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

Endpoint summary

Additional information

In vitro

Gene mutation in bacteria

Genetic toxicity in bacteria was evaluated in a study performed according to GLP and OECD guideline 471 (2004-0316-FGM). S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were treated with triallyl isocyanurate dissolved in DMSO at concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation by S9 mix from livers of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone. No increase in revertants compared to the vehicle control was observed in any of the strains at all test concentrations using the preincubation method. Toxicity (growth inhibition) was noted at concentrations of 2500 and 5000 µg/plate in all strains with and without metabolic activation.

In another Ames test performed similar to OECD guideline 471, triallyl isocyanurate was tested in S. typhimurium strains TA 97, TA 98, TA 100, TA 1535 and TA 1537 at concentrations of 33, 100, 333, 1000 and 3333 µg/plate without metabolic activation and at concentrations of 100, 333, 1000, 3333 and 6666 µg/plate with metabolic activation using the preincubation protocol (Zeiger et al., 1992). For metabolic activation cofactor supplemented post-mitochondrial fraction (S9 mix) were used, containing rat or hamster liver enzymes (S9) at a concentration of 10% or 30%, respectively. No genotoxicity was noted in any of the tested strains. Background clearing or reduction in revertants (< 60% compared to control) was observed in TA 1537 at 3333 µg/plate without S9 mix and in strain TA 98 starting at 3333 µg/plate with 10% rat and 10% hamster liver S9. In strain TA 97 a decrease in revertants below 60% compared to control plates was noted at concentrations of 3333 µg/plate and above with 30% rat and 30% hamster liver S9 as well as with 10% hamster liver S9. TA 98 revealed cytotoxicity at the highest concentration tested with 30% rat liver S9.

In conclusion, based on these test results triallyl isocyanurate is not mutagenic in bacteria.

Cytogenicity in mammalian cells

Triallyl isocyanurate was assayed in a number of chromosomal aberration tests in human peripheral lymphocytes and in different cell lines both in the presence and absence of metabolic activation.

In a GLP guideline study according to OECD 473, human peripheral lymphocytes were exposed to the test substance for 4 h with and without S9 mix at concentrations of 250, 500, 1000 and 2000 µg/mL and for 24 h without S9 mix at concentrations of 125, 250, 500 and 1000 µg/mL (2002-0084-DGT). The harvest time was 24 h after start of exposure. In the experiments without S9 mix, only at the pronounced cytotoxic concentrations of 2000 µg/mL medium (4 h exposure) or 1000 µg/mL (24 h exposure) marginal, though not significant, increases were noted in the number of aberrations. Since it is known, that high cytotoxicity causes artefacts in form of aberrations in in vitro chromosomal tests, these marginal increases are considered as artefacts and not test substance-related. No increase in cells with chromosomal aberrations was observed in the test with S9 mix, where cytotoxicity was noted at the highest dose tested.

 

In another chromosome aberration test performed according to OECD guideline 473, CHL cells were incubated with triallyl isocyanurate for short- (6 h with and without S9 mix) and longterm exposure (24 h without S9 mix) and harvested after 24 h (2004-0318-FGM). After short-term exposure without S9 mix at concentrations of 630, 1300 and 2500 µg/mL a significant increase of cells with structural aberrations was observed at 1300 and 2500 µg/mL. However, this incidence was considered not to be biologically significant. Precipitation was observed at a concentration of 1000 µg/mL and above and cytotoxicity was noted starting at concentrations of 2000 µg/mL. No increase in cells with aberrations were found for long-term exposure without S9 mix at concentrations of 250, 500 and 1000 µg/mL and in the experiment performed with metabolic activation at concentrations of 630, 1300 and 2500 µg/mL. The negative result in the experiment with metabolic activation was confirmed by an additional test performed with concentrations of 1500, 2000 and 2500 µg/mL.

 

Triallyl isocyanurate were also tested in Chinese Hamster ovary (CHO) cells for clastogenic activity (Loveday et al., 1990). Negative results in the incidence of chromosome aberrations were obtained after treatment of CHO cells for 2 h with 291, 970 and 2910 µg triallyl isocyanurate/mL (harvest time 12 h) with metabolic activation and for a 8 h treatment (harvest time 10.5 h) at concentrations of 146, 485 and 1460 µg triallyl isocyanurate/mL without S9 mix.

In a further chromosome aberration assay similar to OECD 473 in Chinese Hamster Lung (CHL) cells, inconclusive results were reported after long-term exposure (24 h and 48 h) without metabolic activation in concentrations up to 1000 µg/mL (Sofuni et al., 1990). After an exposure period of 6 h (harvest time: 24 h) with metabolic activation a positive result was obtained for CHL cells. The frequency of aberrant cells was over 30% in test concentrations of 250, 500 and 1000 µg triallyl isocyanurate/mL. Sofuni et al. (1990) compared the results in CHO cells and CHL cells (Loveday et al., 1990) and and concluded, that the positive results determined in CHO cells were presumably due to an experimental set-up of prolonged exposure times. The authors reported on an experiment in CHO cells leading to a positive result when the extended exposure procedure (6 h treatment, 24 h harvest) was followed. However, in these studies by Sofuni et al. (1990) no data on cytotoxicity were presented. An interlaboratory study comparing the two cell lines using different assay protocols for CHO and CHL cells with main differences in the treatment schedule (treatment length and sampling time) also pointed to that problem (Galloway et al., 1997). Additionally, the positive or weak positive results found with or without metabolic activation, respectively, were only obtained at dose levels producing visible precipitates. One laboratory obtained a clear positive result without S9 mix, but only in a cytotoxic concentration range of 1750 to 2250 µg/mL, leading to a false positive assay outcome. The strong positive results starting at a concentration of 250 µg/mL in CHO cells with metabolic activation (Sofuni et al., 1990) could not be reproduced in the interlaboratory study even in the same laboratory.

 

In conclusion, triallyl isocyanurate is considered to have no clastogenic properties based on the negative result obtained in the key studies performed according to current OECD guideline 473 (2002-0086-DGM, 2004-0318-FGM). The reported positive or equivocal results in chromosome aberrations assays may most likely be assigned to confounding factors like precipitation and cytotoxicity. In addition, triallyl isocyanurate did not induce sister chromatid exchanges in CHO cells exposed to triallyl isocyanurate at concentrations up to 605 µg/mL without metabolic activation and at concentrations up to 291 µg/mL with metabolic activation (Loveday et al., 1990).

 

Mutagenicity in mammalian cells

Triallyl isocyanurate was tested in an in vitro mammalian cell gene mutation assay under GLP according to OECD guideline 476 (2002-0086-DGM). In two independent experiments, cultured Chinese hamster fibroblasts (V79 cells, genetic marker HPRT) were treated with triallyl isocyanurate at concentrations of 56.25, 312.5, 625, 1250 and 2500 µg/mL with and without metabolic activation by Aroclor 1254-induced rat liver S9 mix. The exposure duration with triallyl isocyanurate was 24 h in experiments without S9 mix and 4 h in experiments with S9 mix. The mutation frequency of the cultures treated with triallyl isocyanurate in the presence and in the absence were within the normal range of the negative controls, whereas the positive controls caused a pronounced increase in the mutation frequency. Cytotoxicity in form of decreased plating efficiency was noted at all concentrations in the first experiment with and without metabolic activation. In the second experiment cytotoxicity was observed at 1250 and 2500 µg triallyl isocyanurate/mL or from 312.5 µg/mL onwards with and without metabolic activation, respectively. 

Thus, under the present test conditions triallyl isocyanurate was negative in the HPRT-V79 mammalian cell mutagenicity test.

In vivo

The effect of triallyl isocyanurate on chromosome structure in bone marrow cells was investigated in mice following oral administration according to OECD guideline 474 under GLP (2000-0412-DGM). Triallyl isocyanurate at concentrations of 500, 1000 and 1500 mg/kg bw was administered via gavage to 10 (5 per sex) NMRI mice each. The animals were sacrificed 24 h after treatment and samples of the bone marrow were taken for analysis. An additional test group of 5 male and 5 female mice received 1500 mg triallyl isocyanurate/kg bw and was sacrificed 48 h after treatment, before bone marrow cells were examined. Application of 1500 mg/kg bw of the test substance resulted in toxic symptoms like sedation and excitation with tremors in male and female animals 24 h p.a. In addition, the body weight was decreased up to 15.5% in mice treated with the highest concentration. 4 males and 2 females were found dead 24 h or 48 h after treatment with 1500 mg triallyl isocyanurate kg/bw, respectively.

The frequency of detected micronuclei was in the range of the historical negative control in all doses and at any preparation time after application of the test substance. No indication of bone marrow toxicity, as determined by the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) was noted in any group treated with triallyl isocyanurate.

It is concluded, that under the conditions of the test, there was no evidence of an aneugenic or clastogenic effect of triallyl isocyanurate.

Taking all data together, triallyl isocyanurate is considered not to cause genetic damage.

Short description of key information:
In vitro:
- Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA: negative with and without metabolic activation
- Cytogenicity in mammalian cells (OECD 473, Chromosome aberration assays): negative with and without metabolic activation in human lymphocytes and CHL cells
- Gene mutation in mammalian cells (OECD 476, HPRT): negative with and without metabolic activation

In vivo:
-Chromosome aberration (micronucleus assay, OECD 474): negative (3 concentrations: 500, 1000 and 1500 mg/kg bw orally administered to mice; post exposure period: 24 and 48 h)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance 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.