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Additional information

Justification for grouping of substances and read-across

There are no data available on the genetic toxicity (mutagenicity in mammalian cells in-vitro) of Triacetin (CAS 102-76-1). In order to fulfil the standard information requirements set out in Annex VIII, 8.4.3, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance is conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical and toxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of Genetic toxicity

Substance

CAS 102-76-1

EC 905-964-4

Chemical name

Triacetin

Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin

Molecular weight

218.20 g/mol

134.13 - 218.20 g/mol

Genetic toxicity (mutagenicity) in bacteria in-vitro

Experimental result:
not mutagenic

Experimental result:
not mutagenic

Genetic toxicity (cytogenicity) in mammalian cells in-vitro

Experimental result:
not clastogenic

--

Genetic toxicity (mutagenicity) in mammalian cells in-vitro

RA EC 905-964-4

Experimental result:

not mutagenic

(a) The target substance is indicated in bold font.

(b) Reference (read-across) substance is indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of the structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoints for Triacetin (CAS 102-76-1). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

In vitro gene mutation in bacteria

Two reliable studies are available investigating the mutagenic potential of Triacetin (CAS 102-76-12) in bacteria.

A bacterial reverse mutation assay (Ames test) with the substance was performed similarly to OECD guideline 471 and under GLP conditions (Mulky, 1988). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with Triacetin diluted in DMSO using the plate incorporation method with one hour pre-incubation. Five concentrations in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9) were used. The concentration range was determined in a preliminary toxicity assay and was 50, 150, 500, 1500 and 5000 µg/plate for both of two consecutive experiments. The included positive and negative controls in the experiments showed the expected results and were therefore considered as valid. No cytotoxicity was observed as the test material caused no reduction of number in revertant colonies at any concentration. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any test material concentration, either with or without metabolic activation.

Furthermore, a bacterial reverse gene mutation assay (Ames test) with the substance was conducted using the pre-incubation method similarly to OECD guideline 471/472 and under GLP conditions (MHLW, 1998). Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and E. coli WP2 uvrA strain were treated with 313, 625, 1250, 2500, 5000 µg/plate Triacetin in water with and without metabolic activation. Two independent experiments were carried out in triplicate. The included positive and negative controls in the experiments showed the expected results and were therefore considered as valid. No cytotoxicity was observed up to the limit concentration. Triacetin was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA at concentrations up to 5000 ug /plate, with or without an exogenous metabolic activation system.

A further Ames test with the substance performed similar to OECD guideline 471 is available (Wallat, 1982), in which Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with Triacetin concentrations of 0, 4, 20, 100, 500 and 2500 µg/plate diluted in Tween 80 with and without rat liver S9 mix using the plate incorporation method. No mutagenicity was reported at any concentration in any of the used bacteria strains. However, since only a short abstract of the study was available, it was not further taken into account for hazard assessment of the substance.

In conclusion, the available data on genetic toxicity in the selected strains of bacteria demonstrate that the substance is not considered to be mutagenic in vitro.

In vitro cytogenicity in mammalian cells

An in vitro mammalian chromosome aberration test was performed with Triacetin (CAS 102-76-1) in Chinese hamster lung (CHL/IU) cells similarly to OECD Guideline 473 and under GLP conditions (MHLW, 1998). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (S9-mix from rats treated with phenobarbital and 5,6-benzoflavone). Test substance concentrations of 0.55, 1.1 and 2.2 mg/mL were used. Cells were treated continuously for 24 or 48 h without metabolic activation. For short term treatment, cells were treated for 6 h with and without S9 mix and cultivated with fresh media for 18 h. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In the absence of metabolic activation, no chromosomal aberrations were observed at any test substance concentration up to 48 h of continuous exposure. Structural chromosomal aberrations (including gaps) were found following short-term treatment with an exogenous metabolic activation system at the highest dose of 2.2 mg/L in the second experiment. However, at the highest test substance concentration the culture conditions were considered to be not physiological due to a decrease of the pH value which was visible in a change in medium colour. Concomitantly, an increase in cytotoxicity was found at this test concentration. These confounding factors do not allow an interpretation of the observed chromosomal damages as test-substance specific. Cifone et al. (1987) showed that the mutant frequency in L5178Y cells increased sharply for pH values below pH 6.8 and the colonies were found to be of the small-colony phenotype, indicating possible clastogenic activity. Similar pH effects are expected for other mammalian cell culture systems. In addition, in the current study, polyploidy was not induced under any conditions. Without metabolic activation no relevant cytotoxic effects of the test substance were observed.

A further study in Chinese hamster lung (CHL/IU) cells studying the clastogenic potential of Triacetin is available (Kusakabe, 2002), which, however, was not taken into account for hazard assessment due to insufficient documentation of experimental conditions and results.

In summary, the substance was not considered to induce clastogenicity in Chinese hamster lung (CHL/IU) cells in the presence and absence of metabolic activation under the conditions of this assay.

In vitro gene mutation in mammalian cells

No studies are available investigating the in vitro mutagenicity in mammalian cells of Triacetin (CAS 102-76-1). In order to fulfil the standard information requirements set out in Annex VIII, 8.4.3, in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5 read-across from the structurally related substance Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin (EC 905-964-4) is conducted.

Two in vitro Mammalian Cell Gene Mutation Assays according to OECD Guideline 476 and GLP are available for Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin (EC 905-964-4) in mouse lymphoma L5178Y cells (Adams,1996 a,b). In both assay, cells were treated for 3 h with 300, 625, 1250, 2500, 3750 and 5000 µg/mL with and without metabolic activation (Phenobarbital/β-naphtoflavone-induced rat liver S9-mix). The vehicle and positive controls in both studies showed the expected results and were in the range of historical control data. As no cytotoxicity was observed, the highest concentrations ranging from 1250 to 5000 µg/mL were evaluated for mutagenic effects in the absence and presence of S9-mix. A decrease in the pH value was observed from the physiological pH value of 7.4 in controls to a pH value of 6 at the limit concentration of 5000 µg/mL. No significant increase in the mutation frequency at the TK locus was observed after treatment with Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin, neither in the absence nor in the presence of metabolic activation. Based on these results, it was concluded that Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of both studies.

Conclusion for genetic toxicity in vitro

In summary, several bacterial reverse mutation assays are available for Triacetin (CAS 102 -76 -1), which consistently showed negative results. Furthermore, the in vitro mammalian chromosome aberration test with Triacetin was evaluated to be negative. However, at the highest test substance concentration the culture conditions were not considered to be physiological due to a decrease of the pH value which was visible in a change in medium colour. Concomitantly, an increase in chromosomal aberrations and cytotoxicity was found at this test concentration. In addition, two mouse lymphoma assays conducted with the structurally related substance Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin (EC 905-964-4) were found to be negative. Again, the pH value of the treatment culture medium was found to be reduced from pH 7.5 to pH 6.0 at the highest dose of 5000 µg/mL tested.

Ambiguous results in vitro observed at the highest test concentrations were evaluated as negative results as the effects were observed at nonphysiologic pH values. Therefore, the available data do not provide any indications for a potential genetic toxicity, and thus all available in vitro genotoxicity studies with the substance and the structurally related analogue Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin (EC 905-964-4) were judged to be negative.

 

References

Cifone, M.A. et al. (1987). Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/- cells. Mutat Res. 189(1):39-46.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies performed with the substance and the structural analogue Reaction mixture of Glycerol 1,3- di(acetate), glycerol acetate and triacetin (EC 905-964-4) were negative.

Short description of key information:
Negative results in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2, with and without metabolic activation (OECD 471, GLP).
Negative results in mammalian chromosomal aberration test with Chinese hamster lung (CHL/IU) cells, with and without metabolic activation (OECD 473, GLP).
Negative results in mammalian cell gene mutation tests using Chinese hamster ovary cells, with and without metabolic activation (OECD 476, GLP) based on read-across to the structural analogue Glycerol diacetate (technical) (EC 905-964-4).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on in vitro genetic toxicity of the 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.