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

Endpoint summary

Additional information

Justification for grouping of substances and read-across

There are no data available on the genetic toxicity of "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin".

In order to fulfil the standard information requirements set out in Annex VIII, 8.4, 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

CAS #	EC 905-964-4	CAS 102-76-1
Chemical name	"Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin"	Triacetin
Molecular weight	134.13 - 218.20 g/mol	218.20 g/mol
Genetic toxicity (mutagenicity) in bacteria in-vitro	Negative + RA CAS: 102-76-1	Experimental result: Negative
Genetic toxicity (cytogenicity) in mammalian cells in-vitro	RA CAS: 102-76-1	Experimental result: Negative
Genetic toxicity (mutagenicity) in mammalian cells in-vitro	Experimental result: Negative	--

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

In vitro gene mutation study in bacteria

There are 4 studies available investigating the in vitro mutagenicity in bacteria of "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin".

Additionally, a read-across, in accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5, from the structurally related analogue substance Triacetin (CAS 102-76-1) was conducted. There are 4 bacterial reverse mutation assays available conducted with "Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin".

The first bacterial reverse mutation assay was performed according to OECD guideline 471 and under GLP conditions (Kitching, 1997a). Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 were exposed to 50-5000 µg/plate with and without metabolic activation in the first and second experiment and with 500-7500 µg/plate with metabolic activation (10 and 30% S9-mix) only with TA 1535 in the third experiment. The first two experiments were done as plate incorporation assays for 72 h and the third experiment included an additional pre-incubation period of one hour. The included positive and negative controls in the experiments showed the expected results and were therefore considered as valid. The test material did not induce cytotoxicity and was therefore tested up to the limit concentration of 5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains without metabolic activation and for strains TA98, TA100 and TA1537 with metabolic activation with all concentrations tested. However, metabolic activation led to a roughly two-fold increase in revertant colonies for strain TA1535 starting at 2500-5000 µg/plate. This result was confirmed in the independent repeated experiment and with pre-incubation and different concentrations of S9 mix.

The setup of the second Ames test was identical to the first experiment (Kitching, 1997b). Again no increases in the frequency of revertant colonies could be observed for all strains without metabolic activation and for strains TA98, TA100 and TA1537 with metabolic activation. However, strain TA1535 incubated with "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin" with metabolic activation showed a roughly two-fold increase in revertant colonies at the highest concentrations applied.

A third Ames test likewise performed according to OECD guideline 471 and under GLP (Kitching, 1997c) is available. No cytotoxicity could be observed and testing was again carried out up to the limit concentration. No significant increases in the frequency of revertant colonies were recorded for TA100, TA98 and TA1537 with and without metabolic activation with all concentrations tested. However, strain TA1535 incubated with "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin" with and without metabolic activation showed a roughly two-fold increase in revertant colonies at the highest

concentration of 5000 µg/plate.

A fourth Ames test is available conducted according to OECD guideline 471 and under GLP conditions (Richold, 1983). In this experiment 6 different batches of "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin"

(HR1 – HR6) were tested for their mutagenic activity on the TA1535 strain. The first and second experiments were carried out with the following concentrations: 625, 1250, 2500, 5000 and 10000 µg/plate with and without metabolic activation. The third experiment included concentrations of 5000, 7500, 10000, 15000 µg/plate with and without metabolic activation. With 2/6 batches of "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin" an increase in revertant colonies for TA1535 with metabolic activation was observed. HR5 showed a slight concentration-dependent increase in the number of revertant colonies from 5000 µg/plate. HR3 showed only a slight increase at the highest concentration tested, but no concentration-dependency. Repeat experiments with HR3 confirmed this, as again slight increases were only observed at concentrations of 10000 µg/plate and higher. Incubations without metabolic activation did not show any differences. Since only 2 out of 6 different batches of test substance showed a positive result, "Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin" was not considered to have mutagenic potential in bacteria.

In addition, two bacterial reverse mutation assays are available for Triacetin (CAS 102-76-1) and considered for read across in a weight of evidence approach.

The first bacterial reverse mutation assay (AMES) 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.

A second bacterial reverse gene mutation assay (AMES) was conducted using the pre-incubation method similarly to OECD guideline 471/472 and under GLP conditions (MHW, Japan: 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.

In summary, slight increases in the revertant colony number of strain TA1535 were observed at the recommended highest test concentration of 5000 µg/plate and higher especially with metabolic activation in several Ames tests. However, no increase in the number of revertant colonies in the strain TA100 were identified which is sensitive for base-pair substitution type mutations, as well. In further genotoxicity assays with "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin" a decrease of the pH value under the

physiological pH value at concentrations of 5000 µg/mL has been observed (Adams, 1996a, b). Cifone et al. (1987) showed that conditions of non-physiological pH may affect the results of in vitro genetic tests by mechanisms unrelated to the test substance. Especially under test conditions with S9-mix and weak acid conditions the mutant frequency was increased. In the current experiments, an increased number of revertant colonies were especially observed using S9-mix and in concentrations were a lowered pH-value may be relevant. In addition, a genotoxic effect was not confirmed in experiments with the surrogate substance Triacetin. Considering a maximal two-fold increase in the number of revertant colonies in the strain TA1535 alone, the absence of a mutagenic property in all other strains, the absence of a genotoxicity with Triacetin, and the likely influence of a lowered pH-value on the mutant frequency, the effects in the strain TA1535 at high test substance concentrations (≥ 5000 µg/plate) are not evaluated as a positive result.

In vitro cytogenicity in mammalian cells

No studies are available investigating the in vitro cytogenicity in mammalian cells of "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin".

In order to fulfil the standard information requirements set out in Annex VIII, 8.4.2, in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5 read-across from the structurally related analogue substance Triacetin (CAS 102-76-1) is conducted.

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 Japan, 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.

 

In vitro gene mutation in mammalian cells

Two in vitro Mammalian Cell Gene Mutation Assays according to OECD Guideline 476 and GLP were performed with

"Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin" in mouse lymphoma L5178Y cells

(Adams,1996a,b). The 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 the study showed the expected resultsand were in the range of historical control data.No toxicity was observed and concentrations from 1250 to 5000 µg/mLwere evaluated 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" either in the absence or in the presence of S9-mix. It was concluded that "Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin is not mutagenic in the mouse lymphoma L5178Y tests system under the experimental conditions described.

Conclusion for genetic toxicity in vitro

In summary, several bacterial reverse mutation assays are available for "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin" and the structurally related analogue substance Triacetin (CAS 102 -76 -1).

The results of the bacterial reverse mutation assays were evaluated to be negative. However, in strain TA 1535 slightly increased mutant frequencies were observed at the highest recommended test concentrations (and above) and were not evaluated as positive results, as they were likely to be the result of changes in pH . An in vitro mammalian chromosome aberration test was performed with the surrogate substance Triacetin. The results of this assay were evaluated to be negative. 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 chromosomal aberrations and cytotoxicity was found at this test concentration. In addition, two mouse lymphoma assays conducted with "Reaction mixture of glycerol-1,3 -di(acetate), glycerol acetate and triacetin" 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 of

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

 

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from structural analogues. No study was selected, since all available in vitro genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Negative results in Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, with and without metabolic activation (OECD 471, GLP).
Negative results in mammalian chromosomal aberration test with Chinese hamster lung cells (OECD 473, GLP).
Negative results in mammalian cell gene mutation tests using Chinese hamster ovary cells, with and without metabolic activation (OECD 476, GLP).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of "Reaction mixture of glycerol-1,3-di(acetate), glycerol acetate and triacetin"

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