4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

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:

28 June 2018 - 24 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

EC Number:

276-309-1

EC Name:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

Cas Number:

72058-41-4

Molecular formula:

C11H18Cl2N4

IUPAC Name:

4,6-dichloro-N-(2,4,4-trimethylpentan-2-yl)-1,3,5-triazin-2-amine

Test material form:

solid

Details on test material:

Colour: light yellow

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SUQIAN UNITECH CO., LTD; 2018041002
- Purity: 99.29%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: stable at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 1.007 was applied to consider the purity of the test item, with the exception of tester strains TA1535, TA1537 and TA102 tested in experiment I. In this case the highest concentration of 5000 g/mL corresponds to 4965 g/mL active component. This difference was negligible and, in addition, toxicity was observed in the highest concentration. Therefor the recommended maximum dose was reached, since it was tested up to a cytotoxic concentration.

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium: TA98, TA100, TA1535, TA1537, TA102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
- method of preparation of S9 mix : The S9 mix preparation was performed according to the standard Ames method
- concentration or volume of S9 mix and S9 in the final culture medium: 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
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): a)Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene; b)Sterility Test.

Test concentrations with justification for top dose:

Preliminary test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Main test (Experiment I): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Main test (Experiment II): 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (all tester strains except for TA102)
Main test (Experiment II)3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA102)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- 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. 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 dimethylsulfoxide (DMSO) at a 100-fold concentration.

Controls

Untreated negative controls:

yes

Remarks:

Water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine(Without metabolic activation; S. typhimurium: TA98, TA1537); 2-aminoanthracene(With metabolic activation; all strains)

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: Experiment 1: in agar (plate incorporation); Experiment 2: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min at 37 °C
- Exposure duration/duration of treatment: 48 hrs


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) 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:

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 TA102 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 than the reversion rate of the solvent control.

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:

100 μg/plate (-S9); 1000 μg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 μg/plate (-S9); 5000 μg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate (-S9); 1000 μg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate. The following doses were selected for the main experiments:
Main test (Experiment I): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Main test (Experiment II): 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (all tester strains except for TA102)
Main test (Experiment II)3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA102) (Table 1)

STUDY RESULTS
- Concurrent vehicle negative and positive control data: All controls (solvent and negative) gave the appropriate responses.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: None
- Statistical analysis; p-value if any: No statistical analysis performed

Ames test:
- Signs of toxicity: Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II. In experiment I toxic effects of the test item were observed in tester strain TA98 at concentrations of 100 μg/plate and higher (without metabolic activation) and at concentrations of 1000 μg/plate and higher (with metabolic activation). In tester strain TA100 toxic effects of the test item were seen at concentrations of 100 μg/plate and higher (with and without metabolic activation). In tester strains TA1535 and TA1537 toxic effects of the test item were noted at concentrations of 316 μg/plate and higher (with and without metabolic activation). In tester strain TA102 toxic effects of the test item were observed at concentrations of 2500 μg/plate and higher (without metabolic activation) and at a concentration of 5000 μg/plate (with metabolic activation) (Table 2). In experiment II toxic effects of the test item were noted in tester strains TA98, TA100 and TA1535 at concentrations of 316 μg/plate and higher (with and without metabolic activation). In tester strain TA1537 toxic effects of the test item were seen at concentrations of 316 μg/plate and higher (without metabolic activation) and at a concentration of 1000 μg/plate (with metabolic activation). In tester strain TA102 toxic effects of the test item were observed at a concentration of 5000 μg/plate (with and without metabolic activation) (Table 3).
- Individual plate counts: Yes, refer to Tables 2 and 3
- Mean number of revertant colonies per plate and standard deviation: Yes, refer to Tables 2 and 3

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Yes historical control data from 2015-2017 (Tables 5, 7)
- Negative (solvent/vehicle) historical control data: Yes historical control data from 2015-2017 (Tables 4, 6)

Applicant's summary and conclusion

Conclusions:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is considered to be nonmutagenic in this bacterial reverse mutation assay with or without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (183809), strains of S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 102 were exposed to 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in DMSO at concentrations of 3.16 - 5000 µg/plate (plate incorporation), 0.316 - 1000 µg/plate (pre-incubation; all strains except for TA102) and 3.16 - 5000 µg/plate (pre-incubation; TA102) in the presence and absence of mammalian metabolic activation (phenobarbital and β-naphthoflavone-induced rat liver S9).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in the S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 100 strains using the plate incorporation or pre-incubation methods in the presence or absence of metabolic activation.