N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]was not mutagenic in the strains S. typhimurium TA 98, TA 100, TA1535, TA1537 and E. coli WP2uvrA (pKM 101) in the presence and absence of phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation. (OECD 471/GLP).

Chromosome aberration (in vitro cytogenicity/micronucleus study): N4,N4’-hexane-1,6- diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] did not induce any chromosome damage or damage to the cell division apparatus in Chinese hamster V79 cells in the presence or absence of phenobarbital and ß-naphthoflavone-induced rat liver S9 (OECD 487/GLP).

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in mouse lymphoma L5178Y cells exposed to N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]in the presence and absence of phenobarbital and

ß-naphthoflavone-induced rat liver S9 metabolic activation (OECD 490/GLP)

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/12/2019-12/05/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: SUQIAN UNITECH CORP., LTD; 190701
- Expiration date of the lot/batch:24 July 2021
- Purity: 91%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing : The test item was dissolved in ethanol, processed by ultrasound for 3 min at 37 °C and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

[E. coli WP2 uvrA (pKM 101)]

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 :Trinova Biochem GmbH, Gießen, Germany. Male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone.

- method of preparation of S9 mix : 100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of: 8 mM MgCl2/33 mM KCl/5 mM glucose-6-phosphate/4 mM NADP. This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:co-factor solution 9.5 parts/liver preparation 0.5 parts
During the experiment the S9 mix is stored on ice.

- concentration or volume of S9 mix and S9 in the final culture medium :
The protein concentration in the S9 preparation was 40.5 mg/mL (Lot: 3952, expiry date: 26.04.2020) or 39.2 mg/mL (Lot: 4180, expiry date: 12.12.2021), respectively and was adjusted to 30 mg/mL.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

Pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate. Due to strong toxicity, observed in the pre-experiment, 316 μg/plate was selected as the maximum concentration for the main experiment I.

Experiment I: 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 μg/plate (TA98, TA100)
0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 μg/plate (TA1535, TA1537, E. coli WP2 uvrA (pKM 101))

Experiment II:
0.0158, 0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100.0 μg/plate (all strains)

Vehicle / solvent:

- Solvent(s) used: ethanol

- Justification for choice of solvent: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative - water; Solvent - ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine: S. typhimurium: TA98, TA1537 - S9; 2-aminoanthracene: all strains +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation): In case of severe toxicity of the test item or the use of e.g. ethanol, acetone or tetrahydrofuran as the most appropriate solvent, the confirmatory experiment is carried out according to the plate incorporation method with a different spacing between dose levels. So both experiments were via the plate incorporation method.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: incubated at 37 °C for at least 48 h in the dark.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM 101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

Mean and SD

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10μg/plate - S9; 100μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

15.8μg/plate - S9; 100μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10μg/plate - S9; 100μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5μg/plate - S9; 50μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3.16μg/plate - S9; 31.6μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5μg/plate - S9; 50μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.316μgμg/plate - S9; 100μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.158μg/plate - S9; 50μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

31.6μg/plate - S9; 100μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

15.8μg/plate - S9; 50μg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES (if applicable):
The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiments (plate incorporation test; Table 1). Due to strong toxicity, observed in the pre-experiment, 316 μg/plate was selected as the maximum concentration for the main experiment I. The concentration range covered two logarithmic decades.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: All criteria of validity were met.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: None
- Statistical analysis; p-value if any: Not applicable

Ames test:
- Signs of toxicity: Toxic effects of the test item were noted in all tester strains used in experiment I and II:
- In experiment I toxic effects of the test item were observed at concentrations of 0.316 μg/plate and higher (without metabolic activation) and at concentrations of 10.0 μg/plate and higher (with metabolic activation), depending on the particular tester strain.
- In experiment II toxic effects of the test item were noted at concentrations of 0.158 μg/plate and higher (without metabolic activation) and at concentrations of 50.0 μg/plate (with metabolic activation), depending on the particular tester strain.

- Individual plate counts & Mean number of revertant colonies per plate and standard deviation: refer to attached results tables.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Historical control data (+/-S9) for positive and negative controls were provided. Data from 2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM 101). For this tester strain the period was December 2019 to February 2020 (Appendix 1).

Conclusions:

N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 98, TA 100, TA1535, TA1537 and E. coli WP2uvrA (pKM 101) were exposed to N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in ethanol at concentrations of 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 μg/plate (TA98, TA100; experiment 1), 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 μg/plate (TA1535, TA1537, E. coli WP2 uvrA (pKM 101); experiment 1) and 0.0158, 0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100.0 μg/plate (all strains; experiment 2). Both experiments were carried out via the plate incorporation method and metabolic activation was phenobarbital/β-naphthoflavone-induced rat liver S9.

The positive controls of each strain showed a marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain.

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). In experiment I toxic effects of the test item were observed at concentrations of 0.316 μg/plate and higher (without metabolic activation) and at concentrations of 10.0 μg/plate and higher (with metabolic activation), depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 0.158 μg/plate and higher (without metabolic activation) and at concentrations of 50.0 μg/plate (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. Therefore, N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Dec 2019 - 10 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: SUQIAN UNITECH CORP., LTD; 190701
- Expiration date of the lot/batch: 24 July 2021
- Purity: 91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments.
- Suitability of cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells were chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardized culture conditions.

For cell lines:
- Absence of Mycoplasma contamination: Routine checking of mycoplasma infections were carried out before freezing.
- Methods for maintenance in cell culture: For the experiments thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 105 cells per flask were seeded in 15 mL of MEM(minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
- Complete Culture Medium was MEM medium supplemented with: 10 % fetal bovine serum (FBS), 100 U/100 μg/mL penicillin/streptomycin solution, 2 mM L-glutamine, 2.5 μg/mL amphotericin and 25 mM HEPES.
- 5% carbon dioxide atmosphere (95% air)
- 37 °C

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone.

- method of preparation of S9 mix
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP

- concentration or volume of S9 mix and S9 in the final culture medium
The final concentration of S9 mix in the cultures is 5%.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-0-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium
assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

Preliminary experiment 1: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 1500 and 2000 μg/mL
Due to strong cytotoxic effects observed down to the lowest test concentration, the pre-experiment was repeated with the following concentrations:
Preliminary experiment 2: 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 5, 15 and 30 μg/mL

Experiment I with short-term exposure (4h):
without metabolic activation: 0.005, 0.01, 0,025, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.75 and 1.0 μg/mL
with metabolic activation: 1.0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 and 25 μg/mL

Experiment II with long-term exposure (24h):
without metabolic activation: 0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.10, 0.20 and 0.40 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF

- Justification for choice of solvent/vehicle and percentage of solvent in the final culture medium: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium. Therefore the test item was dissolved in tetrahydrofurane (THF) and diluted in cell culture medium to reach a final concentration of 0.5% v/v THF in the samples. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative controls (cell culture medium) and Solvent controls (cell culture medium with 0.5% THF)

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration ：duplicate
- Number of independent experiments：2 (4 hrs with and without S9; 24 hrs -S9)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in cell culture medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: (4 hrs with and without S9; 24 hrs -S9)
- Harvest time after the end of treatment (sampling/recovery times): At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 μg/mL cytochalasin B for 20 h at 37 °C (experiment 1) and 23 h at 37 °C (experiment 2).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: 1.5 μg/mL cytochalasin B for 20 h (experiment 1) and 23 h (experiment 2).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 ml complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for seven minutes at room temperature. After the treatment with the hypotonic solution the cells were fixed for 24h with methanol + glacial acetic acid (3:1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei.

As an exception, less than 2000 binucleated cells in total were scored for the concentration 0.40 μg/mL in experiment I (1105 cells) and II (1750 cells) without metabolic activation as only a few
cells were present on all four slides evaluated. For the solvent control in experiment I without metabolic activation, 2000 binucleated cells were additionally screened to verify the analysis, as significant differences between both slides (generally factor > 2) were observed. The experiment was regarded as valid since the test item caused pronounced cell death but surviving cells showed normal proliferation capacity.

For the solvent control in experiment I with metabolic activation, 1000 binucleated cells were additionally screened to verify the analysis, as significant differences between both slides (generally factor > 2) were observed.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): According to the criteria of Fenech i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells.
- Any supplementary information relevant to cytotoxicity: If cytotoxicity is observed the highest concentration evaluated should not exceed the limit of 55% ± 5% cytotoxicity according to the OECD Guideline 487. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. The other concentrations evaluated should exhibit intermediate and little or no toxicity. However, OECD 487 does not define the limit for discriminating between cytotoxic and non-cytotoxic effects. According to laboratory experience this limit is a value of the relative cell growth of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database;
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control;
- Cell proliferation criteria in the negative/solvent control according to OECD 487 should be fulfilled;
- All experimental conditions are tested unless one resulted in positive results;
- Adequate number of cells and concentrations are analysable;
- Criteria for the selection of top concentration are fulfilled.

Evaluation criteria:

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/solvent control;
- the increase is concentration-related in at least one experimental condition when evaluated with
an appropriate trend test;
- any of the results are outside the distribution of the historical negative/solvent control data (e.g.Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaksand/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of thecriteria mentioned above are met.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 1 4 hrs

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

53% cytostasis at 5.0 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 1 4hrs

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

36% cytostasis at 0.30 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 2 24 hrs

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

36% cytostasis at 0.20 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: For the maximum concentration the osmolality (in comparison to solvent control) and pH value were determined (Refer to table below).

- Precipitation and time of the determination: No precipitate of the test item was noted in any concentration evaluated in experiment I and II without metabolic activation at the end of treatment. In experiment I with metabolic activation precipitated was seen at concentration 10 μg/mL and higher.

RANGE-FINDING/SCREENING STUDIES (if applicable):
According to the used guideline the highest recommended concentration was 2000 μg/mL. The test item was dissolved in THF and rediluted with cell culture medium at a ratio of 1:200 to obtain the final test item concentrations and final THF concentration of 0.5% v/v. A pre-experiment performed in the concentration range between 7.8 and 2000 μg/mL showed strong cytotoxic effects with and without metabolic activation down to the lowest test concentration. The pre-experiment was repeated using the following lower concentrations: 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 5, 15 and 30 μg/mL. The highest dose group evaluated for cytotoxicity in the pre-experiment was 0.10 μg/mL without metabolic activation and 5 μg/mL with metabolic activation. At higher concentrations no or not enough intact cells were found. The cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). Cytostasis was used to describe cytotoxicity. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment (Table 3).

STUDY RESULTS
- Concurrent vehicle negative and positive control data
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.55%) was within the historical control limits of the negative control (0.32% - 1.44%, Table 15) and the micronucleated cell frequency of the solvent control (1.45%) was within the historical control limits of the solvent control (0.37% – 1.49%, Table 15).

In experiment I with metabolic activation the micronucleated cell frequency of the negative control (0.35%) was slightly below the lower historical control limit of the negative control (0.45% – 1.68%,Table 15). The micronucleated cell frequency of the solvent control (0.95%) was within the historical control limits of the solvent control (0.33% – 1.74%; Table 15).

In experiment II without metabolic activation the micronucleated cell frequency of the negative control (0.75%) was within the historical control limits of the negative control (0.32% – 1.44%, Table 15) and the micronucleated cell frequency of the solvent control (0.90%) was within the historical control limits of the solvent control (0.37% – 1.49%, Table 15).

MMS (25 μg/mL) and CPA (2.5 μg/mL) were used as clastogenic controls and colchicine as aneugenic controls (0.08 and 1.5 μg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay. In experiment I without metabolic activation, a statistically significant increase for MMS was only observed compared to the concurrent negative control. The presence of 0.5% (v/v) THF in the solvent control caused a significant increase of the micronuclei frequency compared to the negative control which could explain the lack of a significant response for MMS. However, for the clastogenic control CPA and the aneugenic control colchicine significant increases compared to the solvent control were observed in experiment I that verified the sensitivity of the test system.

In all experiments, the concurrent positive controls induced responses that are compatible with those generated in the laboratory’s historical positive control data base (Table 16).

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 0.20 μg/mL, 36% cytostasis was noted at 0.30 μg/mL and at 0.40 μg/mL a cytostasis of 50% was observed. No increase of the cytostasis above 30% was noted in experiment I with metabolic activation up to a concentration of 2.5 μg/mL. At 5.0 μg/mL a cytostasis of 53% was noted. In experiment II no increase of cytostasis was noted up to a concentration of 0.10 μg/mL. At 0.20 μg/mL a cytostasis of 36% and at 0.40 μg/mL a cytostasis of 68% was observed.

- Genotoxicity results
The following concentrations were evaluated for micronuclei frequencies:
Experiment I with short-term exposure (4 h): without metabolic activation: 0.10, 0.20 and 0.40 μg/mL and with metabolic activation: 1.0, 2.5 and 5.0 μg/mL
Experiment II with long-term exposure (24 h): without metabolic activation: 0.10, 0.20 and 0.40 μg/mL

In experiment I without metabolic activation, the numbers of micronucleated cells found in the groups treated with the test item were within the historical control limits of the solvent control except for the concentration 0.10 μg/mL. The corresponding micronuclei frequency was above the upper historical control limit of the solvent control. However, the increase was not statistically significant compared to the concurrent solvent control and no concentration related increase was observed. Thus the increase was considered not biologically relevant.

In experiment I with metabolic activation, the mean values of micronucleated cells found after treatment with the test item were 0.85% (1.0 μg/mL), 0.80% (2.5 μg/mL) and 0.55% (5.0 μg/mL). The numbers of micronucleated cells were within the historical control limits of the solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
In experiment II without metabolic activation, the mean values of micronucleated cells found after treatment with the test item were 0.75% (0.10 μg/mL), 0.65% (0.20 μg/mL) and 0.65% (0.40 μg/mL). The numbers of micronucleated cells were within the historical control limits of the solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.

No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I without and with metabolic activation (Table 11 and Table 12) and experiment II without metabolic activation (Table 13). The ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II (Table 14).

HISTORICAL CONTROL DATA with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data were provided for positive and negative controls (2014-2019; Tables 15 & 16).

For the maximum concentration the osmolality (in comparison to solvent control) and pH value were determined :

		concentration(μg/mL)	osmolality(mOsm/kg)	PH value
Exp.1	Solvent control	0	349	-
without metabolic activation	Test item	1.0	355	7.4
Exp.1	Solvent control	0	353	-
with metabolic activation	Test item	25	367	7.4
Exp.2	Solvent control	0	367	-
	Test item	0.4	361	7.4

 

Conclusions:

During the study described and under the experimental conditions reported, the test item of N4,N4’-hexane-1,6- diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.

Executive summary:

In an in vitro cytogenicity/micronucleus study (OECD 487/GLP), Chinese hamster V79 cells were exposed to N4,N4’-hexane-1,6- diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in THF. The concentrations without metabolic activation were 0.005, 0.01, 0,025, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.75 and 1.0 μg/mL (first experiment; 4 hrs) and 0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.10, 0.20 and 0.40 μg/mL (second experiment; 24 hrs); the concentrations with metabolic activation were 1.0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 and 25 μg/mL (first experiment; 4 hrs). Metabolic activation provided by phenobarbital and ß-naphthoflavone-induced rat liver S9.

No precipitate of the test item was noted in any concentration evaluated in experiments I and II without metabolic activation at the end of treatment. In experiment I with metabolic activation precipitated was seen at concentration 10 μg/mL and higher. In experiment I without metabolic activation an increase of the cytostasis above 30% was noted at concentrations of 0.30 μg/mL and higher. In experiment I with metabolic activation an increase of the cytostasis above 30% was noted at concentrations of 5.0 μg/mL and higher. In experiment II without metabolic activation an increase of the cytostasis above 30% was noted at concentrations of 0.20 μg/mL and higher.

In the both main experiments with and without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item. The nonparametric Chi-squared Test was performed to verify the results in both experiments. No statistically significant increase (p< 0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I without and with metabolic activation. The Chi-squared Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II. The positive controls, MMS and CPA, gave the appropriate responses.

N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)- 1,3,5-triazine-2,4-diamine] did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2020 - 11 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source : SUQIAN UNITECH CORP., LT, Batch No.: 190701
- Expiration date of the lot/batch: 24 July 2021
- Purity: 91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

Thymidine Kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y mouse lymphoma cells (clone TK+/- 3.7.2C)
- Suitability of cells: L5178Y mouse lymphoma cells (clone TK+/- 3.7.2C) have been used successfully in in vitro experiments for many years.

For cell lines:
- Absence of Mycoplasma contamination: Each cell batch is routinely checked for mycoplasma infection.
- Methods for maintenance in cell culture: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Cell cycle length, doubling time or proliferation index : 10-12 h doubling time of the Eurofins Munich stock cultures
- Modal number of chromosomes: near diploid karyotype (40 ± 2 chromosomes)
- Periodically ‘cleansed’ of spontaneous mutants: Yes To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing in RPMI 1640 supplemented with: 9.0 μg/mL hypoxanthine, 15.0 μg/mL thymidine, 22.5 μg/mL glycine, 0.1 μg/mL methotrexate. The cells are resuspended in medium without methotrexate but with thymidine, hypoxanthine and glycine for 1-3 days.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Complete Culture Medium: RPMI 1640 medium supplemented with10 % HS, 100 U/100 μg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 μg/mL amphotericin B.
Treatment Medium: RPMI 1640 medium supplemented with 5 % HS, 100 U/100 μg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 μg/mL amphotericin B.
Selective Medium: RPMI 1640 medium supplemented with 20 % HS, 100 U/100 μg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 μg/mL amphotericin B, 5 μg/mL TFT.

- 5% carbon dioxide atmosphere (95% air)
- 37 °C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone.
- method of preparation of S9 mix: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to achieve the below concentrations in 100 mM sodium-phosphate-buffer pH 7.4: 8 mM MgCl2, 33 mM KCl,5 mM Glucose-6-phosphate, 5 mM NADP.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 mix in the cultures was 5%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.3 μg/mL - 15 μg/mL with/without metabolic activation

Main test without metabolic activation (4 hrs): 0.05 μg/mL, 0.1 μg/mL, 0.3 μg/mL, 0.6 μg/mL
Main test with metabolic activation (4 hrs): 1 μg/mL, 2 μg/mL, 2.5 μg/mL, 3 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF (Supplier: Merck; Lot: 10984207)

- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL.
Based on the results of the solubility test, THF was used as solvent (0.25% THF v/v). Different dilutions of test item stock solution were prepared and added to the
samples. The solvent was compatible with the survival of the cells and the S9 activity. The measured pH value of the test item was within the physiological range (pH 7.0 ± 0.4).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative controls (treatment medium) and solvent controls (0.25% THF v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Single cultures for test item; Negative and/or solvent controls are tested in duplicate.
- Number of independent experiments: 1 (4 hrs with and without S9).

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hrs

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days. Cloning efficiency for cytotoxicity: at least 7 days
- Selection time (if incubation with a selective agent): about 12 days
- Method used: microwell plates
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 5 μg/mL trifluorothymidine (TFT) [Sigma-Aldrich (Lot No.: BCCCD1998)] in selective medium for about 12 days at 37 °C in 5% CO2/95% humidified air.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Viability: 1.6 cells/well in two 96-well plates
Mutants: Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 μL selective medium with TFT. The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection.
Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to –ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in non-selective medium)). The mutant frequency is usually expressed as “mutants per 106 viable cells”.

Mutant frequency = -ln [NW/TW (selective medium)] / -ln [NW/TW (non-selective medium)] x 800
NW: number of negative wells
TW: number of total wells

- Criteria for small (slow growing) and large (fast growing) colonies: The size of the colonies is usually characterised as follows: Small colonies approximately ≤ ¼ of well diameter; large colonies approximately > ¼ of well diameter. Size is the key factor and morphology should be secondary.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG). The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture.
- Any supplementary information relevant to cytotoxicity: The maximum concentration is based on cytotoxicity, therefore the highest concentration should aim to achieve between 20 and 10% RTG.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF was calculated to be 126 mutants/106 cells for the microwell method.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10+6 cells and
- a concentration-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF or the trend of the test is negative.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to determine if there are statistical differences between the mutant frequencies of the treated test groups compared to the solvent controls.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

15.8% RTG at 3μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

9.6% RTG at 0.6μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The measured pH value of the test item in THF was within the physiological range (pH 7.0 ± 0.4).
- Precipitation and time of the determination: No precipitation of the test item was noted in the experiment.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 15 μg/mL, based on the results of the study STUGC19AA2353-3 (In vitro Mammalian Micronucleus Assay in Chinese Hamster V79 Cells). Seven concentrations (0.3 μg/mL to 15 μg/mL) were tested without and with metabolic activation. The experimental conditions in these pre-experiments were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. 0.6 μg/mL (without metabolic activation) and 3 μg/mL (with metabolic activation) were selected as the highest concentrations.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
In the main experiment without and with metabolic activation all validity criteria were met. The negative and solvent controls showed mutant frequencies within the acceptance range of 50-170 mutants/10+6 cells, according to the IWGT criteria. In all experiments the percentage of small colonies in the negative and solvent controls, was found to be lower than 40%.
The positive controls EMS, MMS and B[a]P showed distinct responses in mutation frequency, thus demonstrating the ability of the test system to detect potential mutagenic effects. The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥ 40%), thus confirming the ability of the test system to indicate potential clastogenic effects (Table 6, Table 9).

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value if any: p < 0.05
- Any other criteria: GEF = 126 mutants/10+6 cells

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Growth inhibition was observed in the experiment without and with metabolic activation. The relative total growth (RTG) was 9.6% (without metabolic activation) and 15.8% (with metabolic activation) for the highest concentration evaluated. (Table 4, Table 7).

- Genotoxicity results:
The induced mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF) and a statistical analysis (Table 10, Table 11) was performed. In experiment I and II higher numbers of mutants were noted after treatment with the test item (without and with metabolic activation) when compared with the concurrent control values. However, the Global Evaluation Factor was not exceeded by the induced mutant frequency at any concentration. Therefore, any differences observed in mutant frequency between the treated and concurrent control groups were concluded upon as not biologically relevant. The mutant frequencies induced by the test item did not show any biologically relevant increase. The GEF of 126 mutants/106 cells was not exceeded in any of the concentrations (Table 5, Table 8). A statistical analysis displayed that some of the mutant frequencies were significantly increased over those of the solvent controls (Table 10, Table 11).

Colony size was counted for all concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication of potential clastogenic effects and/or chromosomal aberrations. Thus based on the non-mutagenic effects of N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6- tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine], an assessment of clastogenicity was not feasible.
HISTORICAL CONTROL DATA with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data: Historical data for mutant frequencies are shown in Table 12 (Appendix). All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich (2014-2019).

Conclusions:

In conclusion, the test item N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5 - triazine-2,4-diamine] is considered to be non-mutagenic in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

In a mammalian cell gene mutation assay [MLA; OECD 490/GLP], mouse lymphoma L5178Y cells cultured in vitro were exposed to N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in THF for 4 hours without metabolic activation at concentrations of 0.05, 0.1, 0.3 and 0.6 μg/mL and in the presence of phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation at concentrations of 1, 2, 2.5, 3 µg/mL.

No precipitation of the test item was noted in the experiment. The RTG was 9.6% (without metabolic activation) and 15.8% (with metabolic activation) for the highest concentration evaluated. The positive and negative controls induced the appropriate response. No biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The GEF of 126 mutants/10^6 cells was not exceeded by the induced mutant frequency at any concentration. Therefore, N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is considered to be non-mutagenic in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

There is one gene mutation study (Bacterial Reverse Mutation Assay/Ames test) with the test item available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 98, TA 100, TA1535, TA1537 and E. coli WP2uvrA (pKM 101) were exposed to N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in ethanol at concentrations of 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 μg/plate (TA98, TA100; experiment 1), 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 μg/plate (TA1535, TA1537, E. coli WP2 uvrA (pKM 101); experiment 1) and 0.0158, 0.050, 0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 100.0 μg/plate (all strains; experiment 2). Both experiments were carried out via the plate incorporation method and metabolic activation was phenobarbital/β-naphthoflavone-induced rat liver S9. The positive controls of each strain showed a marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain. No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). In experiment I toxic effects of the test item were observed at concentrations of 0.316 μg/plate and higher (without metabolic activation) and at concentrations of 10.0 μg/plate and higher (with metabolic activation), depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 0.158 μg/plate and higher (without metabolic activation) and at concentrations of 50.0 μg/plate (with metabolic activation), depending on the particular tester strain. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. Therefore, N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is considered to be non-mutagenic in this bacterial reverse mutation assay.

Chromosome aberration (in vitro cytogenicity/micronucleus study):

There is one in vitro cytogenicity/micronucleus study available.

In an in vitro cytogenicity/micronucleus study (OECD 487/GLP), Chinese hamster V79 cells were exposed to N4,N4’-hexane-1,6- diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in THF. The concentrations without metabolic activation were 0.005, 0.01, 0,025, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.75 and 1.0 μg/mL (first experiment; 4 hrs) and 0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.10, 0.20 and 0.40 μg/mL (second experiment; 24 hrs); the concentrations with metabolic activation were 1.0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 and 25 μg/mL (first experiment; 4 hrs). Metabolic activation provided by phenobarbital and ß-naphthoflavone-induced rat liver S9. No precipitate of the test item was noted in any concentration evaluated in experiments I and II without metabolic activation at the end of treatment. In experiment I with metabolic activation precipitated was seen at concentration 10 μg/mL and higher. In experiment I without metabolic activation an increase of the cytostasis above 30% was noted at concentrations of 0.30 μg/mL and higher. In experiment I with metabolic activation an increase of the cytostasis above 30% was noted at concentrations of 5.0 μg/mL and higher. In experiment II without metabolic activation an increase of the cytostasis above 30% was noted at concentrations of 0.20 μg/mL and higher. In the both main experiments with and without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item. The nonparametric Chi-squared Test was performed to verify the results in both experiments. No statistically significant increase (p< 0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I without and with metabolic activation. The Chi-squared Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II. The positive controls, MMS and CPA, gave the appropriate responses. N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)- 1,3,5-triazine-2,4-diamine] did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.

 

Gene mutation (mammalian cell gene mutation assay):

There is one gene mutation (mammalian cell gene mutation assay; MLA) available.

In a mammalian cell gene mutation assay (OECD 490/GLP), mouse lymphoma L5178Y cells cultured in vitro were exposed to N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in THF for 4 hours without metabolic activation at concentrations of 0.05, 0.1, 0.3 and 0.6 μg/mL and in the presence of phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation at concentrations of 1, 2, 2.5, 3 µg/mL. No precipitation of the test item was noted in the experiment. The RTG was 9.6% (without metabolic activation) and 15.8% (with metabolic activation) for the highest concentration evaluated. The positive and negative controls induced the appropriate response. No biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The GEF of 126 mutants/10^6 cells was not exceeded by the induced mutant frequency at any concentration. Therefore, N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is considered to be non-mutagenic in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Justification for classification or non-classification

Based on the available information in the dossier, the substance N4,N4’-hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (CAS No.83420-16-0 ) does not need to be classified for germ cell mutagenicity when the criteria outlined in CLP Regulation (Annex I of 1272/2008/EC) are applied.