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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects on fertility were observed in a screening study for reproductive toxicity (OECD 422, GLP) at the nominal dose of 1000 mg/kg bw (analytically determined average 840 mg/kg bw).

Link to relevant study records

Reference

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

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)

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

Specific details on test material used for the study:

Physical state/ appearance: solid / rose
Storage conditions: room temperature
Homogeneity: given visual

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

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).

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.

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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

Critical effects observed:

no

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

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.

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)

Critical effects observed:

no

Reproductive effects observed:

no

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

Conclusions:

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

840 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance was administered daily by gavage as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) at nominal doses of 0, 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals were dosed daily with the vehicle only (0.5% Carboxy-methylcellulose suspension in drinking water). The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Additional treated but not mated animals (recovery animals) was maintained for a subsequent period of at least 14 days of no test substances administration in order to observe reversibility of the findings.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 14 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents of main groups and recovery groups were determined regularly once weekly before mating period as well as males and females of the recovery groups until sacrifice. In dams during gestation days 7,14 and 20 and lactation days 4, 7, 10 and 13 the food consumption was also determined. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areola anlagen were counted. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations.

After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. At necropsy on PND 13, one selected male and one female pup per litter were 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 and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. The remaining 5 animals per sex of test groups 10 and 11 (recovery animals) were maintained for a 2-week recovery period after the administration period without test substance exposure.

Effects on developmental toxicity

Description of key information

- OECD 414; GLP; Wistar rats; 100, 300, 1000 mg/kg bw/day (nominal); no adverse effects observed; NOAEL (maternal/prenatal development) 881 mg/kg bw/day (actual measured) (2020)

- No adverse effects were observed in rats in a screening study for developmental toxicity (OECD 422, GLP) at the nominal dose of 1000 mg/kg bw (analytically determined average 840 mg/kg bw).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2019 - Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jun 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch number of test material: 0016595783
- Expiration date of the lot/batch: 05 Feb 2022
- Purity: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: Guaranteed
- Stability under test conditions: at least 7 days
- Homogeneity: Confirmed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

FORM AS APPLIED IN THE TEST: solid

Species:

rat

Strain:

Wistar

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: 10-12 weeks
- Weight at study initiation: 175.4 - 224.0g
- Fasting period before study: no
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (illumination 6 am to 6 pm)

IN-LIFE DATES: From: 15/16/17 May (Cohort 1/2/3, resp.) 2019 To: 4/5/6 Jun (Cohort 1/2/3, resp.) 2019

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Beginning of administration and thereafter at regular intervals
- Volume applied: 10 ml/kg bw/day
- Concentration: 1.00, 3.00, 10.00 g/100 ml for test groups 100, 300, 1000 mg/kg bw/day, respectively

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent three times (at the beginning of administration [samples 3-9] and due to equivocal results after the end of administration
[samples 3R-5R and 7R-9R, as well as samples 11-18 and 20-27]) to the analytical laboratory for verification of the concentrations.
Reserve samples were described by the suffix “R” in the report.
The concentrations of the test substance in the samples were calculated by means of their nitrogen content. The concentrations were in accordance with the expected values.

Details on mating procedure:

Time-mated
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

daily

Duration of test:

from implantation to one day prior to the expected day of parturition

Dose / conc.:

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

Remarks:

nominal 100 mg/kg bw

Dose / conc.:

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

Remarks:

nominal 300 mg/kg bw

Dose / conc.:

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

Remarks:

nominal 1000 mg/kg bw

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Result of OECD 422 study (2017).
- Rationale for route: The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Due to technical reasons, the study was carried out in 3 cohorts.
- Reason for species selection: The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked: morbidity, pertinent behavioral changes and/or signs of overt toxicity

CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
- Clinical observations checked: mortality

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD20
- Blood samples obtained: Yes, from all animals
- Anesthesia: Yes (Isoflurane)
- Organs examined: Thyroid glands (with parathyroid glands), All paired organs were weighed together (left and right).
- Histopathology: Yes, Thyroid glands

THYROID HORMONES
- Parameters checked: Total triiodothyronine (T3), Total thyroxine (T4), Thyroid stimulating hormone (TSH)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance: Yes
- Fetuses were sexed, weighed, condition of placenta checked and umbilical cords, fetal membranes, and fluids were examined.

Statistics:

DUNNETT-test, FISHER'S EXACT test, WILCOXON-test, KRUSKAL-WALLIS test.
* for p < 0.05
** for p < 0.01

Indices:

The conception rate, The preimplantation loss, The postimplantation loss, anogenital index

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In dams at GD21 (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of serum T3, T4 and TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. No weight changes were noted in in the thyroid glands.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One spontaneous finding was noted in one low-dose female (100 mg/kg bw/d), i.e. a dilated renal pelvis.
No further necropsy findings which could be attributed to the test substance were seen in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (1000 mg/kg bw/d), the minimal follicular cell hypertrophy/hyperplasia in 5 out of 25 dams and the minimal altered colloid in 4 out of 25 dams were assumed to be treatment-related when compared to the incidence of these findings in the control group and test groups 1 and 2 (100 and 300 mg/kg bw/day, resp.). These changes were assumed to be treatment-related but not adverse due to their low magnitude and the absence of treatment-related hormonal changes in this test group.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Concerning serum thyroid hormone measurements, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, no weight changes were noted in in the thyroid glands. In test group 3 (1000 mg/kg bw/d), histopathology of the thyroid glands revealed a minimal follicular cell hypertrophy/hyperplasia in 5 out of 25 dams and minimally altered colloid in 4 out of 25 dams. These changes were assumed to be treatment-related but not adverse due to their low magnitude and the absence of treatment-related hormonal changes in this test group. All other histopathological findings in the thyroid glands 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.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in the test groups 0, 1 and 2 (0, 100 and 300 mg/kg bw/d) and 100 % in test group 3 (1000 mg/kg bw/d).

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- The statistically significantly decreased mean number of corpora lutea in test group 1 (mean 12.1* [* = p ≤ 0.05 Dunnett-test]) is assessed as incidental and biologically not relevant as this value is well within the historical control range (mean 11.7 [10.5 - 13.8]) and there is no doseresponse.
- No effects observed in mean number of implantation sites

Details on maternal toxic effects:

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.
Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Dose descriptor:

NOAEL

Remarks:

maternal developmental

Effect level:

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

Based on:

test mat.

Remarks:

actual dose (nominal 1000 mg/kg bw)

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

An apparent shift in the sex ratio, visible as a statistically significantly decreased mean number of live female fetuses in test group 3 is assessed as incidental and biologically not relevant as the overall sex ratio in this test group (45% live females and 55% live males) was within the historical control range (live males [41.2% – 55.9%] and live females [44.1% – 58.8%])

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No external malformations and no external variation occurred in treated animals. One unclassified external observation was recorded. Placentae fused were seen in one litter
in test group 2 (300 mg/kg bw/d). This finding was not considered biologically relevant, since it was a single event and can be found in the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in two fetuses of the control group and one fetus in the high-dose group (1000 mg/kg bw/d). The isolated finding in one fetus of the high-dose group (‘misshapen basisphenoid’) was not assessed as treatment-related and adverse, since it can be found in the historical control data at comparable incidences.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.
Affected fetuses per litter in control, 100, 300, and 1000 mg/kg bw/day groups were 97.2%, 93.8%, 95.9%, and 96.3%, respectively.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus each of test group 1 and 3 (100 and 1000 mg/kg bw/d) had a situs inversus. This finding was not related to dose and single events in individual fetuses. The mean values of affected fetuses per litter were within the range of historical control data. Thus it is not considered as treatment-related and adverse.
Four soft tissue variations were detected: short innominate and absent lung lobe (lobus inferior medialis) in one single high-dose fetus, dilated renal pelvis and dilated ureter in test groups 1-3 (100-1000 mg/kg bw/day). The incidences of ‘dilated renal pelvis’ and the concomitant finding ‘dilated ureter’ (0.0/4.2*/7.5**/1.3 mean% affected fetuses/litter, respectively) were statistically significantly increased in test groups 1 and 2 (100 and 300 mg/kg bw/d). However, due to lack of dose-response relationship and the fact that incidences were within the historical control data, increases were considered as spontaneous and not treatment-related

Other effects:

no effects observed

Description (incidence and severity):

- The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.
- The anogenital distance and anogenital index of all male and female fetuses in all test groups was comparable to the concurrent control values.

Details on embryotoxic / teratogenic effects:

There were noted external, soft tissue and skeletal malformations in two fetuses of the control, one fetus of the low-dose (100 mg/kg bw/d) and two fetuses of the high-dose groups (1000 mg/kg bw/d).
One fetus carried more than one malformation: female control fetus No. 15-06 had an anal atresia and acaudate which was confirmed during the skeletal examination (absent sacral vertebra, absent caudal vertebra).
Further malformations, i.e. situs inversus, misshapen basisphenoid and malpositioned and bipartite sternebra were observed in individual fetuses, unrelated to dose and all of them can be found in the historical control data.
All these findings were single cases, no ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment.
One external variation, four soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dosing. The majority of individual variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency. The incidences of two soft tissue variations, i.e. ‘dilated renal pelvis’ and the concomitant finding ‘dilated ureter’ (0.0/4.2*/7.5**/1.3 mean% affected fetuses/litter, respectively), were statistically significantly increased in test groups 1 and 2 (100 and 300 mg/kg bw/d). The addition of those soft tissue variations resulted in an increased total affected fetuses per litterincidence in test groups 1-3 which attained statistical significance. However, due to the lack of dose-response relationship and the fact, that all incidences of test groups 1-3 were within the historical control range (HCD: mean% 4.0 [0.7 - 12.6]), while the control incidence was even below the historical control range, these increases in soft tissue variations are considered to be spontaneous in origin and not treatment-related.
Unclassified soft tissue observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external and skeletal cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.
Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to 1000 mg/kg bw/d.

Dose descriptor:

NOAEL

Remarks:

prenatal development

Effect level:

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

Based on:

test mat.

Remarks:

nominal 1000 mg/kg bw

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Analytical determination of the test material in the vehicle

• The stability of the test substance in 0.5% CMC in drinking water over a period of 7 days at room temperature was demonstrated.

• The homogeneous distribution of the test substance in the vehicle was shown.

Most values during concentration control analyses did not meet the in-house quantity criteria of 100% +/- 10%. Thus, the target dose levels were corrected using i) the calculated mean of the recovery values from the measured mid-dose samples, i.e. 86.3%, ii) and the calculated mean value for the high-dose level, i.e. 88,1%.

Only pregnant dams were used for the calculations of mean maternal water consumption, food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, corrected (net)

body weight gain and summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):

• Female No. 17 - not pregnant

Test group 1 (100 mg/kg bw/d):

• Female No. 49 - not pregnant

Test group 2 (300 mg/kg bw/d):

• Female No. 66 - not pregnant

Table 1: Histopathology maternal animals, summary

Thyroid glands	Female animals
Test group (mg/kg bw/d)	0 (0)	1 (100)	2 (228)	3 (881)
No. of dams	24	24	24	25
Hypertrophy/hyperplasia, follicular	0	2	1	5
·       Grade1		2	1	5
Altered colloid	2	2	2	4
·       Grade1	2	2	2	4

Table 2: Total external malformations, fetus

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 228 mg/kg bw/d	Test group 3 881 mg/kg bw/d
Litter Fetuses	N N	24 290	24 262	24 275	25 291
Fetal incidence	N (%)	1 (0.3)	0.0	0.0	0.0
Litter incidence	N (%)	1 (4.2)	0.0	0.0	0.0
Affected fetuses/litter	Mean%	0.5	0.0	0.0	0.0

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

Table 3: Soft tissue malformations, fetus

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 228 mg/kg bw/d	Test group 3 881 mg/kg bw/d
Litter Fetuses	N N	24 138	24 125	24 131	25 138
Fetal incidence	N (%)	0.0	1 (0.8)	0.0	1 (0.7)
Litter incidence	N (%)	0.0	1 (4.2)	0.0	1 (4.0)
Affectedfetuses/litter	Mean%	0.0	0.8	0.0	0.7

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

Table 4: Skeletal malformations, fetus

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 228 mg/kg bw/d	Test group 3 881 mg/kg bw/d
Litter Fetuses	N N	24 152	24 137	24 144	25 153
Fetal incidence	N (%)	2 (1.3)	0.0	0.0	1 (0.7)
Litter incidence	N (%)	2 (8.3)	0.0	0.0	1 (4.0)
Affectedfetuses/litter	Mean%	1.5	0.0	0.0	1.0

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

Conclusions:

The no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 881 mg/kg bw/d (actual dose received; 1000 mg/kg bw nominal).

Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at nominal doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose suspension [CMC] in deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24 - 25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including sampling of thyroid glands (with parathyroid glands) and weight determinations of the thyroid glands (with parathyroid glands), unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. No test substance-related adverse effects on dams, gestational parameters or fetuses were observed at nominal doses of 100, 300, and 1000 mg/kg bw/day. Most values during concentration control analyses did not meet the in-house quantity criteria of 100% +/- 10%. Thus, the target dose levels were corrected using i) the calculated mean of the recovery values from the measured mid-dose samples, i.e. 86.3%, ii) and the calculated mean value for the high-dose level, i.e. 88,1%.  

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 881 mg/kg bw/d (nominal 1000 mg/kg bw/d) provided no evidence of maternal or developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 881 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

881 mg/kg bw/day

Species:

rat

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 test substance was tested for its prenatal developmental toxicity in Wistar rats according to OECD 414 and in compliance with GLP regulations (2020). The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at nominal doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. After analytical verification of the dosing solutions, the actual ingested doses were recalculated to be 100. 228 and 881 mg/kg bw. The control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose suspension [CMC] in deionized water) in parallel. Food consumption and body weights were recorded regularly. On GD 20, blood samples were obtained from all females. After blood sampling, all females were sacrificed and assessed by gross pathology and weight determinations of the thyroid glands (with parathyroid glands), unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

Concerning serum thyroid hormone measurements, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, no weight changes were noted in in the thyroid glands. In test group 3 (1000 mg/kg bw/d), histopathology of the thyroid glands revealed a minimal follicular cell hypertrophy/hyperplasia in 5 out of 25 dams and minimally altered colloid in 4 out of 25 dams. These changes were assumed to be treatment-related but not adverse due to their low magnitude and the absence of treatment-related hormonal changes in this test group. All other histopathological findings in the thyroid glands 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. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

There were noted external, soft tissue and skeletal malformations in two fetuses of the control, one fetus of the low-dose (100 mg/kg bw/d) and two fetuses of the high-dose groups (1000 mg/kg bw/d). All these findings were single cases, no ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment.

The incidences of two soft tissue variations, i.e. ‘dilated renal pelvis’ and the concomitant finding ‘dilated ureter’ (0.0/4.2*/7.5**/1.3 mean% affected fetuses/litter, respectively), were statistically significantly increased in test groups 1 and 2 (100 and 300 mg/kg bw/d). The addition of those soft tissue variations resulted in an increased total affected fetuses per litter incidence in test groups 1-3 which attained statistical significance. However, due to the lack of dose-response relationship and the fact, that all incidences of test groups 1-3 (10-1000 mg/kg bw/day) were within the historical control range (HCD: mean% 4.0 [0.7 - 12.6]), while the control incidence was even below the historical control range, these increases in soft tissue variations are considered to be spontaneous in origin and not treatment-related. Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to 1000 mg/kg bw/d. 

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d provided no evidence of maternal or developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 1000 mg/kg bw/d nominal / 881 mg/kg bw/d (actual ingested).

Justification for classification or non-classification

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 on fertility or development were observed in a screening study in rats (OECD 422/421) and no adverse effects were observed in a teratogenicity/developmental toxicity study in rats (OECD 414). 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 fourteenth time in Regulation (EC) No. 2020/217.

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