N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

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:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

EC Number:

401-990-0

EC Name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Cas Number:

106990-43-6

Molecular formula:

C132 H250 N32

IUPAC Name:

N2-[2-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]amino)ethyl]-N2-[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]-N4,N6-dibutyl-N4,N6-bis(1,2,2,6,6-pentamethylpiperidin-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: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks (females), 11 weeks (males)
- Weight at study initiation: 201-241 g for virgin females, at least 328 g for males
- Housing: no more than 5 of one sex before and after mating in polysulfone solid bottomed cages, measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided. During mating, 1 male and 1 female were kept in polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor Tecniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy) with certified dietary levels of phytooestrogen (genistein less 350 μg), ad libitum
- Water: drinking water ad libitum
- Acclimation period: 26 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of powder of the test item was weighed (correcting for purity) and then transferred to a mortar and with the aid of pestle, the granularity of the test item was reduced to a small size; a small amount of the vehicle was added to the powder and mixed to forma smooth
paste; the paste was transferred to a suitable container and rinsed the mortar with additional
vehicle (at least 3×); the washings was transferred to the main container and diluted to volume with vehicle; the obtained suspensions was then homogenised (by Silverson) for approximately
5 minutes and left overnight on a magnetic stirrer (for at least 16 hours) at room temperature prior to use and up until the time of dosing of the last animal.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily or weekly

VEHICLE
- Concentration in vehicle: 0.5%
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Females were paired overnight, 1:1
- the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

once daily

Duration of test:

on GD 20, all surviving females were sacrificed

Doses / concentrations

Remarks:

Doses / Concentrations:
75, 225 and 750 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of the dose levels of 75, 225 and 750 mg/kg bw/day was based on information from a preliminary non-GLP compliant study
- 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: twice a day on working days, weekends and Public Holidays throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day for individual animals

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 9, 12, 15, 18 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GD 3, 6, 9, 12, 15, 18 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Brain, mesenteric lymph node, spleen, thyroid
- Tissue Fixation: Mesenteric lymph node, spleen, thyroid
- Histophathology: Abnormalities, Thyroid

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, internal foetal sex determination in each foetuses allocated to the skeletal examination, anogenital distance (AGD) in all live foetuses, number of intra-uterine deaths, gross evaluation of placentae

Blood sampling:

- Serum: Yes
- Volume collected: 0.8 mL of blood samples. The serum obtained was divided in two aliquots (first with 250 µL and the second with the remaining amount)

Fetal examinations:

- Weight of each fetus: Yes
- Sex: Yes
- Gross evaluation of placentae: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter

Statistics:

- Dunnett’s test or a modified t test: for continous variables
- Kruskal-Wallis test: for non-continuous variables
- non-parametric version of theWilliams test: for intergroup differences between the control and treated groups
- The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

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

Maternal developmental toxicity

Details on maternal toxic effects:

- MORTALITY:
No mortality occurred.

- CLINICAL SYMPTOMS:
Piloerection was the principal treatment-related clinical sign noted in females receiving 750 mg/kg bw/day. The sign appeared after approximately one week of treatment within 15-30 minutes of dosing. In some cases it was persisted and then recorded also before dosing. In addition, six females showed hunched posture during the second part of the gestation period for a maximum of six consecutive days. Hairloss was noted in two females. Scabs (one female) and hairloss on head (4 females) were evident at 225 mg/kg bw/day. With the exception of one female in which these signs occurred simultaneously (from Day 11 post coitum onwards). In the other case hairloss were sporadically observed. Scabs and hairloss on head were noted in one female for three consecutive days (Days 18 to 20) dosed at 75 mg/kg bw/day.

- FOOD CONSUMPTION:
Food consumption was statistically significant decreased in females receiving 750 mg/kg bw/day from Days 9 to 20 post coitum (-26 % to -37 % when compared with controls). Compared to the control and significant, at statistical analysis, a reduction in food consumption was also seen from Days 9 to 15 post coitum at 225 mg/kg bw/day (-11 % to -19 %) and limited to Day 12 in females receiving 75 mg/kg bw/day (-11%).

- BODY WEIGHT:
A slight, but statistically significant decrease in mean body weights (not greater than 10%, compared to the controls), was observed between Days 12 to 20 post coitum in females receiving 750 mg/kg bw/day.
Reduced body weight gain was consistently noted at 750 mg/kg bw/day, from Days 9 to 20 post coitum, with the significance at statistical analysis evident (-32% to -88%). Compared to the control group the biggest difference was seen on Day 9 post coitum (-88%). This trend indicated a maternal toxicity at this dose level. No relevant differences in body weight and body weight gain were noted at the dose levels
of 225 and 75 mg/kg bw/day.
- TERMINAL BODY WEIGHT, CORRECTED BODY WEIGHT AND BODY WEIGHT GAIN:
Terminal body weight and corrected maternal body weight were statistically significant decreased in females dosed at 750 mg/kg bw/day, when compared to the control group (not greater than -11%). The effect on maternal body weight loss was better evident in the corrected body weight gain (terminal body weight at necropsy minus gravid uterine weight, minus body weight at Day 6 post coitum) which was decreased (significant, at statistical analysis) in females of the 750 mg/kg bw/day group. No changes were noted in the lower dose levels tested. The decrease in corrected body weight gain noted at 225 mg/kg bw/day was without significance, at statistical analysis, it was dose-unrelated and then considered to be fortuitous (they were ascribed to body weight losses observed in three females).

- THYROID HORMONE:
No differences between control and treated animals were recorded. T4 values were slightly, but statistically significantly lower at 750 mg/kg bw/day. Based on the minimal difference and in absence of any other differences (T3, TSH, thyroid weight and histopathology), this is not considered to be toxicologically relevant.
- NECROPSY FINDINGS:
> ORGAN WEIGHTS: Statistically significant increase in absolute and relative mesenteric nodes weight was observed in females at 750 mg/kg bw/day (+29 % relative to brain weight) and 225 mg/kg bw/day (+53 % relative to brain weight) when compared to the controls. Any organ weight changes other than those listed above were within the range of occasionally observed and expected spontaneous changes in rats of the same age and considered unrelated to treatment.
> MACROSCOPIC OBSERVATIONS: Treated females did not show relevant macroscopic changes. Any macroscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
> MICROSCOPIC OBSERVATIONS: No treatment-related changes were noted in thyroid as well as on all abnormalities. Any microscopic observations had a comparable incidence in control and treated groups
and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

- REPRODUCTION DATA:
Refer to Table 1 (Section: Any other information on results incl tables)

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

225 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity: Clinical signs (piloerection, hunched posture), reduced (corrected) body weight & (corrected) body weigt gain & terminal body weight & food consumption, increased mesenteric lymph node weight (abs/rel)

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

- LITTER DATA: All reproductive outcomes of treated females, including the number of corpora lutea, number of implantations sites, live foetuses or uterine deaths, as early and late resorptions, pre-implantation loss and in the sex ratio of the fetuses did not show significant differences against the control. Higher post-implantation loss (mostly due to the loss at early stage of development) was noted at all dose levels when compared to the control groups. However in view of that the values were well within the range of our historical control data and limited in magnitude, the difference was not considered to be toxicologically relevant. Moreover, litter weight and mean foetal weight (for each sexes, as well as for both sexes combined) of treatment groups were comparable to the control group.

- ANOGENITAL DISTANCE: No relevant differences were noted in the mean values of the AGD (anogenital distance normalised for the cube root of the body weight performed on Day 20 post coitum) of foetuses of both sexes maternally exposed at all dose levels compared to the control group.

- EXTERNAL EXAMINATIONS:
At the external examination of the foetuses minor alterations were noted as follows: control group: 4 foetuses small in size (body weight lower than 2.7 g) in 3 litters; 75 mg/kg bw/day: 2 foetuses small in size in 2 litters; 225 mg/kg bw/day: 5 foetuses small in size in 5 litters . Two foetuses in one litter showed abnormal shape of the hindlimbs; 750 mg/kg bw/day: 8 foetuses small in size in 6 litters. An increase incidence in percentage of foetuses and litters affected were noted in the two upper dose levels, when compared to the control group. However the differences of the percentages found between the dose level of 225 and 750 mg/kg/day were low of magnitude. This indicated a poor correlation between dose levels and the anomaly although the incidences of this finding were above our background range.
Major findings included: umbilical hernia in one control foetus, small in size (female no. 1). Multiple alterations like as umbilical hernia, polydactyly and subcutaneous oedema (generalised) was noted in one foetus (female no. 53) maternally exposed at 75 mg/kg bw/day. The occurrence of these major findings in foetuses were considered incidental.

- FIXED VISCERAL EXAMINATIONS:
There was no visceral malformation associated with treatment with the test item at any dose levels.
Two control foetuses in two different litters showed major abnormalities: one (female no. 1) as confirmation of that observed during the external examination of the foetus (umbilical hernia) and the other (female no. 43) on the urinary system including an extreme pelvic dilatation of the kidney (unilateral) associated to the extreme dilatation of the ureter (unilateral).
Overall the minor alterations (variations and anomalies) recorded on the kidneys and ureters (e. g. pelvic dilatation and/or ureters enlarge, slight to moderate), on the testis (displaced) or on the abdominal and thoracic cavities (haemorrhagic) occurred with similar incidence between control and treated groups.
Consequently, under the conditions described for this study the test item did not reveal teratogenic potential up to and including the dose level of 750mg/kg bw/day.

- SKELETAL EXAMINATIONS: Major abnormalities, that mainly included defects in the ossification of the rib, sternebrae and thoracic vertebrae, were recorded in foetuses maternally exposed at 75 (3 foetuses in 3 litters), 225 (2 foetuses in one litter) and 750 mg/kg bw/day (one foetus in one litter) as well as in the control group (one foetus in one litter). In particular, the malformations were related to the absence of one rib in three foetuses, one control (female no. 1) and two (female nos. 59, 77) at 75 mg/kg bw/day. In addition one foetus maternally exposed at 75 mg/kg bw/day (female no. 53) showing polydactyly at
external examination, have one additional metatarsal in the hindpaw, at skeletal examination. Ribs fused and thoracic haemivertebrae were seen in the same foetus at 225 mg/kg bw/day. Cleaved sternum was found in one foetus at 750 mg/kg bw/day. The incidence of malformations were not dose related, suggesting no conclusive correlation with the treatment.
However, the skeletal assessment revealed instances of an increase incidence of irregular sternebral ossification in the high dose foetuses (750 mg/kg bw/day) compared to control. These included ossification findings in the sternal elements as asymmetrical cleaved, bipartite, bifurcate distally or with additional ossification site. Development and ossification of the sternum occurs also in the perinatal period and the alterations noted can be considered as a transient delay of ossification that will resolve shortly after birth rather than as teratogenicity sign. Finally these foetal alterations occurred at the dose level of 750 mg/kg bw/day where maternal toxicity was evident.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

225 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

increase incidence of irregular sternebral ossification (transient delay)

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

225 mg/kg bw/day

Treatment related:

no

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Any other information on results incl. tables

Table 1: Reproductive data

Group (mg/kg bw/day)	1(0)	2 (75)	3 (225)	4 (750)
Unilateral implantation	0	0	0	1
Not pregnant	0	0	2	0
Pregnant females at term	25	25	23	25

 

Table 2: Postimplantation loss

Groups (mg/kg/day)	Group 1 0 mg/kg/day	Group 2 75 mg/kg/day	Group 3 225 mg/kg/day	Group 4 750 mg/kg/day	HCD mean
Post implantation loss	0.51	1.81	2.49	2.40	3.71

 

Table 3: External malformation of foetuses

		Group 1 0 mg/kg/day		Group 2 75 mg/kg/day			Group 3 225 mg/kg/day		Group 4 750 mg/kg/day		HCD mean
Number of foetuses examined		285		281			260		272		849
Number of litter examined		25		25			23		25		70
Incidence of foetuses/litter with		N	%	N	%	N		%	N	%	N	%
Anomalies
Small foetuses Body weight <2.7g	Foetus Litter	4 3	1.4 12.0	2 2	0.71 8.0	5 5		1.9 21.7	8 6	2.9 24.0	4 4	0.5 5.7

HCD = data from 3 studies (OECD 414 inWistar rats) conducted at the Test Facility in years 2014-2018.

 

Table 4: Skeletal examinations of foetuses

		Group 1 0 mg/kg/day		Group 2 75 mg/kg/day		Group 3 225 mg/kg/day		Group 4 750 mg/kg/day
Number of fetuses examined		149		147		136		140
Number of litter examined		25		25		23		25
Incidence of fetuses/litter with		N	%	N	%	N	%	N	%
Malformation
Rib absent	Foetus Litter	1 1	0.67 4	2 2	1.36 8	0 0	0 0	0 0	0 0
Rib fused	Foetus Litter	0 0	0 0	0 0	0 0	1 ^a 1	0.74 4	0 0	0 0
Thoracic Haemivertebrae	Foetus Litter	0 0	0 0	0 0	0 0	1 ^a 1	0.74 4	0 0	0 0
Hindpaw Additional metatarsal	Foetus Litter	0 0	0 0	1 1	0.67 4	0 0	0 0	0 0	0 0
Cleaved sternum	Foetus Litter	0 0	0 0	0 0	0 0	0 0	0 0	1 1	0.71 4

^a Same foetus

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in the study, the NOAEL for maternal toxicity and for developmental toxicity could be set at 225 mg/kg bw/day.