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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

Reference

Reference 1

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source 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:

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.

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

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

316 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

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

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)

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.

Reference 2

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

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source 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:

- 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 ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al..

- method of preparation of S9 mix: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.

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

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

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.

Reference 3

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

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source 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

Description of key information

In vivo mammalian somatic cell study (In vivo Mammalian Alkaline Comet Assay): the substance 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration to rats. (OECD 489/GLP).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

In vivo Mammalian Alkaline Comet Assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet 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; Unitechem-20211231
- Purity: 99.29%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Samples were prepared according to a previous formulation study (Eurofins Munich Study No. 183815)

Species:

rat

Strain:

other: Wistar rats, Crl: WI(Han) (Full Barrier)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at study initiation: 222-258g (first administration; males)
- Housing: The animals will be kept in groups of 2-3 animals / sex / group / cage in IVCs (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
According to a previous studies including a formulation study (Eurofins Munich Study No. 183815), the test item was prepared in Corn Oil and diluted prior to treatment. All animals received a single volume orally of 10 mL/kg bw. The solvent was chosen according to its relative non-toxicity for the animals. The prepared formulations were transferred to BSL Munich together with the control formulations under consideration of stability.

- Purity:

Details on exposure:

Test item and control formulations were prepared at Eurofins Munich (Eurofins Munich Study No.:STUGC22AA0720-2) and transferred to BSL Munich under consideration of the respective stability (Eurofins Munich Study No. 183815) to enable administration of all designated animals before expiration of the test item.

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

0 and 24 hrs

Post exposure period:

Target organs are sampled at 4 hours after the last dose.

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

LD

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

MD

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

HD

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control item ethylmethanesulfonate (EMS) was administered only once via gavage 4 hours before animal sacrifice at a dose of 250 mg/kg bw

Tissues and cell types examined:

Isolation of primary hepatocytes:
A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept for not more than 15 seconds until bigger fragments of the liver settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of small intestine, upper and lower part of colon cells:
The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. The duodenum was cut open into two halves. One half of the duodenum was minced with a pair of scissors, the other one was kept for histopathology. The cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of glandular stomach cells:
The stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pestle to release single cells. The suspension was kept for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of germ cells from seminiferous tubules
The end of the epithelial capsule of the gonad was punctured to squeeze out the seminiferous tubules. Prepared cell pellets were used for preparing comet slides. Comet slides of the male gonads were stored at room temperature, under dry conditions and protected from light.

One part of the liver, glandular stomach, duodenum and one male gonad was preserved in 10% neutral-buffered formalin for histopathological evaluation

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A dose range-finding study on acute toxicity was performed with the same strain in both sexes to identify the maximum tolerated dose, defined as the dose inducing slight toxic effects at the end of the of the study period.

Under consideration of available data, a starting dose was chosen with the aim to elicit slight toxic symptoms. Based on acute oral toxicity data for female Wistar rats (Eurofins Munich / BSL Munich Study No. 183811), which showed moderate to strong signs of toxicity at a dose level of 2000 mg/kg bw, a limit dose of 1000 mg/kg bw was used in the dose range-finding experiment for female rats. As there is no information about the acute toxicity in male rats, a starting dose of 2000 mg/kg bw was used for male rats in the pre-experiment.

Due to the results obtained in the pre-experiment the highest dose for male rats was 1000 mg/kg bw and 500 mg/kg bw for female rats. After consultation of the sponsor, male rats were chosen for the main experiment as they showed the higher maximum tolerated dose.


DETAILS OF SLIDE PREPARATION:
Once single cells were obtained, the basic steps of the assay included:

Slide preparation:
The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).

Lysis:
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow for the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.

Unwinding of DNA and electrophoresis:
Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. An incubation period of 20 min was generally considered appropriate for alkali unwinding. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order.

Neutralisation and dehydration of slides:
After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.

DNA staining:
Following dehydration, the cells were stained by applying 75 µL gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS:
Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated. Each slide was screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet might be adjusted manually if they were not set correctly automatically. All cells of the visual field were scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, were considered to be heavily damaged cells). Therefore, cells were classified into three categories scorable, non-scorable and “hedgehog”. To avoid artefacts only scorable cells and at least 150 cells per sample on two slides (75 cells per slide) were scored. The third back-up slide was scored in case of discordant results. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.

Evaluation criteria:

Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the % tail intensity in at least one dose group at a single sampling time in comparison with the negative control value.

Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).

To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.

Statistics:

All slides, including those of positive and vehicle controls were independently coded and blinded before microscopic analysis. The median % tail intensity for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.

For each tissue type, the mean of the individual animal means was then determined to give a group mean % of tail intensity. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control .

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Duodenum; at all doses

Toxicity:

yes

Remarks:

LD - piloerection MD - spontaneous activity, piloerection, prone position and hunched posture; four out of five male rats lost weight HD - spontaneous activity, piloerection, diarrhea and hunched posture; a reduction of body weight was noted in each male.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Glandular stomach; at all doses

Toxicity:

yes

Remarks:

LD - piloerection MD - spontaneous activity, piloerection, prone position and hunched posture; four out of five male rats lost weight HD - spontaneous activity, piloerection, diarrhea and hunched posture; a reduction of body weight was noted in each male.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Liver; at all doses

Toxicity:

yes

Remarks:

LD - piloerection MD - spontaneous activity, piloerection, prone position and hunched posture; four out of five male rats lost weight HD - spontaneous activity, piloerection, diarrhea and hunched posture; a reduction of body weight was noted in each male.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY

In the pre-experiment, one male rat received a single limit dose of 2000 mg/kg bw orally and showed severe signs of toxicity such as reduced spontaneous activity, prone position, piloerection, hunched posture and half eyes closed after the first administration of the test item. After the second administration of the test item, the same clinical signs were present but more severe in addition to moving the bedding.

Therefore, the dose was reduced to 1500 mg/kg bw and administered orally to one male rat. Most of the former effects were observed including reduced spontaneous activity, piloerection, hunched posture and half eyes closed after the first administration of the test item. After the second administration of the test item, the same clinical signs were present in addition to diarrhea.

Due to this observation, the dose was again reduced to 1000 mg/kg bw and administered orally to one male rat. After the first administration of the test item piloerection, hunched posture and reduced spontaneous activity were observed. Piloerection and diarrhea were still present before the second administration of the test item. During the observation period of 4h, reduced spontaneous activity, diarrhea and hunched posture were additionally observed. Due to the results obtained, two additional male rats were treated with a dose of 1000 mg/kg bw. Reduced spontaneous activity, piloerection, prone position and diarrhea were observed after the first administration of the test item. During the observation period of 4h, reduced spontaneous activity, diarrhea and piloerection were observed (see Appendix 2: Study-related Toxicity Data; Table 10).
In the pre-experiment, one female rat received a single dose of 1000 mg/kg bw orally and showed clinical signs such as reduced spontaneous activity, hunched posture and piloerection after the first administration of the test item. After the second administration of the test item, piloerection was observed. Therefore, two additional female rats were dosed with 1000 mg/kg bw. Both female rats showed reduced spontaneous activity and piloerection after the first administration of the test item. After the second administration of the test item, the females showed more severe clinical signs, such as reduced spontaneous activity, piloerection, hunched posture, diarrhea and half eyes closed (only one female; (see Appendix 2: Study-related Toxicity Data; Table 10).

Therefore, the amount of the test item was reduced to 500 mg/kg bw/d and administered orally to one female rat. After the first administration of the test item piloerection and reduced spontaneous activity were observed. Piloerection and diarrhea were present before the second administration of the test item. During the observation period of 4h, reduced spontaneous activity, hunched posture and piloerection were observed. The toxicity signs noted in female rats after the application of 500 mg/kg bw/d were classified as slight to moderate toxic effects, therefore two additional female rats received the same dosing to confirm the concentration of 500 mg/kg bw/d as HD for female rats. Piloerection and reduced spontaneous activity were observed after the first administration of the test item and were also present after the second administration of the test item.

Due to the results obtained in the pre-experiment the highest dose for male rats was 1000 mg/kg bw and 500 mg/kg bw for female rats. After consultation of the sponsor, male rats were chosen for the main experiment as they showed the higher maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY

Clinical signs and body weight
All animals treated with the highest dose (HD) showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection, diarrhea and hunched posture after the first and second application (Appendix 2: Study-related Toxicity Data; Table 12). Animals treated with a dose of 500 mg/kg bw showed spontaneous activity, piloerection, prone position and hunched posture after the first and second application (see Appendix 2: Study-related Toxicity Data; Table 11). Rats treated with 200 mg/kg bw (LD) showed piloerection after the second administration of the test material.

Three out of five male rats in the vehicle control group (corn oil) lost weight at the end of the experiment. A reduction of body weight was noted in each male animal of the highest dose group (1000 mg/kg bw) for male rats at the end of the experiment. At the lower concentrations tested, four out of five male rats in the MD group (500 mg/kg bw) lost weight. All male rats in the lowest does group (200 mg/kg bw) gained weight by the end of the experiment.

GENOTOXICITY
Liver
The group mean value obtained in the main experiment for the vehicle control was 1.03 % in the case of male liver cells (Table 3). The tail intensity of the LD (200 mg/kg bw; 1.18 %), MD (500 mg/kg bw; 1.08 %) and HD (1000 mg/kg bw; 0.87 %) were within the historic control limits of the testing facility (0.07– 3.82%; 16.1 Appendix 1: Laboratory Historical Control Data). No significant increase compared to the vehicle control (corn oil) was noted for any dose group evaluated for male liver cells (Table 6). No concentration-dependency was noted for the tail intensities in male cells of the liver (Table 7). Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 11.88% derived from male liver cells. This demonstrates the validity of the assay.

Glandular stomach
In glandular stomach cells, the group mean value obtained for the vehicle control was 3.26% (Table 4). The tail intensity of the dose groups at 200 mg/kg bw (LD; 4.61%), 500 mg/kg bw (MD; 3.70%) and 1000 mg/kg bw (HD; 3.07%) were within the historic vehicle control range of the testing facility (1.30- 5.46%, 16.1 Appendix 1: Laboratory Historical Control Data). No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male glandular stomach cells (Table 6). No concentration-dependency was noted for the tail intensities in male cells of the glandular stomach (Table 7). Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 12.19% derived from male glandular stomach cells

Duodenum
In the duodenum, the group mean value obtained for the negative control was 2.39% (Table 5). The group mean tail intensities of the test item concentrations 200 mg/kg bw (LD; 2.15%), 500 mg/kg bw (MD; 3.95%) and 1000 mg/kg bw (HD; 2.39%) were within the range of the historic vehicle control with tail intensities 0.89- 4.06% (16.1 Appendix 1: Laboratory Historical Control Data). No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male duodenum cells (Table 6). No concentration-dependency was noted for the tail intensities in male cells of the duodenum (Table 7). Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control. In vivo treatment with EMS revealed a significant increase in DNA damage with tail intensity values of 11.08% derived from male duodenum cells. This demonstrates the validity of the assay.

No increase of hedgehogs was detected in all organs evaluated (see Table 13, Table 14 and Table 15).

STATISTICS
Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control.
A statistically significant increase was determined for the positive control group of each organ. No significances compared to the vehicle control were determined in any of the dose groups of each organ (Table 6).

For the determination of a statistically significant concentration-related increase, the mean values of each animal per dose group were evaluated with a linear trend test at the 5 % level (p<0.05). The p-value was used as a limit in judging for significance levels.
No concentration-related increase was noted in any of the analysed organs in male rats (Table 7).

As the results in all 3 tissues were clearly negative and target tissue exposure was confirmed, the male gonad slides were not evaluated.

Less than 150 cells were scored for one male in the HD group (animal 37 (149 cells) in glandular stomach cells as not enough cells were present for evaluation (see Table 26). Nevertheless, this did not affect the validity of the assay, as still 149 cells (animal 2) were scored and these values were in the same range as the tail intensities of the animals 1, 3,4 and 5 of the same dose group.

 

Conclusions:

In an in vivo Mammalian Alkaline Comet Assay of liver, glandular stomach and duodenum cells in male Wistar rats, the test item did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration to rats.

Executive summary:

In a Wistar rat comet assay, groups of 5 male rats were treated by oral gavage with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in corn oil at doses of 200, 500 and 1000 mg/kg bw. The animals were dosed on 2 consecutive days (0, 24 ± 1 hrs) and samples of the liver, glandular stomach, duodenum and male gonads were removed four hours after the last administration of the test item. The positive control was ethyl methanesulfonate (250 mg/kg bw). A total of 150 cells/animal/tissue (75 cells per slide) were evaluated and DNA migration during electrophoresis was determined and expressed as % tail intensity. Systemic toxicity was evaluated via clinical signs and body weight changes.

 

All animals treated with the highest dose (HD) showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection, diarrhea and hunched posture after the first and second application. Animals treated with a dose of 500 mg/kg bw showed spontaneous activity, piloerection, prone position and hunched posture after the first and second application. Rats treated with 200 mg/kg bw (LD) showed piloerection after the second administration of the test material. A reduction of body weight was noted in each male animal of the highest dose group (1000 mg/kg bw) for male rats at the end of the experiment. At the lower concentrations tested, four out of five male rats in the MD group (500 mg/kg bw) lost weight. All male rats in the lowest does group (200 mg/kg bw) gained weight by the end of the experiment. Therefore, the highest dose (1000 mg/kg bw) showed clinical signs that were strong enough to demonstrate systemic exposure without evidence of study-limiting toxicity.

 

The group mean value obtained in the main experiment for the vehicle control was 1.03 % in the case of male liver cells. The tail intensity of the LD (200 mg/kg bw; 1.18 %), MD (500 mg/kg bw; 1.08 %) and HD (1000 mg/kg bw; 0.87 %) were within the historic control limits of the testing facility (0.07– 3.82%). No significant increase compared to the vehicle control (corn oil) was noted for any dose group evaluated for male liver cells. No concentration-dependency was noted for the tail intensities in male cells of the liver. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 11.88% derived from male liver cells.

 

In glandular stomach cells, the group mean value obtained for the vehicle control was 3.26%. The tail intensity of the dose groups at 200 mg/kg bw (LD; 4.61%), 500 mg/kg bw (MD; 3.70%) and 1000 mg/kg bw (HD; 3.07%) were within the historic vehicle control range of the testing facility (1.30- 5.46%). No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male glandular stomach cells. No concentration-dependency was noted for the tail intensities in male cells of the glandular stomach. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 12.19% derived from male glandular stomach cells.

 

In the duodenum, the group mean value obtained for the negative control was 2.39%. The group mean tail intensities of the test item concentrations 200 mg/kg bw (LD; 2.15%), 500 mg/kg bw (MD; 3.95%) and 1000 mg/kg bw (HD; 2.39%) were within the range of the historic vehicle control with tail intensities 0.89- 4.06%. No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male duodenum cells. No concentration-dependency was noted for the tail intensities in male cells of the duodenum. In vivo treatment with EMS revealed a significant increase in DNA damage with tail intensity values of 11.08% derived from male duodenum cells.

 

No increase of hedgehogs was detected in all organs evaluated. No concentration-related increase was noted in any of the analysed organs in male rats. As the results in all 3 tissues were clearly negative and target tissue exposure was confirmed, the male gonad slides were not evaluated.

 

Therefore, the test item did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration to rats.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test)

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

 

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 98, TA 100, 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.

 

In vivo mammalian somatic cell study (In vivo Mammalian Alkaline Comet Assay):

In a Wistar rat comet assay, groups of 5 male rats were treated by oral gavage with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in corn oil at doses of 200, 500 and 1000 mg/kg bw. The animals were dosed on 2 consecutive days (0, 24 ± 1 hrs) and samples of the liver, glandular stomach, duodenum and male gonads were removed four hours after the last administration of the test item. The positive control was ethyl methanesulfonate (250 mg/kg bw). A total of 150 cells/animal/tissue (75 cells per slide) were evaluated and DNA migration during electrophoresis was determined and expressed as % tail intensity. Systemic toxicity was evaluated via clinical signs and body weight changes.

 

All animals treated with the highest dose (HD) showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection, diarrhea and hunched posture after the first and second application. Animals treated with a dose of 500 mg/kg bw showed spontaneous activity, piloerection, prone position and hunched posture after the first and second application. Rats treated with 200 mg/kg bw (LD) showed piloerection after the second administration of the test material. A reduction of body weight was noted in each male animal of the highest dose group (1000 mg/kg bw) for male rats at the end of the experiment. At the lower concentrations tested, four out of five male rats in the MD group (500 mg/kg bw) lost weight. All male rats in the lowest does group (200 mg/kg bw) gained weight by the end of the experiment. Therefore, the highest dose (1000 mg/kg bw) showed clinical signs that were strong enough to demonstrate systemic exposure without evidence of study-limiting toxicity.

 

The group mean value obtained in the main experiment for the vehicle control was 1.03 % in the case of male liver cells. The tail intensity of the LD (200 mg/kg bw; 1.18 %), MD (500 mg/kg bw; 1.08 %) and HD (1000 mg/kg bw; 0.87 %) were within the historic control limits of the testing facility (0.07– 3.82%). No significant increase compared to the vehicle control (corn oil) was noted for any dose group evaluated for male liver cells. No concentration-dependency was noted for the tail intensities in male cells of the liver. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 11.88% derived from male liver cells.

 

In glandular stomach cells, the group mean value obtained for the vehicle control was 3.26%. The tail intensity of the dose groups at 200 mg/kg bw (LD; 4.61%), 500 mg/kg bw (MD; 3.70%) and 1000 mg/kg bw (HD; 3.07%) were within the historic vehicle control range of the testing facility (1.30- 5.46%). No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male glandular stomach cells. No concentration-dependency was noted for the tail intensities in male cells of the glandular stomach. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 12.19% derived from male glandular stomach cells.

 

In the duodenum, the group mean value obtained for the negative control was 2.39%. The group mean tail intensities of the test item concentrations 200 mg/kg bw (LD; 2.15%), 500 mg/kg bw (MD; 3.95%) and 1000 mg/kg bw (HD; 2.39%) were within the range of the historic vehicle control with tail intensities 0.89- 4.06%. No significant increases compared to the vehicle control (corn oil) was noted for any dose group evaluated in male duodenum cells. No concentration-dependency was noted for the tail intensities in male cells of the duodenum. In vivo treatment with EMS revealed a significant increase in DNA damage with tail intensity values of 11.08% derived from male duodenum cells.

 

No increase of hedgehogs was detected in all organs evaluated. No concentration-related increase was noted in any of the analysed organs in male rats. As the results in all 3 tissues were clearly negative and target tissue exposure was confirmed, the male gonad slides were not evaluated.

 

Therefore, the test item did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration to rats.

 

 

 

 

Justification for classification or non-classification

Based on the available information in the dossier, the substance 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (CAS No. 72058-41-4) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.