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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was not mutagenic in the strains S. typhimurium TA 98, TA 100, TA102, TA 1535 and TA 1537 in the presence and absence of phenobarbital and beta-naphthoflavone S9 metabolic activation. (OECD 471/GLP).

Chromosome aberration (in vitro cytogenicity/micronucleus study): 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine 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 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine in the absence of phenobarbital and beta-naphthoflavone-induced rat liver S9 metabolic activation. However, there was a statistically and biologically relevant increase in mutant colonies over background in the presence of metabolic activation. Overall, 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is mutagenic in the MLA assay.

Link to relevant study records

Referenceopen allclose all

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

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2018 - 11 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
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 in room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
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 and re-diluted in cell culture medium to reach a final concentration of 1% v/v DMSO in the samples and to achieve the final test item
concentrations. Upon re-dilution in cell culture medium, the test item precipitated again. The solvent was compatible with the survival of the cells and the S9 activity.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
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. Routine checking of mycoplasma infections were carried out before freezing.

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
Cytokinesis block (if used):
Cytochalasin B
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 experiment:
Without metabolic activation (24 hrs): 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL (experiment was terminated at preparation since no intact cells were left in any culture)
Without metabolic activation (24 hrs): 0.005, 0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 2.5 and 5.0 μg/mL (repetition, reported in this study)
With metabolic activation (4 hrs): 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL

Experiment I:
Without metabolic activation (4 hrs): 0.10, 0.25, 0.5, 0.8, 0.9, 1.0 and 1.25 μg/mL
With metabolic activation (4 hrs): 2, 4, 8, 9, 10, 12 and 14 μg/mL
Experiment II (not reported, evaluation of proliferation index of three highest concentrations showed that cytotoxicity criteria according to OECD 487 were not met)
Without metabolic activation (24 hrs): 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.25 μg/mL
Experiment II (repetition, reported for this study):
Without metabolic activation (24 hrs): 0.5, 1.25, 2.5, 5.0, 7.5, 10, 12.5, 15, 20, 30 and 40 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: 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 and re-diluted in cell culture medium to reach a final concentration of 1% v/v DMSO in the samples and to achieve the final test item concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
other: Colchicine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 2
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):Experiment I: Approx. 50 000 cells were seeded per cell culture flask, containing 5 mL complete culture medium; Experiment II: Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm2 cell culture flasks.
- Test substance added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment I: 4hrs +/-S9; Experiment II: 24 hrs +/-S9

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): In Experiment 1, after 4 hrs test item exposure and washing, 1.5 μg/mL cytochalasin B was added for 20 h at 37 °C. In Experiment 2, 1 hr after the test item was added, 1.5 μg/mL cytochalasin B was added and the cells were incubated for 23 h at 37 °C.

- 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 some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems). After the treatment with the hypotonic solution the cells were fixed 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.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): All slides, including those of positive and negative controls were independently coded before microscopic analysis. For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei 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, e.g.: The cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). Cytostasis was used to describe cytotoxicity.
- 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.
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 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 breaks and/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 the criteria mentioned above are met.
Statistics:
Statistical significance: statistical significant difference in micronucleated cells frequency compared to solvent control (nonparametric χ² test, p < 0.05).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment 1
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
12 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment 1
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
0.9 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment 2
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
7.5 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH & osmolality: For the maximum concentration without metabolic activation the osmolality (in comparison to solvent control) and pH value were determined. The test item had no effect on either.
- Precipitation and time of the determination: The test item was dissolved in DMSO and re-diluted in cell culture medium (MEM medium). Precipitation of the test item was noted at 31.3 μg/mL and higher with metabolic activation in the pre-experiment but not without metabolic activation and no precipitate of the test item was noted in any concentration group evaluated in main experiment I and II at the end of treatment since all the concentrations used were below the solubility limit.

RANGE-FINDING/SCREENING STUDIES (if applicable):
According to the used guideline the highest recommended concentration is 2000 μg/mL. The test item was dissolved in DMSO and re-diluted with cell culture medium to achieve the final test item concentrations. Precipitation of the test item was noted at 31.3 μg/mL with metabolic activation but not without metabolic activation. The following concentrations were tested:
without metabolic activation: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL (experiment was terminated at preparation since no intact cells were left in any culture);
without metabolic activation: 0.005, 0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 2.5 and 5.0 μg/mL (repetition, reported in this study);
with metabolic activation: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL
The highest dose group evaluated in the pre-experiment was 2.5 μg/mL without and 15.6 μg/mL with metabolic activation. At higher concentrations microscopic evaluation was not possible since no intact cells were present on the slides (Table 3).

STUDY RESULTS
- Concurrent vehicle negative and positive control data: MMS (20 and 25 μg/mL) and CPA (2.5 μg/mL) were used as clastogenic controls and colchicine as aneugenic control (0.08 and 2.0 μg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: The χ² 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).
- Statistical analysis; p-value if any: 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 and II with and without metabolic activation (Table 11, Table 12, Table 13).

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells:
In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to 0.5 μg/mL. At 0.9 μg/mL a cytostasis of 41% and at 1.25 μg/mL a cytostasis of 51% was noted (Table 4).

In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to 10 μg/mL. At 12 μg/mL a cytostasis of 38% and at 14 μg/mL a cytostasis of 49% was observed (Table 4).

In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 2.5 μg/mL. At 7.5 μg/mL a cytostasis of 48% and at 10 μg/mL a cytostasis of 61% was noted(Table 7).

o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells):
Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate:
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (1.35%) was within the historical control limits of the negative control (0.37% – 1.37%, Table 15) and the micronucleated cell frequency of the solvent control (1.10%) was within the historical control limits of the solvent control (0.47% – 1.48%, Table 15). The mean values of micronucleated cells found after treatment with the test item were 0.75% (0.5 μg/mL), 0.75% (0.9 μg/mL) and 0.60% (1.25 μg/mL) (Table 5). 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 I with metabolic activation the micronucleated cell frequency of the negative control (1.45%) was within the historical control limits of the negative control (0.42% – 1.64%, Table 15) and the micronucleated cell frequency of the solvent control (1.55%) was within the historical control limits of the solvent control (0.35% – 1.75%, Table 15). The mean values of micronucleated cells found after treatment with the test item were 1.35% (10 μg/mL), 0.65% (12 μg/mL) and 0.85% (14 μg/mL) (Table 6). 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 micronucleated cell frequency of the negative control (1.30%) was within the historical control limits of the negative control (0.37% – 1.37%, Table 15) and the micronucleated cell frequency of the solvent control (1.05%) was within the historical control limits of the solvent control (0.47% – 1.48%, Table 15). The mean values of micronucleated cells found after treatment with the test item were 0.75% (2.5 μg/mL), 0.65% (7.5 μg/mL) and 0.50% (10 μg/mL) (Table 8). 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.


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: Provided from 2012-2017 (Table 16)
- Negative (solvent/vehicle) historical control data: Provided from 2012-2017 (Table 15)
Conclusions:
4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is considered to be nonmutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test, with or without metabolic activation.
Executive summary:

In an in vitro cytogenicity/micronucleus study (183810), Chinese hamster V79 cells were exposed to 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in DMSO. The concentrations without metabolic activation were 0.10, 0.25, 0.5, 0.8, 0.9, 1.0 and 1.25 μg/mL (first experiment; 4 hrs) and 0.5, 1.25, 2.5, 5.0, 7.5, 10, 12.5, 15, 20, 30 and 40 μg/mL (second experiment; 24 hrs); the concentrations with phenobarbital and ß-naphthoflavone-induced rat liver S9 were 2, 4, 8, 9, 10, 12 and 14 μg/mL (first experiment; 4 hrs).

Cytotoxicity was noted without metabolic activation at 0.9 ug/mL (4 hrs) and 7.5 ug/mL (24 hrs); cytotoxicity was noted with metabolic activation at 12 ug/mL (4 hrs). Positive controls induced the appropriate response. There was no evidence of any chromosome damage or damage to the cell division apparatus induced over background.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 December 2018 - 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. certificate)
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 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was dissolved in dimethylsulfoxide at a 100-fold concentration and re-diluted in cell culture medium to reach a final concentration of 1% v/v DMSO in the samples and to achieve the final test item concentrations. Upon re-dilution in cell culture medium, the test item precipitated again. Different test item stock solutions were prepared and added to the samples. The solvent was compatible with the survival of the cells and the S9 activity.
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10 - 12 h doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40  2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.
To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells 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.
Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich. This allows the repeated use of the same cell batch in experiments. Each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
Additional strain / cell type characteristics:
other: clone TK+/- -3.7.2C
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: 0.10, 0.25, 0.50, 2, 5, 15, 40 and 100 µg/mL
Main test Experiment I (4hrs, -S9): 0.50, 0.75, 2, 3, 4, 5 and 10 µg/mL
Main test Experiment I (4hrs, +S9): 5, 7.5, 11, 13, 15, 20 and 22.5 µg/mL
Main test Experiment II (4hrs, +S9) 5, 11, 12, 14, 16, 17 and 18 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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, duplicate, triplicate): single for test items and positive controls; duplicates for negative/solvent controls
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4hrs +/-S9

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 12 days (7 days in non-selective medium for cloning efficiency)
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: TFT at 5 µg/mL in RPMI 1640 medium (selective medium) for 12 days
- Method used: microwell plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Viability: 1.6 cells/well in two 96-well plates
Mutant cells: 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
- Criteria for small (slow growing) and large (fast growing) colonies:
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) (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: RTG = RSG x RCE /100)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
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

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 arrived to be 126 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 106 cells and
- a dose-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.

A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the solvent controls. Mutant frequencies of the solvent controls were used as reference. Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Species / strain:
mouse lymphoma L5178Y cells
Remarks:
4 hrs
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
4 hrs (Expt 1)
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
11µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
4 hrs (Expt 2)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
12µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH/osmolality: The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration was 469 mOsm/kg
- Precipitation and time of the determination: No precipitation of the test item was noted in the pre-experiment / main experiments.

RANGE-FINDING/SCREENING STUDIES (if applicable): Based on the results obtained in the in vitro Mammalian Micronucleus Assay (Eurofins Munich Study No. 183810) and due to the toxicity that occurred, the maximum tested concentration was 100 µg/mL. Eight concentrations [0.10, 0.25, 0.50, 2, 5, 15, 40 and 100 µg/mL] were tested without and with metabolic activation. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. 10 µg/mL (without metabolic activation) and 22.5 µg/mL (with metabolic activation) were selected as the highest concentrations (Table 3, 4).

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
In the experiments I (without and with metabolic activation) and experiment II (with metabolic activation) all validity criteria were met. The negative and solvent controls showed mean mutant frequencies within the acceptance range of 50 -170 mutants/106 cells, according to the IWGT criteria.
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 proving the ability of the test system to indicate potential clastogenic effects (Table 7, Table 10). All positive controls were within the historical control data range (Table 18).

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible/Statistical analysis:
Experiment I without metabolic activation: None
Experiment I with metabolic activation: In experiment I with metabolic activation an increase of mutants was found after treatment with the test item. The GEF was exceeded by the induced mutant frequency at concentrations of 15 µg/mL and higher. Moreover, a statistically significant concentration-response relationship was observed. According to the OECD guideline 490 care should be taken, if an increase of mutant frequency was only observed between 10 and 20% RTG. As in this study the GEF was exceeded at concentrations within this mentioned toxicity range (15 µg/mL) or even below 10% RTG (20 and 22.5 µg/mL), thus the test item was considered in the first conducted experiment as equivocal.
Experiment II with metabolic activation: In experiment II with metabolic activation the mutant frequencies induced by the test item showed a distinct biologically relevant increase. The GEF of 126 was exceeded at concentrations of 16 µg/mL and higher (201.9 mutants/106 cells). A statistical analysis displayed that the corresponding values of the mutant frequencies were significantly increased over those of the solvent controls. Additionally, a statistically significant concentration-related increase was determined in the ² test for trend.
- Any other criteria: GEF = 126

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG)
Growth inhibition was observed in the experiment I (without and with metabolic activation) and in experiment II (with metabolic activation):
In experiment I the relative total growth (RTG) was 7.8% (without metabolic activation) for the highest concentration evaluated (10 µg/mL) and 15.3% for concentration 15 µg/mL, 6.8% for concentration 20 µg/mL and 3.4% for concentration 22.5 µg/mL (with metabolic activation) (Table 5, Table 8).
In experiment II the relative total growth (RTG) was 10.9% (with metabolic activation) for the highest concentration evaluated, 17.2 % for concentration 17 µg/mL, 20.1% for concentration 16 µg/mL and 40.9% for concentration 14 µg/mL (Table 11)

- Genotoxicity results:
In experiment I without metabolic activation the mutant frequencies induced by the test item did not show any biologically relevant increase. The GEF of 126 was not exceeded in any of the concentrations (Table 6). A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the solvent controls (Table 14). 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.
In experiment I with metabolic activation an increase of mutants was found after treatment with the test item. The GEF was exceeded by the induced mutant frequency at concentrations of 15 µg/mL and higher (Table 9). Additionally, a statistical analysis displayed that this increase was statistically significant and a significantly concentration-related increase was determined in the ² test for trend (Table 15, Table 17).

According to the OECD guideline 490 care should be taken, if an increase in mutant frequency was only observed between 10 and 20% RTG. As in this study the GEF was exceeded at concentrations within this mentioned toxicity range (15 µg/mL) or even below 10% RTG (20 µg/mL and 22.5 µg/mL), the test item was considered as equivocal under the experimental conditions reported.

Due to the equivocal result in experiment I with metabolic activation the test item was evaluated in a second experiment with closer spaced concentrations in the relevant range.

In experiment II with metabolic activation the mutant frequencies induced by the test item showed a distinct biologically relevant increase (Table 12). The GEF of 126 was exceeded at concentrations of 16 µg/mL (201.9 mutants/106 cells), 17 µg/mL (335.8 mutants/106 cells) and 18 mg/mL (397.5 mutants/106 cells). A statistical analysis displayed that the corresponding values of the mutant frequencies were significantly increased over those of the solvent controls (Table 16). Even the next lower concentration 14 µg/mL displayed a statistically significantly increase in mutant frequency with 119.4 mutant/106 cells and thus very close to the GEF of 126. Additionally, a statistically significant concentration-related increase was determined in the ² test for trend (Table 17).

Historical data for mutant frequencies are shown in Table 18 (Appendix). In experiment I (without and with metabolic activation) the mutant frequencies of the negative controls were slightly increased (without metabolic activation: 159.0 and 161.1 mutants/106 cells; with metabolic activation: 155.5 and 143.7 mutants/106 cells) and outside the historic control range (i.e. LCL and UCL; without metabolic activation: 32.5 - 140.0 mutants/106 cells; with metabolic activation: 34.0 - 135.8 mutants/106 cells). Nevertheless, these values were considered acceptable for inclusion in the historical control data set as they were only slightly increased and were still within the range for the Min and Max values of the historical data set.

Colony sizing was performed for the highest 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 for potential clastogenic effects and/or chromosomal aberrations. Thus based on the non-mutagenic effects of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine in experiment I without metabolic activation, an assessment of clastogenicity was not feasible (Table 7).

In the experiments I and II with metabolic activation the percentage of small colonies in the negative controls and in the solvent controls, was found to be lower than 40%. Due to the increased number of small colonies and corresponding mutagenicity in the highest concentrations of the test item (experiment I: 15 µg/mL and 22 µg/mL; experiment II: 16, 17 and 18 µg/mL), these concentrations were considered as potential clastogenic (Table 10, Table 13).

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: Data provided for January 2011 to December 2018 (Table 18)
- Negative (solvent/vehicle) historical control data: Data provided for January 2011 to December 2018 (Table 18)
Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is considered to be non-mutagenic in the experiment without metabolic activation, but in the experiment with metabolic activation the test item was concluded to be mutagenic. Overall the test item response was concluded to be mutagenic in the 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: TK+/-; 188279], mouse lymphoma L5178Y cells cultured in vitro were exposed to 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%)in DMSO for 4 hours without metabolic activation at concentrations of 0.50, 0.75, 2, 3, 4, 5 and 10 µg/mL (first experiment) and in the presence of phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation at concentrations of 5, 7.5, 11, 13, 15, 20 and 22.5 µg/mL (first experiment) and 5, 11, 12, 14, 16, 17 and 18 µg/mL (second experiment).

Cytotoxicity was noted at 4 ug/mL without metabolic activation, at 11 ug/mL with metabolic activation (first experiment) and 12 ug/ml (second experiment).  The positive controls induced the appropriate response.  

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is considered to be non-mutagenic in the experiment without metabolic activation.  In experiment I with metabolic activation an increase of mutants was found after treatment with the test item. The GEF was exceeded by the induced mutant frequency at concentrations of 15 µg/mL and higher. Additionally, a statistical analysis displayed that this increase was statistically significant and a significantly concentration-related increase was determined in the ² test for trend. According to the OECD guideline 490 care should be taken, if an increase in mutant frequency was only observed between 10 and 20% RTG. As in this study the GEF was exceeded at concentrations within this mentioned toxicity range (15 µg/mL) or even below 10% RTG (20 µg/mL and 22.5 µg/mL), the test item was considered as equivocal under the experimental conditions reported. Due to the equivocal result in experiment I with metabolic activation the test item was evaluated in a second experiment with closer spaced concentrations in the relevant range.

In experiment II with metabolic activation the mutant frequencies induced by the test item showed a distinct biologically relevant increase. The GEF of 126 was exceeded at concentrations of 16 µg/mL (201.9 mutants/106 cells), 17 µg/mL (335.8 mutants/106 cells) and 18 mg/mL (397.5 mutants/106 cells). A statistical analysis displayed that the corresponding values of the mutant frequencies were significantly increased over those of the solvent control. Even the next lower concentration 14 µg/mL displayed a statistically significantly increase in mutant frequency with 119.4 mutant/106 cells and thus very close to the GEF of 126. Additionally, a statistically significant concentration-related increase was determined in the Chi² test for trend. In the experiments I and II with metabolic activation the percentage of small colonies in the negative controls and in the solvent controls, was found to be lower than 40%. Due to the increased number of small colonies and corresponding mutagenicity in the highest concentrations of the test item (experiment I: 15 µg/mL and 22 µg/mL; experiment II: 16, 17 and 18 µg/mL), these concentrations were considered as potential clastogenic.

In conclusion, the test item 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is considered to be non-mutagenic in the experiment without metabolic activation, but in the experiment with metabolic activation the test item was concluded to be mutagenic. Overall the test item response was concluded to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

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, 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.


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 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in DMSO. The concentrations without metabolic activation were 0.10, 0.25, 0.5, 0.8, 0.9, 1.0 and 1.25 μg/mL (first experiment; 4 hrs) and 0.5, 1.25, 2.5, 5.0, 7.5, 10, 12.5, 15, 20, 30 and 40 μg/mL (second experiment; 24 hrs); the concentrations with phenobarbital and ß-naphthoflavone-induced rat liver S9 were 2, 4, 8, 9, 10, 12 and 14 μg/mL (first experiment; 4 hrs). Cytotoxicity was noted without metabolic activation at 0.9 ug/mL (4 hrs) and 7.5 ug/mL (24 hrs); cytotoxicity was noted with metabolic activation at 12 ug/mL (4 hrs). Positive controls induced the appropriate response. There was no evidence of any chromosome damage or damage to the cell division apparatus induced over background.

Gene mutation (mammalian cell gene mutation assay):

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

In a mammalian cell gene mutation assay [MLA: TK+/-; OECD 490/GLP], mouse lymphoma L5178Y cells cultured in vitro were exposed to 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%)in DMSO for 4 hours without metabolic activation at concentrations of 0.50, 0.75, 2, 3, 4, 5 and 10 µg/mL (first experiment) and in the presence of phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation at concentrations of 5, 7.5, 11, 13, 15, 20 and 22.5 µg/mL (first experiment) and 5, 11, 12, 14, 16, 17 and 18 µg/mL (second experiment). Cytotoxicity was noted at 4 ug/mL without metabolic activation, at 11 ug/mL with metabolic activation (first experiment) and 12 ug/ml (second experiment).  The positive controls induced the appropriate response.  

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is considered to be non-mutagenic in the experiment without metabolic activation.  In experiment I with metabolic activation an increase of mutants was found after treatment with the test item. The GEF was exceeded by the induced mutant frequency at concentrations of 15 µg/mL and higher. Additionally, a statistical analysis displayed that this increase was statistically significant and a significantly concentration-related increase was determined in the ² test for trend. According to the OECD guideline 490 care should be taken, if an increase in mutant frequency was only observed between 10 and 20% RTG. As in this study the GEF was exceeded at concentrations within this mentioned toxicity range (15 µg/mL) or even below 10% RTG (20 µg/mL and 22.5 µg/mL), the test item was considered as equivocal under the experimental conditions reported. Due to the equivocal result in experiment I with metabolic activation the test item was evaluated in a second experiment with closer spaced concentrations in the relevant range. In experiment II with metabolic activation the mutant frequencies induced by the test item showed a distinct biologically relevant increase. The GEF of 126 was exceeded at concentrations of 16 µg/mL (201.9 mutants/106 cells), 17 µg/mL (335.8 mutants/106 cells) and 18 mg/mL (397.5 mutants/106 cells). A statistical analysis displayed that the corresponding values of the mutant frequencies were significantly increased over those of the solvent control. Even the next lower concentration 14 µg/mL displayed a statistically significantly increase in mutant frequency with 119.4 mutant/106 cells and thus very close to the GEF of 126. Additionally, a statistically significant concentration-related increase was determined in the Chi² test for trend. In the experiments I and II with metabolic activation the percentage of small colonies in the negative controls and in the solvent controls, was found to be lower than 40%. Due to the increased number of small colonies and corresponding mutagenicity in the highest concentrations of the test item (experiment I: 15 µg/mL and 22 µg/mL; experiment II: 16, 17 and 18 µg/mL), these concentrations were considered as potential clastogenic. In conclusion, the test item 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is considered to be non-mutagenic in the experiment without metabolic activation, but in the experiment with metabolic activation the test item was concluded to be mutagenic. Overall the test item response was concluded to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Due to the result of the MLA, no final conclusion can be made on the genetic toxicity; therefore an in vivo Comet assay (OECD 489) or Transgenic Rodent (TGR) Somatic and Germ Cell Gene Mutation Assays (OECD 488) is proposed.


Justification for classification or non-classification

Based on the available information in the dossier, it is not possible to reach a final conclusion on germ cell mutagenicity for the substance 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (CAS No. 72058-41-4), when the criteria outlined in Annex I of 1272/2008/EC are applied. An in vivo Comet assay (OECD 489) or Transgenic Rodent (TGR) Somatic and Germ Cell Gene Mutation Assays (OECD 488) is proposed.