test group 3 (1000 mg/kg bw/d).

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Observations up to postnatal day 4 (screening study only)

Reproductive effects observed:

not specified

Conclusions:

No adverse effects on reproduction were observed at the limit dose of 1000 mg/kg bw (OECD 422).

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:

GLP, OECD 422

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats the test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals). The dose levels were 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Corn oil was used as a vehicle. The substance was suspended since it is insoluble in any vehicle compatible with gavage dosing. Regarding clinical examinations, signs of general systemic toxicity were not observed in male and female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire study period. Fertility indices for male and female animals were not impaired by test-substance administration even at the limit dose of 1000 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not impaired by test-substance administration up to the highest dose tested (1000 mg/kg bw/d). Regarding clinical pathology no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, there were no treatment-related findings regarding organ weights, macroscopy or histpathology. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The NOEL for fertility and reproductive performance was 1000 mg/kg bw/d. The NOEL for developmental toxicity in the F1 progeny of the test substance-treated group was found to be 1000 mg/kg bw/d.

Short description of key information:
No adverse effects on fertility were observed in a GLP and OECD 422 compliant gavage study in rats at dose levels of 100, 300 and 1000 mg/kg bw.

Effects on developmental toxicity

Description of key information

Information is available at the screening level (OECD 422). No adverse effects on developmental parameters were observed at the limit dose of 1000 mg/kg bw.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Screening information (OECD 422)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available screening study is considered reliable and suitable for classification purposes under 67/548/EEC. No adverse effects were observed up to the limit dose of 1000 mg/kg bw. As a result the substance is not considered to be classified for fertility or developmental toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG. 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008.

No adverse effects were observed up to the limit dose of 1000 mg/kg bw.

As a result the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC944/2013.

During the four days covered in the screening study, no effects via lactation were observed.