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Toxicological information

Genetic toxicity: in vitro

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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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
Due to migration, the value was transferred to one of the current document's attachments
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 resultsopen allclose all
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
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:
100 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
316 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
316 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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:
316 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
316 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
316 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
316 μg/plate (-S9); 1000 μg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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.