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

Endpoint summary

Description of key information

In a subacute study with rats (Covance, 1997) the test substance did not cause any adverse effects up to the highest dose tested (1000 mg/kg), therefore, the NOAEL was set at 1000 mg/kg bw.

In a subchronic study with rats (BASF SE, 2022) the test substance did not cause any adverse effects up to the highest dose tested (1000 mg/kg), therefore, the NOAEL was set at 1000 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

Specific details on test material used for the study:

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

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

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

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.

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".

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

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

Critical effects observed:

no

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP-compliant subchronic toxicity study (OECD 408), the test substance in 0.5% methylcellulose was administered daily by gavage to 10 female and 10 male Wistar rats at dose levels of 0 (test group 0, vehicle control), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (test group 3) over a period of 3 months. There were no test substance-related, relevant effects observed concerning clinical examinations, clinical pathology and pathology in all groups tested. No animal died prematurely in the present study. 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. 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. During the open-field observations 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. Regarding the examination of thyroid hormones, 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. Concerning the relative organ weights, the thyroid gland was statistically significant decreased in test group 3 (-15%) , when compared with the control group 0 (=100%). 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. 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.

 

In a GLP-compliant subacute toxicity study (OECD 407), the test substance in 0.5% methylcellulose was administered daily by gavage to five Sprague-Dawley rats per sex and dose group at 0, 10, 100, and 1000 mg/kg body weight for a period of 28 days. Five additional male and female animals were assigned to the control and the high dose group for a 14-day recovery period. All animals survived to their scheduled sacrifice. There were no test substance-related clinical observations and no effects on body weight, food consumption, organ weights, and macroscopic/microscopic appearance of tissues. Some variations in hematology parameters in group 4 reached the level of statistical significance but were only present at the end of the recovery period and were therefore considered incidental and of no toxicological relevance. Some significant changes were noted during clinical chemistry observation, however they could not be attributed to the administration of the test material due to the low magnitude of the changes, the lack of dose dependence, and/or the occurrence of the difference only at recovery Day 49. No target organ was identified. Therefore, based on the results of this study and under the conditions chosen, the NO(A)EL was set at 1000 mg/kg bw in males/females.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study which is well documented and meets generally accepted scientific principles.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.