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]

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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]

EC Number:

835-183-3

Cas Number:

83420-16-0

Molecular formula:

C56 H102 Cl2 N14

IUPAC Name:

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]

Test material form:

solid: particulate/powder

Details on test material:

light yellow powder

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.

Method

Species / strain

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.

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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

Test results 7

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

Test results 8

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

Test results 9

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

Test results 10

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).

Applicant's summary and conclusion

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.