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

Endpoint summary

Additional information

In vitro

Gene mutation in bacteria

The genetic toxicity of TMT in bacteria was evaluated in study performed similar to OECD Guideline 471 (81-0088-DKM). S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2 uvr A (pKM 101) were treated with TMT 15 dissolved in water at concentrations of 378, 1130, 3780, 11300, 37800 and 113300 µg/plate (corresponding to 57, 170, 567, 1695, 5670 and 16995 µg anhydrous TMT/plate) with and without metabolic activation by S9 mix from livers of male rats treated with Aroclor 1254. No increase in revertants compared to the vehicle control was observed in any of the strains at all test concentrations using the plate incubation method. Toxicity (thin background lawn) was noted at the highest test concentration. Based on the test results TMT is not mutagenic in bacteria.

 

Gene mutation in mammalian cells

TMT 55 (analytical purity: 57%) was tested in an in vitro mammalian cell gene mutation assay under GLP according to OECD Guideline 476 (2012-0084-DGM). In two independent experiments L5178Y mouse lymphoma cells (genetic marker: thymidine kinase) were treated with 156.3, 312.5, 625, 1250 and 2500 µg TMT 55/mL (equivalent to 89.1, 183.3, 356.3, 712.5 and 1425 µg anhydrous TMT/mL) or 312.5, 625, 1250, 2500 and 5000 µg TMT 55/mL (equivalent to 183.3, 356.3, 712.5, 1425 and 2850 µg anhydrous TMT/mL) without or with metabolic activation by Aroclor 1254-induced rat liver S9 mix, respectively. The exposure duration was 3 and 24 h in experiments without S9 mix and 3 h in experiments with S9 mix. The mutation frequency of the cultures treated with TMT 55 in the presence and in the absence of S9 mix was within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells, whereas the positive controls caused a pronounced increase in the mutation frequency. Cytotoxicity in form of decreased survival was noted at the top concentration of 2500 µg TMT 55/mL (equivalent to 1425 µg/mL anhydrous TMT) in the absence of metabolic activation or at the top concentration of 5000 µg TMT 55/mL (equivalent to 2850 µg anhydrous TMT/mL) in the presence of metabolic activation. No change was noted in the ratio of small to large mutant colonies.

Thus, based on the test result TMT was not mutagenic in the mouse lymphoma forward mutation assay and did not exhibit clastogenic potential at the concentration-range investigated.

 

In vivo

The effect of TMT on chromosome structure in bone marrow cells was investigated in mice following oral administration similar to OECD Guideline 474 under GLP (89-0017-DGM). TMT 15 (analytical purity: 17% wt.) was administered once via gavage to 21 male and female mice at a concentration of 2870 mg/kg bw in water corresponding to 488 mg anhydrous TMT/kg bw. The applied test concentration was determined in an orientating study of the acute toxicity and was considered to be the maximum tolerated dose. Control animals received a single dose of isotonic saline solution (0.9%). 24, 48 and 72 h after treatment mice were sacrificed and bone marrow cells were prepared for micronuclei analyses. Since 4 of 7 females provided for the 72 h test point died before sacrifice, the test was repeated for this sampling time with 15 females. 4 females of this repetition group died and the first seven animals showing clinical symptoms of toxicity were sacrificed and used for bone marrow preparation.

Clinical symptoms were registered in many of the test animals and consisted of hypokinesia, tremor, clonic convulsions, tonic convulsions of hind legs, stilted gait, decrease of muscle tone, loss of righting reflex, loss of pinna, pain and corneal reflex, ptosis, lacrimation, piloerection, sunken sides and reduced body surface temperature. Necropsy of the dead animals revealed reddening of the glandular stomach or the small intestine and filling of the small intestine with yellowish or black-reddish fluid.

Five animals per sex and group were used for evaluation. No significant test substance related increase in micronucleated polychromatic erythrocytes (PCEs) was noted in either male or female animals, respectively males and females combined, when compared with the negative control group. A reduction in the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) was observed in both single male and female test group animals at all sampling times indicating toxicity of the test material TMT. The positive control group animals administered cyclophosphamide revealed a significant increase in the number of micronucleated polychromatic erythrocytes.

Thus, under the conditions of the erythrocyte micronucleus test TMT did not induce chromosome mutations in mice.

 

In conclusion, TMT is considered not to cause genetic damage, since all in vitro and in vivo genetic toxicity studies revealed negative results.

 

Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro and in vivo genetic toxicity studies were negative.

Short description of key information:
In vitro:
- Gene mutation in bacteria (similar to OECD 471, Ames test): S. typhimurium TA 100, TA 1535, TA 98, TA 1537, TA 1538 and E. coli WP2 uvrA pKM 101: negative with and without metabolic activation
- Gene mutation in mammalian cells (OECD 476, TK): negative with and without metabolic activation
In vivo:
-Chromosome aberration (similar to OECD 474, micronucleus assay): negative (maximum tolerable dose: 488 mg anhydrous TMT/kg bw orally administered to mice; post exposure period: 24, 48 and 72 h)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of TMT do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.