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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 Jun 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

Specific details on test material used for the study:

- Physical state/appearance: Solid/ off-white
- Storage condition of test material: At room temperature

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Fasting period before study: no
- Housing: Group housing (5 animals per cage) in H-Temp polysulfonate cages type 2000P (floor area about 2065 cm2). Dust-free wooden bedding was used in this study.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, meal (Granovit AG, Kaiseraugst, Switzerland), ad libitum.
- Water (e.g. ad libitum): drinking water, ad libitum.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
- The supplier assayed the food used in the study for chemical and microbiological contaminants.
- The drinking water was regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.

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

IN-LIFE DATES: From: 07.04.2021 To: 09.07.2021

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was s 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% Sodium carboxymethyl cellulose (CMC) suspension in deionized water was filled up to the desired volume, subsequently mixed using an Ultra-turrax.
- The test-substance preparations were produced daily until study day 41. From study day 42 onwards the test-substance preparations were produced at least once a week and stored at refrigerator. During administration the testsubstance preparations were kept homogeneous using a magnetic stirrer. The administration volume was 10 mL/kg body weight.


VEHICLE
- Concentration in vehicle: 0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water
- Amount of vehicle (if gavage): 10 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in 0.5% Sodium carboxymethyl cellulose suspension in deionized water at room temperature over a period of up to 7 days was proven during the application period.
Homogeneity was verified in 3 samples in the highest and lowest concentration (were used as
a concentration control at the same time) at the beginning and towards the end of the administration period; additional concentration control analyses were verified in the mid concentration. Furthermore, samples were taken twice during the administration period.
Since equivocal analytical results in main samples from the beginning and the end of the
administration period in the low and mid dose occurred, retain samples of these samples were
analyzed.
Retain samples, described by the suffix “R”, were stored together with samples taken during
the administration period at the laboratory Mechanistic Toxicology (at -20 °C). Following
finalization of the report, all remaining analytical samples, including retain samples, will be
discarded

Duration of treatment / exposure:

93 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses/Concentration: 0, 100, 300, 1000 mg/kg bw/day

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels of 100,300 and 1000 mg/kg bw/day were chosen based on the results of an OECD 421 study (BASF project no. 80R0252/12X194) with a no observed adverse effect level (NOAEL) of 1000 mg/kg bw/d.
- Fasting period before blood sampling for clinical biochemistry: about 16 to 20 hours

Positive control:

none.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed in all animals prior to the administration period and thereafter at weekly intervals.
- Parameters examined: refere to "Any other information on materials and methods incl. tables".

BODY WEIGHT: Yes
- Time schedule for examinations: was determined on study day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION: Yes
- Time schedule: weekly (as representative value over 7 days) for each cage.

WATER CONSUMPTION: Yes
- Time schedule: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined prior to the start of the administration period. At the
end of the administration period, i.e. study day 90, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes
- How many animals: all
- Parameters: refere to "Any other information on materials and methods incl tables".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters: refere to "Any other information on materials and methods incl tables".

BLOOD AND SERUM: Yes
- Time of blood sample collection: In the morning
- Animals fasted: Yes
- How many animals: all
- Thyorid hormone determination: Yes (including T3, T4, TSH determination)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Animals fasted: Yes
- Parameters examined: refere to "Any other information on materials and methods incl. tables".

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional observation battery was performed in all animals at the end of the administration period starting in the morning.
Parameters examined: refere to "Any other information on materials and methods incl. tables".

-The motor activity assessment was also measured in the early afternoon onwards on the same day as the FOB was performed.
Parameters examined: refere to "Any other information on materials and methods incl. tables".

ESTROUS CYCLE DETERMINATION: YES
- Time schedule for examinations: Vaginal smears for terminal vaginal cytology examinations were prepared in the morning of the day of sacrifice.

HEMATOLOGY: YES
- Time schedule: end of the administration period
- Parameters examined: refere to "Any other information on materials and methods incl. tables".

CLINICAL CHEMISTRY: YES
- Time schedule: end of the administration period
- Parameters examined: refere to "Any other information on materials and methods incl. tables".

Sacrifice and pathology:

PATHOLOGY: Yes (organs examined: refere to "Any other information on materials and methods incl. tables".
- Organ weights
- Fixation
- Histopathology

Statistics:

Please refere to table 1-3 in the section "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse, substance related clinical findings were observed in male and female animals of all test groups.
Female animal No. 62 of test group 2 (300 mg/kg bw/d) had an injury in the left mouth region from study day 7 until study day 11 caused by an accident with the food hopper. On the day of the accident (study day 7) the animal showed piloerection. From study day 8 onwards an injury and opacity of the right eye was observed. The injury was seen last on study day 11, the opacity lasted until study day 41. This finding is assessed neither substance nor treatment related.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse, test-substance related changes of mean body weights and mean body weight change values in both sexes were observed.
In female animals of test group 3 (1000 mg/kg bw/d) body weight change values were increased over the entire study period between 11.1% and 28.5% in comparison to the control group, with statistically significant values on study days 7, 35, 56 to 70, 84 and 91. It cannot be excluded that the increased body weight gain was test-substance related, but it was assessed as non-adverse.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related alterations of T3, T4 and TSH levels were observed.
At the end of the administration period, in males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) T4 values were significantly lower compared to those of the study controls. But when comparing the values with the historical control range T4 values of the study controls were above, whereas those of the three dose groups were within this range (T4 males, 44.65-73.22 nmol/L). Moreover, the T4 change in males of test groups 1, 2 and 3 was not dose dependent. In females of test group 1 (100 mg/kg bw/d) T4 values were significantly lower compared to study controls, but this change was also not dose dependent. Therefore, the T4 alterations were regarded as incidental and not treatment related.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FOB:
- Home cage observations: No test-substance related adverse effects were observed.
- Open field observations: No test-substance related adverse effects were observed. One male animal (No. 10) of test group 0 (0 mg/kg bw/d, control) showed a reduced exploration of the area (activity/ arousal level). This single finding was assessed as spontaneous in nature.
- Sensorimotor tests/ reflexes: No test-substance related effects were observed.
Quantitative parameters: No test-substance related effects were observed.

Motor activity measurement:
Regarding the overall motor activity as well as single intervals, no test-substance related deviations were noted for male and female rats.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Absolute organ weights: All mean absolute weight parameters did not show significant differences when compared to the control group 0.
- Relative organ weights: When compared with control group 0 (=100%), the following mean relative organ weights were significantly decreased in test group 3 (statistically significant changes refer to Table 5 in "Any other information on results incl tables"). The significant decrease of the mean relative weight of the thyroid glands did not show dose dependency and was not associated to histopathological changes and is therefore regarded as incidental without any relation to treatment.
All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 5: Relative organ weights

	Female animals
Test group (mg/kg)	1 (100)	2 (300)	3 (1000)
Thyroid glands	-11.4%	+3.1%	-15.0%*

* : p <= 0.05, **: p <= 0.01

Applicant's summary and conclusion

Conclusions:

The administration of the test item by gavage to male and female Wistar rats for 3 months caused no signs of toxicity. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d for male and female Wistar rats.

Executive summary:

The following test substance-related, relevant findings were noted:
Test group 3: 1000 mg/kg bw/d
Clinical Examinations, Clinical Pathology and Pathology
• No test substance-related, relevant effects were observed.
Test group 2: 300 mg/kg bw/d
Clinical Examinations, Clinical Pathology and Pathology
• No test substance-related, relevant effects were observed.
Test group 1: 100 mg/kg bw/d
Clinical Examinations, Clinical Pathology and Pathology
• No test substance-related, relevant effects were observed.