Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine

EC Number:

425-020-0

EC Name:

Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine

Cas Number:

191680-81-6

Molecular formula:

C50H77N11O2-C168H230N32O8

IUPAC Name:

N2-(2-{[4,6-bis({butyl[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]amino})-1,3,5-triazin-2-yl](3-{[4,6-bis({butyl[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]amino})-1,3,5-triazin-2-yl]amino}propyl)amino}ethyl)-N2-(3-{[4,6-bis({butyl[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]amino})-1,3,5-triazin-2-yl]amino}propyl)-N4,N6-dibutyl-N4,N6-bis[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]-1,3,5-triazine-2,4,6-triamine

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ordered from Charles River Italia S.p.A., Calco (Lecco), Italy and supplied by Charles River Laboratories, France.
- Age at study initiation: 9 weeks (females), 11 weeks (males)
- Weight at study initiation: 200-225g (females), 336.3 g (males)
- Housing: Before mating for all animals and after mating for males, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). During the mating period, one male rat was housed with one female rat in clear polysulfone cages measuring 42.5×26.6×18.5 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a
stainless steel mesh lid and floor. After mating, the mated females were housed individually in clear polysulfone cages measuring 42.5×26.6×18.5 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags. Nesting material was changed at least 2 times a week
- Diet (e.g. ad libitum): drinking water, ad libitum
- Water (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy), ad libitum
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTION:
The required amount of test item was weighed and the required amount of vehicle was added. The mixture with a Silverson (medium head) was treated for 5 minutes and the resulting suspension was left under magnetic stirring for at least 3 hours prior to
dosing or analysis.
The formulation was prepared daily or up to a weekly interval (concentrations of 10, 30
and 100 mg/mL) according to the stability data. Concentrations were calculated and expressed in terms of test item as supplied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations. Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department at ERBC. The software used for this activity was Empower® 2 Build No. 2154 (Waters).
Results of the analyses were within the acceptability limits stated in ERBC SOPs (85-115%
for concentration and CV < 10% for homogeneity).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Females were paired one to one in the home cage of the male and left overnight.
- M/F ratio per cage: 1:1
- Length of cohabitation: over night
- Proof of pregnancy: sperm in the vaginal smear or the presence of a copulation plug, referred to as Day 0 of gestation.

Duration of treatment / exposure:

GD6-19

Frequency of treatment:

once daily

Duration of test:

on GD 20, all surviving females were sacrificed

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 mated females per group

Control animals:

yes

Details on study design:

- Dose levels were selected by the Sponsor.
- The oral route was selected as it is a possible route of exposure of the test item in man.
- The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Clinical signs were recorded daily.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: on days 3, 6, 9, 12, 15, 18 and 20 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs weight and fixated: Thyroid, Brain
- Histopathology: Thyroid

HORMONE ANALYSIS: Yes
- Time schedule: on Day 20 post coitum.

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
- Other: number, sex and weight of all live foetuses, number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); number of intra-uterine deaths; gross evaluation of placentae.

Blood sampling:

- Serum: Yes
- Volume collected: approximately 1 mL. The serum obtained was divided in two aliquots (300 µL in the aliquot A, the remaining in the aliquot B)

Fetal examinations:

- Weight of each fetus: Yes
- Sex: Yes
- Gross evaluation of placentae: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: Approximately one-half of the foetuses
- Skeletal examinations: Yes: in the remaining foetuses
- Anogenital distance (AGD): Yes: each live foetus on Day 20 post cioitum

Statistics:

For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Indices:

Pre-implantation loss % = no. of corpora lutea−no. of implantations/no. of corpora lutea x 100

Post-implantation loss % = no. of implantations−no. of live foetuses/ no. of implantations x 100

Total implantation loss % = no. of corpora lutea−no. of live foetuses/no. of corpora lutea x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Mortality:

no mortality observed

Description (incidence):

All females survived to the final sacrifice on gestation Day 20.

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects: no effects

Details on maternal toxic effects:
- MORTALITY:
All females survived to the final sacrifice on gestation Day 20.

- CLINICAL SYMPTOMS:
No treatment-related clinical signs were recorded throughout the study. Signs of hairloss were observed in single animals regardless the treatment groups. Therefore, this sign was not attributed to treatment with the test item.


- FOOD CONSUMPTION:
Food consumption was comparable between treated and control groups throughout the study.


- BODY WEIGHT:
Maternal body weight and body weight gain were unaffected by treatment.

- TERMINAL BODY WEIGHT:
No test-substance related findings.

- UTERUS WEIGHT:
No test-substance related findings.

- HORMONE DETERMINATION:
Serum levels of T3, T4 and TSH did not differ between treated and control groups.

- NECROPSY FINDINGS:
Organ weights: There was no effect on terminal body weight or on the absolute and relative (to brain)
thyroid gland weights in treated females when compared to controls. Any organ weight variations were within the range of expected variations in Wistar rats of the same age and considered incidental and unrelated to treatment.
Macroscopic and microscopic observations: At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item up to 1000 mg/kg/day, when compared to controls. No treatment-related changes were noted in thyroid gland of females receiving the test item up to 1000 mg/kg/day, when compared to controls.

- REPRODUCTION DATA:
Two controls (nos. 3 and 19) and one low dose female (no. 63) were not pregnant. At necropsy, the uterus from one mid-dose female (no. 139) showed unilateral implantation (left horn) and the right one was not pregnant. The final number of females with live foetuses on gestation Day 20 was:
– 23 in the control group (0 mg/kg/day)
– 24 in the low dose group (100 mg/kg/day)
– 25 in the mid-dose group (300 mg/kg/day)
– 25 in the high dose group (1000 mg/kg/day)

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- SEX DISTRIBUTION:
No treatment-related effects.

- WEIGHT OF FETUSES:
No treatment-related effects.

- ANOGENITAL DISTANCE:
The anogenital distance did not show differences between groups both in female and male foetuses.

- FETAL EXTERNAL EXAMINATIONS:
No abnormalities were recorded at external examination in all groups with exception of isolated cases of small foetuses (<2.7g) in treated groups.

- FETAL SKELETAL EXAMINATIONS:
The absence of one rib (13th) was noted in one low dose female. This major finding occurred as single case and it was considered incidental. Minor abnormalities or variations occurred in all groups and included for example altered ossification (asymmetrical, incomplete or no ossification) of several bones of the skull, sternebrae, thoracic/sacral vertebrae and the presence supernumerary ribs (14th). The incidence of the affected litters in treated groups was similar or even lower than observed in the control group or without dose relation. Therefore, the findings were considered unrelated to treatment.

- FETAL VISCERAL EXAMINATIONS:
No major abnormalities were found. The incidences of foetuses or litters with anomalies or variations did not suggest any test item effect.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest tested dose

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.

Executive summary:

In a GLP compliant prenatal developmental toxicity study (OECD 414), the test item (in 0.5%CMC) and administered by oral gavage at 100, 300 and 1000mg/kg/day to a total of 25 females per group. The animals were treated throughout the gestation period from Day 6 up to Day 19 post coitum. Control females received the vehicle (0.5 % CMC) at the same dose volume during the same treatment period.

No mortality occurred in the study. All females were sacrificed at termination and were pregnant with the exception of 2 controls and one female at 100 mg/kg/day. No treatment-related effects for observed for clinical signs, maternal body weight and body weight gain, food consumption, thyroid hormone determination, terminal body weight, uterus weight and absolute weigh gain, organ weight, litter data and sex ratio, anogenital distance and foetal examinations (macroscopic, microscopic, external, skeletal, visceral). 

Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.