1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471): - negative with and without S9 in all tested strains

CA in vitro (OECD 473): - negative in CHL/IU cells after continous treatment with S9, ambigous after short-term treatment with and without S9 and short-term treatment with S9 and pH adjustment

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

- Factors modulating mutagenicity in microbial tests, Matsushima T, Sugimura T, Nagao M, Yahagi T, Shirai A, Sawamura M, in Short-term Test Systems for Detecting Carcinogens, Norpoth KH, Garner RC eds., Springer, Berlin-Heidelberg-New York (1980) pp.273-285
- Revised methods for the Salmonella mutagenicity test, Maron DM and Ames BN, Mutation Research 113: 173-215 (1983)
- Mutation testing using Trp reversion in Eschrichia coli, Green MHL, in Handbook of Mutagenicity Test Procedures, Kilbey BJ, Legator M, Nichols W, Ramel C eds., Elsevier, Amsterdam (1984) pp. 161-187
- Chemical Substance Research Section, Industrial Safety and Health Department, Labor Standards Bureau, Ministry of Labor Supervision: Mutagenicity study of existing chemical substances data collection based on Industrial Safety and Health Law Toxicity Investigation System, Japan Chemical Industry Ecology-Toxicology & Information Center, Tokyo, p.177 (1986)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: histidin requirement
E. coli: tryptophan requirement

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: UV sensitivity, film mutation (rfa), ampicillin resistance factor pKM101 (plasmid)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat)

Test concentrations with justification for top dose:

313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (5-nitro-2-furyl)acrylamide (AF-2), sodium azide (SA), 9-aminoacridine (9 AA), 2-aminoanthracene (2 AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st and 2nd main experiment: pre-incubation method

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours (37°C)

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY: growth inhibition

Evaluation criteria:

When dosage dependence or repeatability in the absence and presence of S9 mix of more than 1 kind of assay bacteria, among the 5 kinds of assay bacteria used, was identified to be increasing, and the average value of the number of mutant colonies on the plate that includes the test material increased more than 2-fold of negative control value, it was determined (positive) as possessing gene mutagenesis in the study test system.

Statistics:

Statistic methodology was not used to determine the results.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Increase of more than 2-fold of the negative control value was not identified in any of the assay bacteria used with and without S9 mix.
Deposits originated from the test material were not identified in any of the dosages used either in the absence or the presence of S9 mix.

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Under conditions tested, the test item did not induce gene mutations by base pair changes or frame-shifts in the genome of the tester strains used.
Thus, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's MEM media, supplemented with 10% calf serum, L-glutamine and NaHCO3
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9 mix: 0.36, 0.53, 0.8 mg/mL
24h treatment without S9 mix: 0.14, 0.21, 0.31 mg/mL
6h treatment with S9 mix (confirmation test): 0.36, 0.53, 0.8 mg/mL
6h treatment with S9 mix (confirmation test, pH adjusted): 0.53, 0.8, 1.2 mg/mL

Vehicle / solvent:

Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide, 10 µg/mL, + S9 mix; mitomycin C, 0.1 µg/mL, - S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24 hours
- Expression time (cells in growth medium): 6h treatment: 24 hours; 24h treatment: 24 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.1 µg/mL final concentration)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; mitotic index of 500 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


OTHER: adjustment of pH in one short-term treatment experiment with S9 mix by addition of NaOH (1M) until the color of the treatment solution turned into the same level of color as MEM media with 10% calf serum

Statistics:

Fisher's exact probability test (P<0.01, unilateral) and Cochran-Armitage trend test (P<0.01, unilateral)

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

short-term treatment, only high concentration

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

54.5 % cell growth

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

short-term treatment, only high concentration

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

24h continous treatment

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Remarks:

pH adjusted

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

short-term treatment; only high concentration

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
When adding the test substance to the culture solution in concentrations up to 1.2 mg/mL, the media color changed to orange at the beginning of the treatment. At a concentration of 1.2 mg/mL and above the media color changed to yellow and pH decreased to 5.79-6.04. However, after 6 h of treatment, the yellowing of culture solution was recognized only at a test concentration of 1.8 mg/mL. At 0.80 mg/mL, the highest concentration for which chromosome analysis was performed and which yielded in a positive response with and without S9 mix, no change in culture solution color was noted at the end of the treatment time (6 h). Therefore, the possibility of structural abnormalities of chromosomes being induced due to acidification of culture solution was considered to be low.
However, a pH adjustment treatment group was included and a confirmation test (treatment time: 6 h) was performed in the presence of S9 mix to examine the influence of acidification of the treatment solution. In both experiments a statistically significant increase in chromosome aberrations was noted at each high dose concentration, at 0.8 mg/mL in the non-adjusted pH group and at 1.2 mg/mL in the adjusted pH group, respectively. The pH value of the test solutions, where structural abnormalities of chromosomes were induced, was 6.73 for the non-adjusted group and 7.27 for the adjusted group at the beginning of the treatment. Since the color change of the culture solution was no longer identified at the end of the treatment, chromosome aberrations induced in these short term treatment groups in the presence of S9 mix were test substance related and not due to acidification of the culture solution.

- Precipitation:
Precipitation was observed at the beginning of treatment in the medium by the naked eye for the following concentrations:
6h with and without S9 mix: 1.2 and 1.8 mg/mL
24h without S9 mix: 0.47 and 0.70 mg/mL
6 h with S9 mix (confirmation test): 0.53, 0.80, 1.2 and 1.8 mg/mL
6h with S9 mix (confirmation test, pH adjusted): 0.53, 0.80, 1.2 and 1.8 mg/mL


RANGE-FINDING/SCREENING STUDIES:
To determine the treatment concentrations of the test material used for the chromosomal aberration test, influence on the cell growth of the test material was examined. The test substance inhibited the growth of CHL cells in a concentration-dependent manner under all treatment conditions. Maximum treatment concentration in the short-term treatment (6 h) experiments was established to be 1.8 mg/mL with and without S9 mix and 0.70 mg/mL in the continuous treatment (24 h) without S9 mix. 5 concentrations separated by a 1.5 ratio were used for the chromosomal aberration test:
6h treatment: 0.36, 0.53, 0.80, 1.2 and 1.8 mg/mL
24h treatment: 0.14, 0.21, 0.31, 0.47 and 0.70 mg/mL
Based on the result of cell proliferation rate and mitotic index, chromosome analysis was made for 3 test concentrations.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cell growth < 50% was observed in the test concentrations not analyzed for chromosome aberrations:
6h treatment with and without S9 mix (including confirmation test, pH non-adjusted): 1.2 and 1.8mg/mL
24h treatment without S9 mix: 0.47 and 0.7 mg/mL
6h treatment with S9 mix (confirmation test, pH adjusted): 1.8 mg/mL

In the confirmation tests (6 h treatment, with S9-mix) regardless of pH adjustment or non-adjustment, increases of polyploidy cells (occurrence ratio: 1.5 (highest dose) – 2.5% (mid dose)) was identified and also among them endoreplication of cells was determined.

Conclusions:

Interpretation of results:
ambigous

Under the condition of this test, the test substance is probably considered to induce chromosome aberrations in Chinese hamster cells.

Additional information

Ames Test (OECD 471):

With the bacterial mutagenicity test (Ames Test) according to OECD 471, a reproducible mutagenic activity of the test compound to any of the tester strains TA 98, TA 100, TA 1535, TA 1537 or E.coli WP2 was not observed with and without metabolic activation. Thus, Taicros TMT is considered non-mutagenic in this bacterial reverse mutation assay.

CA in vitro (OECD 473):

In the in vitro chromosomal aberration test using CHL/IU cells no dose-response relationship concerning induction of chromosomal aberration could be seen. Only the highest tested concentration without a high degree of cytoxicity showed a positive finding in the number of cells with aberrations. However, the positive findings were reported at concentrations where pH effects might have influenced the outcome, as precipitation (6h with S9, 6h with S9 and pH adjustment) and/or color changes of medium were present (at all conditions with positive findings). Furthermore, no historical control data were available to verify the positive results in this cell line after treatment with test substance in the presence of precipitation and color changes of media. Therefore, the positive findings were interpreted as ambiguous.

In vivo mammalian erythrocyte micronucleus test (OECD 474):

As published by the Matsumoto et al. (2015) in an assement of 1,3,5-triazine-2,4,6(1H,3H,5H)-trithione, the in vivo micronucleus study (OECD TG 474) was negative up to the maximum tolerated dose (100 0 mg/kg bw/day for 2 days) in mice.

Justification for classification or non-classification

Based on the available data on genotoxicity, the test substance Taicros TMT does not require classification for mutagenicity accroding to CLP Regulation (EC) No. 1272/2008.