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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
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)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction products of N6,N6'-hexane-1,6-diylbis[N2,N4-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine] and allylbromide, subsequently reacted with ethaneperoxoic acid, hydrogenated
EC Number:
812-927-5
Cas Number:
1902936-62-2
Molecular formula:
not availabale
IUPAC Name:
Reaction products of N6,N6'-hexane-1,6-diylbis[N2,N4-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine] and allylbromide, subsequently reacted with ethaneperoxoic acid, hydrogenated
Test material form:
solid
Details on test material:
Appearance: - physical state: solid; - color: rose
Specific details on test material used for the study:
Physical state/ appearance: solid / rose
Storage conditions: room temperature
Homogeneity: given visual

Test animals

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: 13 - 14 weeks (females) and 15 -16 weeks (males)
- Weight at study initiation: 215 - 218 g (females) and 407 -413 g (males)
- Housing:
during pre-treatment: 5 animals per sex and cage
during mating: 1male/1 female per cage
during rearing up to PND 13: 1 dam with her litter

- 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, 0.5% Carboxymethylcellulose suspension in drinking water was filled up to the desired volume and intensely mixed with a homogenizer. During administration, the preparations was kept homogeneous with a magnetic stirrer. The test substance preparations were produced weekly, at least.
- The administration volume was 10 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 post natal day (PND) 13.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water was demonstrated over a period of 7 days at room temperature.
Considering the low relative deviation of results within the three samples of one test substance preparation in the homogeneity analysis, it can be concluded that the test material was distributed homogeneously in 0.5% Carboxymethylcellulose suspension in drinking water.

The concentrations of the test material in 0.5% Carboxymethylcellulose suspension in drinking water were found to be in the range of 78 to 91% of the nominal concentrations. The analytical method used the solid test material as standard to assess the concentration in the frozen aqueous formulations. Spiked controls to assess the influence on test material storage and loss via absorption were not included. The determined rangeis partially below the strict specification of the test facility (90-110%), but the overall average of 84% was within generally accepted specification for concentration analysis in complex matrixes like the diet (80-120%). The authors assessed the overall accuracy of the prepared concentrations as given.
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 13 days of lactation in females followed by an additional treatment until one day before sacrifice.
Females: up to 63 days
Males: 28 -31 days
Frequency of treatment:
daily
Details on study schedule:
- The females were allowed to litter and rear their pups until day 13 after parturition.
On PND 13, all pups were sacrificed under Isoflurane anesthesia with CO2 and examined.

On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contained 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing).
Doses / concentrationsopen allclose all
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
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 14-day range-finding study

14-day recovery group included for high dose and control group (each 5 animals per sex and dose group)
Estrous cycle determination prior to treatment was performed in a pool of up to 60 non randomized female animals. Only animals with regular estrous cycle were selected for randomization before the st
art of the treatment period. Females are nulliparous and non pregnant at the beginning of the study.

Examinations

Parental animals: Observations and examinations:
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.

Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality were evaluated before the beginning of the administration period.

In all parental females of the main groups estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.
Sperm parameters (parental animals):
testis weight, epididymis weight, stages of spermatogenesis
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 - 13 (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 13.
- 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), PND 4, 7 and 13.
• “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.

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were done in a blind randomized fashion, using a measuring ocular an all live male, female and uncertain pups on PND 1.

All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen were counted.

GROSS EXAMINATION OF DEAD PUPS:
• All surviving pups, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
Postmortem examinations (parental animals):
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

The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKl's method).
Postmortem examinations (offspring):
SACRIFICE

On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically .

On PND 13 all pups were sacrificed under isoflurane anesthesia with CO2 with the exception below.

On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations . Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution.

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.



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
- anogenital index = anogenital distance [mm] / cubic root of pup weight [g]
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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The stages of spermatogenesis in the testes of males of the high dose were comparable to those of the controls.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Thyroid hormones
In parental males in test groups 1, 2, 3 and 11 (100, 300, 1000 and 1000 mg/kg bw/d recovery) no treatment-related alterations of T4 levels were observed.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
nominal (analytically verified 840 mg/kg bw)
Sex:
male/female
Remarks on result:
other: No adverse effects on parent animals

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
One male pup (Animal No. 131-05) of test group 3 (1000 mg/kg bw/day) and two male pups (Animal No. 123-07 and 125-04) of test group 2 (300 mg/kg bw/d) were stillborn. This finding
was spontaneous in nature. All other F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4, resp. PND 13.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weights of all pups in all test groups were comparable to the control group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Thyroid hormones
In parental males in test groups 1, 2, 3 and 11 (100, 300, 1000 and 1000 mg/kg bw/d) and in male and female pups at PND13 in test groups 21, 22 and 23 (100, 300 and 1000 mg/kg
bw/d), no treatment-related alterations of T4 levels were observed.

Nipple retention
The percentage of male pups having nipple/areola anlagen were significantly increased of test groups 2 (300 mg/kg bw/d; 32.8%) and 3 (1000 mg/kg bw/d; 36.1%) when examined on
PND 13. These values did not show a clear dose-dependency and were within the historical control range from 8.7 to 67.0%, therefore, these statistical significant deviations were assessed as incidental and not treatment related.

Anogenital distance
Significantly increased anogenital distance and anogenital index cubic root were noted in male and female pups of test group 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d). In male pups of the current control, values for both parameters were close to the upper limit within the historical control range and in female pups below the lower limit of the historical control range. Because the relative small increases above the current control levels in both sexes were within the relative variability of these two parameters and no dose-dependency was given, these deviations were assessed as incidental and not treatment-related.

Necropsy
Only one male pup of test group 2 (300 mg/kg bw/d) showed an empty stomach. This finding occurred without any relation to dosing and can be found in the historical control data at
comparable or even higher incidences. All other F1 pups of any test group (0-3) did not show adverse findings during necropsy.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
nominal (analytically verified 840 mg/kg bw)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: observed until PND 13 (screening study)

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Summary mating report

    control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
No. of females mated N 10 10 10 10
- Inseminated N 10 f- 10 10 10
Female mating index % 100.0 100.0 100.0 100.0
-- Pregnant N 9 f- 9 10 9
Female fertility index % 90.0 90.0 100.0 90.0
No. of males mated N 10 10 10 10
- With inseminated females N 10 f- 10 10 10
Male mating index % 100.0 100.0 100.0 100.0
- With pregnant females N 9 f- 9 10 9
Male fertility index % 90.0 90.0 100.0 90.0
Females with defined Day 0 pc N 9 10 8 10
Mating days until Day 0 pc Mean 6.2 x+ 2.3 2.1 2.3
S.d. 4.9 1.2 1.0 0.9
Days 0 To 4 N 6 10 8 10
% 66.7 100.0 100.0 100.0
Days 5 To 9 N 0 0 0 0
% 0.0 0.0 0.0 0.0
Days 10 To 14 N 3 0 0 0
  % 33.3 0.0 0.0 0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

Summary Pregnancy Status Report - Reproduction (females)

    control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
No. of females at start N 10 10 10 10
No. of females mated N 10 10 10 10
Without evidence of mating N 1 0 2 0
- Pregnant N 1 0 2 0
- Not pregnant N 0 0 0 0
Females with defined Day 0 pc N 9 10 8 10
Pregnant N 9 9 10 9
- sacrificed scheduled N 9 9 10 9
Not pregnant N 1 1 0 1
- sacrificed scheduled N 1 1 0 1
Pregnant, not delivering N 0 0 0 0
Delivering N 9 9 10 9
-- With liveborn pups N 9 9 10 9
% 100.0 100.0 100.0 100.0
-- With all pups stillborn N 0 0 0 0
  % 0.0 0.0 0.0 0.0

Summary delivery report (females)

    control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
No. of females at start N 10 10 10 10
No. of females mated N 10 f- 10 10 10
% 100.0 100.0 100.0 100.0
Pregnant N 9 f- 9 10 9
% 90.0 90.0 100.0 90.0
Dead N 0 0 0 0
Without delivery N 1 1 0 1
- Pregnant N 0 0 0 0
- Not pregnant N 1 1 0 1
-- Delivering N 9 f- 9 10 9
% 100.0 100.0 100.0 100.0
-- With liveborn pups N 9 f- 9 10 9
Gestation Index % 100.0 100.0 100.0 100.0
Gestation days Mean 22.1 n 22.3 22.1 22.0
S.d. 0.4 0.5 0.4 0.0
N 8 9 8 9
-- With stillborn pups N 0 f+ 0 2 1
% 0.0 0.0 20.0 11.1
-- With all pups stillborn N 0 f+ 0 0 0
  % 0.0 0.0 0.0 0.0

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; n=DUNNETT

Summary Litter Report - Pup Status

    control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
Total Number of Pregnant Females N 9 9 10 9
Total number of litters N 9 9 10 9
With liveborn pups N 9 f- 9 10 9
% 100.0 100.0 100.0 100.0
With stillborn pups N 0 f+ 0 2 1
% 0.0 0.0 20.0 11.1
With all pups stillborn N 0 f+ 0 0 0
% 0.0 0.0 0.0 0.0
Corpora Lutea N 0 0 0 0
Mean 0 X 0.0 X 0.0 X 0.0 X
S.d.
N 0 0 0 0
Implantation Sites N 116 101 125 114
Mean 12.9 x- 11.2 12.5 12.7
S.d. 1.5 2.3 2.1 1.2
  N 9 9 10 9

Preimplantation Loss Mean% 0 X 0.0 X 0.0 X 0.0 X
S.d.
N 0 0 0 0
Pups delivered N 111 99 110 113
Mean 12.3 x- 11.0 11.0 12.6
S.d. 2.0 2.3 3.2 1.1
N 9 9 10 9
Postimplantation Loss Mean% 4.6 x+ 2.0 12.3 0.8
S.d. 7.2 4.1 19.9 2.4
N 9 9 10 9
Pups liveborn N 111 99 108 112
% 100.0 100.0 98.2 99.1
Mean 12.3 x- 11.0 10.8 12.4
S.d. 2.0 2.3 3.1 1.1
N 9 9 10 9
Pups stillborn N 0 0 2 1
% 0.0 0.0 1.8 0.9
Mean 0 x+ 0.0 0.2 0.1
S.d. 0.0 0.0 0.4 0.3
N 9 9 10 9
Perinatal Loss Mean% 0 x+ 0.0 1.6 0.9
S.d. 0.0 0.0 3.4 2.6
N 9 9 10 9

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test

(one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

Summary Litter Report - Dead Pups

    control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
Litters with liveborn pups N 9 9 10 9
Pups delivered N 111 99 110 113
stillborn / Dead N 0 0 2 1
% 0.0 0.0 1.8 0.9
Alive / Alive N 111 99 108 112
% 100.0 100.0 98.2 99.1
sacrificed scheduled / Dead N 72 71 75 72
% 64.9 71.7 68.2 63.7
culled / Dead N 39 28 33 40
% 35.1 28.3 30.0 35.4
Litters not surviving Day 13 N 0 0 0 0
  % 0.0 0.0 0.0 0.0

Summary Litter Report - Pups Died

    control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
Litters with liveborn pups N 9 9 10 9
Pups delivered N 111 99 110 113
Day 0 N 0 0 0 0
% 0 0 0 0
Days 1 To 4 N 0 0 0 0
% 0 0 0 0
Days 5 To 7 N 0 0 0 0
% 0 0 0 0
Days 8 To 13 N 0 0 0 0
% 0 0 0 0
Pups surviving days 0 To 4 N 111 99 108 112
Viability Index Mean% 100 x- 100.0 100.0 100.0
S.d. 0.0 0.0 0.0 0.0
N 9 9 10 9
Pups surviving days 4 To 13 N 72 71 75 72
Survival Index Mean% 100 x- 100.0 100.0 100.0
S.d. 0.0 0.0 0.0 0.0
  N 9 9 10 9

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test

(one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

Summary Litter Report - Live Pups/Litter

    control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
Litters with liveborn pups N 9 9 10 9
Pups delivered N 111 99 110 113
% Live male Day 0 Mean% 55.7 x 60.9 46.0 54.6
S.d. 14.9 18.9 23.1 13.6
N 9 9 10 9
% Live female Day 0 Mean% 44.3 x 39.1 54.0 45.4
S.d. 14.9 18.9 23.1 13.6
N 9 9 10 9
% Live male Day 13 Mean% 51.4 x 58.1 49.6 52.8
S.d. 7.5 16.1 19.9 8.3
N 9 9 10 9
% Live female Day 13 Mean% 48.6 x 41.9 50.4 47.2
S.d. 7.5 16.1 19.9 8.3
  N 9 9 10 9

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test

(one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

Applicant's summary and conclusion

Conclusions:
No indication of reproductive toxicity was observed in the screening study (OECD 422, version of 2016) at the limit dose.