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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jun 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2018

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-Triazine-2,4,6-triamine, N2,N2’-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-, N-allyl derivs., oxidized, hydrogenated

EC Number:

812-927-5

Cas Number:

1902936-62-2

Molecular formula:

not availabale

IUPAC Name:

1,3,5-Triazine-2,4,6-triamine, N2,N2’-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-, N-allyl derivs., oxidized, hydrogenated

Test material form:

solid

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

nominal 100 mg/kg bw

Doses / concentrations 2

Dose / conc.:

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

Remarks:

nominal 300 mg/kg bw

Doses / concentrations 3

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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.