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Administrative data

Key value for chemical safety assessment

Additional information

Justification for grouping of substances and read-across

There are no data available on the genetic toxicity of Glycerides, mixed C8-C10 and succinyl (CAS 91744-56-8). In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances 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 physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, Glycerides, mixed decanoyl and octanoyl (73398-61-5), Glycerides, C8-21 and C8-21-unsatd. mono- and di-, acetates (CAS 97593-30-1), glycerol (CAS 56-81-5), heptanoic acid (111-14-8), fumaric acid (CAS 110-17-8), succinic acid (CAS 110-15-6), sodium hydrogen succinate (CAS 2922-54-5), disodium succinate (CAS 150-90-3) and glutaric acid (CAS 110-94-1) are selected as reference substances for assessment of the in vitro genetic toxicity of Glycerides, mixed C8-C10 and succinyl.

The read-across is based on structural similarity between the source and target substances, as the substance Glycerides, mixed C8-C10 and succinyl is composed of esters of glycerol with succinic acid (a short-chain dicarboxylic acid) and fatty acids with carbon chain lengths of C8 and C10, and the ester bond is anticipated to undergo enzymatic hydrolysis. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Overview of genetic toxicity in vitro

CAS#

Chemical name

Molecular weight (range)

In vitro gene mutation in bacteria

In vitro cytogenicity

In vitro gene mutation in mammalian cells

91744-56-8 (a)

Glycerides, mixed C8-C10 and succinyl

470.68-1211.64

WoE:

RA: CAS 73398-61-5

RA: CAS 97593-30-1

RA: CAS 110-15-6

RA: CAS 2922-54-5

RA: CAS 150-90-3

WoE:

RA: CAS 56-81-5

RA: CAS 111-14-8

RA: CAS 110-15-6

RA: CAS 150-90-3

 

WoE:

RA: CAS 56-81-5

RA: CAS 111-14-8

RA: CAS 110-17-8

RA: CAS 110-94-1

73398-61-5 (b)

Glycerides, mixed decanoyl and octanoyl

470.69-554.85

Experimental result:

negative

--

--

97593-30-1

Glycerides, C8-21 and C8-21-unsatd. mono- and di-, acetates

358.47-498.74

Experimental result:

negative

--

--

56-81-5

Glycerol

92.10

--

Experimental result:

negative

Experimental result:

negative

111-14-8

Heptanoic acid

130.19

--

Experimental result:

negative

Experimental result:

negative

110-17-8

Fumaric acid

116.07

--

--

Experimental result:

negative

110-15-6

Succinic acid

118.09

Experimental result:

negative

Experimental result:

negative

--

2922-54-5

Sodium hydrogen succinate

141.08

Experimental result:

negative

--

--

150-90-3

Disodium succinate

162.05

Experimental result:

negative

Experimental result:

negative

--

110-94-1

Glutaric acid

132.12

--

--

Experimental result:

negative

(a) The substance subject to the REACh Phase-in registration deadline of 31 May 2013 is indicated in bold font. Only for this substance a full set of experimental results and/or read-across is given.

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

 

Gene mutation in bacteria

CAS No. 73398-61-5

In a GLP-conform study performed according to EU method B.13/14, the mutagenicity of Glycerides, mixed decanoyl and octanoyl in bacteria was investigated (Schöberl, 1994). S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested in two independent experiments at concentrations from 8 to 5000 µg/plate both in the presence or absence of metabolic activation (phenobarbiturate-induced rat liver microsomes). No cytotoxicity was observed in any of the tester strains up 5000 µg/plate, but precipitation of the test substance was observed at this concentration both in the presence of absence of the metabolic activation system. Exposure to the test substance in the presence or absence of metabolic activation did not increase the mean number of revertants in any strain of bacteria tested. The positive and negative controls included in the assay yielded the expected results. Under the conditions of this bacterial mutation assay, the test substance was found to be non-mutagenic in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

CAS No. 97593-30-1

A bacterial gene mutation assay was performed with Glycerides, C8-21 and C8-21-unsatd. mono- and di-, acetates in compliance with OECD guideline 471 and GLP (Herbold, 2007). In two independent experiments, S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to concentrations of 50-5000 µg/plate in the presence or absence of metabolic activation system (S9-mix). In the first experiment cytotoxic effects were evident in the presence of S9-mix at ≥ 1581 (TA 100 and TA 1537) and at 5000 µg/plate (TA 1535). In the second experiment, cytotoxicity was observed in all strains at ≥ 1581 µg/plate (with S9-mix), except for TA 1537 which showed cytotoxicity already at ≥ 500 µg/plate in the presence of S9-mix and at 5000 µg/plate in the absence of metabolic activation. Precipitation of the test substance was noted at ≥ 1581 in the first experiment and at 5000 µg/plate in the second experiment, respectively. No increase in the mean number of revertants was observed in any tester strain in the two independent experiments independent of the metabolic activation. The negative (solvent) controls were within the historical range and the positive controls showed the expected results. Based on the results of both experiments, the test substance did not induce mutagenic effects in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

CAS No. 110-15-6

To assess the mutagenic effects of succinic acid in bacteria, the S. typhimurium strains TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98 were investigated similarly to OECD Guideline 471 (Ishidate et al., 1984). In the presence and absence of metabolic activation, all tester strains were exposed to concentrations up to 5000 µg/plate. No increase in the mean number of revertant colonies per plate and no cytotoxicity were observed in any strain up to the highest concentration tested. Based on the study results, the test substance was considered to have no mutagenic activity in the selected S. typhimurium strains with and without metabolic activation.

CAS No. 2922-54-5

The potential mutagenicity of sodium hydrogen succinate was investigated in a bacterial mutation assay with S. typhimurium TA 97 and TA 102 (Fujita et al., 1994). In the presence and absence of metabolic activation, both tester strains were exposed to concentrations ranging from 100 to 5000 µg/plate. No cytotoxicity was observed up to the highest concentration tested. In both tester strains, the mean number of revertants per plate was not increased at any concentration tested. The solvent and positive control values included revealed the expected results. Based on the results of this study, the test substance did not induce mutagenicity in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

CAS No. 150-90-3

A bacterial gene mutation assay with disodium succinate was conducted in compliance with OECD guideline 471 and GLP (Ozaka, 2002). Two independent experiments were conducted both in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were treated with concentrations ranging from 0.305 to 5000 µg/plate. In the second experiment, concentrations ranging from 156 to 5000 µg/plate were tested. In both experiments, no cytotoxicity was observed up to the highest concentration tested. The mean number of revertants was not increased at any concentration tested. The positive and solvent controls included revealed the expected results. Under the experimental conditions reported, the test substance did not induce mutations in the selected strains of S. typhimurium and E. coli with or without metabolic activation.

 

Cytogenicity in vitro

CAS No. 56-81-5

The clastogenic activity of glycerol was investigated in an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473 (Doolittle, 1988). Chinese hamster Ovary (CHO) cells were treated with the test substance at 100, 200, 400, 600, 800 and 1000 µg/mL for a period of 10 and 14 h (without metabolic activation) or 2 h (with metabolic activation) followed by a 10 and 14 h recovery period, respectively. After treatment, no cytotoxicity was observed at any test concentration compared to controls. A statistically significant increase in the frequency of cells with chromosomal aberrations was observed in cells treated with 200 µg/mL for2 h in the presence of S9, followed by a 10 h recovery period. Due to the lack of any concentration-response relationship, this isolated result was considered to be spurious and not biologically significant. No increase in the number of metaphases with aberrations was observed at any other preparation time and concentration. The positive controls showed the expected increase in the number of cells with chromosomal aberrations. Under the conditions of this assay, the test substance was not clastogenic in Chinese hamster Ovary (CHO) cells in the presence or absence of metabolic activation.

CAS No. 111-14-8

The clastogenic potential of heptanoic acid was assessed in an in vitro mammalian chromosome aberration test in cultured human lymphocytes performed according to OECD guideline 473 and under GLP conditions (Sarlang, 2010). The study was performed in two independent experiments, both in the presence and in the absence of metabolic activation. In the first experiment, cells were treated at concentrations ranging from 0.16 to 10 mM for a period of 3 h (short-term-treatment). No significant reduction in mitotic index was observed at any concentration in the absence of S9 mix, whereas in the presence of S9 mix a moderate cytotoxicity was observed at 10mM, as shown by a 52% decrease in mitotic index compared to controls. Based on these results, concentrations of 2.5, 5 and 10 mM were selected for chromosome analysis in the presence and absence of metabolic activation. In the second experiment, short-term treatment for 3 h with S9 mix and continuous treatment for 20 and 44 h without S9 mix was performed with the test substance at concentrations ranging from 0.63 to 10 mM. Short-term treatment for 3 h with S9 mix resulted in a moderate decrease in mitotic index (53%) at 10 mM. Therefore, concentrations of 5, 7.5 and 10 mM were chosen for chromosome analysis. After the 20-h treatment in the absence of S9 mix, a 50% reduction in mitotic index compared to control was observed at 2.5 µM, which was selected as the highest concentration for chromosome analysis, followed by concentrations of 1.25 and 0.63 µM. Based on the cytotoxicity observed after continuous treatment for 44 h, 2.5 mM and 10 mM were chosen as only concentrations for chromosome analysis. No increase in the number of cells with chromosomal aberrations was observed compared to controls at any test concentration and treatment duration. The positive controls included during short-term and continuous exposure showed the expected results. Under the conditions of this experiment, the test substance was not clastogenic in cultured human lymphocytes in the presence and absence of metabolic activation.

CAS No. 110-15-6

The clastogenic activity of succinic acid was investigated in an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473 (Ishidate et al., 1984). Based on a preliminary cytotoxicity experiment, Chinese hamster lung (CHL) fibroblasts were treated with 3 concentrations of the test substance up to 1000 µg/mL for 24 and 48 h in the presence and absence of metabolic activation, respectively. No increase in the number of cells with chromosomal aberrations and polyploidy was observed at any test concentration compared to controls. The negative and solvent controls showed the expected results. Under the conditions of this assay, the test substance was not clastogenic in Chinese hamster lung (CHL) fibroblasts in the presence and absence of metabolic activation.

CAS No. 150-90-3

Disodium succinate was assessed in an in vitro mammalian chromosome aberration test in Chinese hamster lung cells (CHL/IU) according to OECD guideline 473 and under GLP conditions (Ozaki, 2002). Concentrations of 313, 625, 1250, 2500 and 5000 µg/mL µg/mL were selected for chromosome analysis after short-term exposure (6 h) in the presence and absence of S9 mix and continuous exposure (24 and 48 h) in the absence of S9 mix, respectively. No increase in the incidence of cells with chromosomal aberrations, polyploidy or endoreplication and no cytotoxicity were observed compared to controls after short-term and continuous treatment. The positive and solvent controls included during short-term and continuous exposure showed the expected results. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung cells (CHL/IU) in the presence and absence of metabolic activation.

 

Gene mutation in mammalian cells

CAS No. 56-81-5

Glycerol was investigated in a HPRT gene mutation assay in cultured mammalian cells (Chinese hamster ovary (CHO) cells) similarly to OECD guideline 476 (Doolittle, 1988). Gene mutations at the HPRT locus were investigated in the presence and absence of metabolic activation (rat liver S9-mix) with concentrations of 100, 200, 400, 600, 800 and 1000 µg/mL after an exposure period of 5 h. No significant cytotoxicity was reported. An at least threefold increase in mutant frequency compared to controls was observed at 800 and 1000 µg/mL in the absence of metabolic activation. However, this increase was not considered biologically significant and did not fulfil the criteria for a positive response due to the lack of a dose-response relationship. No increase in mutant frequency was noted at any other concentration tested, neither with nor without S9 mix. The positive controls significantly increased the mutant frequency. Therefore, it was concluded that under the conditions of the study, the test material was not mutagenic at the HPRT locus of Chinese hamster ovary cells in the absence and presence of metabolic activation.

CAS No. 111-14-8

An in vitro mammalian cell gene mutation assay with heptanoic acid was performed in mouse-lymphoma L5178Y cells according to OECD guideline 476 (Sarlang, 2010). In a preliminary experiment, the cytotoxicity of the test substance was investigated with six concentrations ranging from 0.02 to 10 mM in the presence and absence of metabolic activation under the same exposure conditions as described in the main experiment. No cytotoxicity was observed up to the highest concentration tested. Based on these results, mutations at the TK locus of mouse-lymphoma L5178Y cells were investigated in two independent experiments at test substance concentrations ranging from 0.313 to 10 mM. In the first experiments cell were exposed for 3 h in the presence and absence of S9 mix, whereas periods of 3 h with S9 mix and 24 h were chosen for treatment without S9 mix in the second experiment. After an expression period of 48 h in the presence of 5-trifluorothymidine (TFT) selective medium, the test substance did not induce a significant increase in the mutant frequency at any test substance concentration in both experiments. Slight to moderate cytotoxicity, as indicated by a reduction in relative total growth of 39-50%, was noted in the first experiment after 3-h treatment with S9 mix at concentrations of 2.5 and 5 mM. In the second experiment, moderate to severe cytotoxicity, as indicated by a reduction in total growth of 53-91%, was observed after 24-h treatment without S9 mix at concentrations ≥ 5mM. No cytotoxic effects were seen at any test concentration after 3-h treatment without S9 mix in the first experiment and after 3-h treatment with S9 mix in the second experiment. The positive controls significantly increased mutant frequency. In conclusion, the test substance did not induce gene mutation in mouse-lymphoma L5178Y cells in the presence and absence of metabolic activation.

CAS No. 110-17-8

An in vitro mammalian cell gene mutation assay with fumaric acid was performed similar to OECD guideline 476 (Seifried et al., 2006). In the presence and absence of metabolic activation, mutations at the TK locus of mouse-lymphoma L5178Y cells were investigated at concentrations of 2856, 3887, 4913, 5943, 6971 and 8000 µg/mL for a period of 4 h. After a selection period of 48 h in the presence of 5-trifluorothymidine (TFT) selective medium, the test substance did not induce a significant increase in the mutant frequency up to 4913 µg/mL with and without S9-mix. At concentrations ≥ 5943 µg/mL, an increase in mutant frequency compared to control was observed with and without S9 mix. However, the test substance induced a concentration-related increase in cytotoxicity (expressed as a decrease in relative total growth) both in the presence and absence of metabolic activation. The relative total growth was decreased to values below 20% from 5943 and 6971 µg/mL without and with S9 mix, respectively. Furthermore, the increasing concentrations may have contributed to a shift in the pH of the medium to lower values due to the acid properties of the test substance. Weak acid conditions (pH 6.0 - 6.8) have been shown to induce increases in mutant frequencies in L5178Y TK+/- cells with and without S9 mix. Therefore, the observed cytotoxic and mutagenic effects at the highest concentrations are likely to be related to a pH shift into non-physiological values. The positive and vehicle controls showed the expected results. In conclusion, the test substance clearly did not induce mutations in mouse-lymphoma L5178Y cells at concentrations up to 4913 µg/mL, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions. At concentrations ≥ 5943 µg/mL the result for mutagenicity in mouse-lymphoma L5178Y cells were ambiguous and considered to be false positive due to the strong effects on cytotoxicity and pH values, respectively.

CAS No. 110-94-1

An in vitro mammalian cell gene mutation assay with Glutaric acid was performed in mouse-lymphoma L5178Y cells in three independent experiments in the presence and absence of metabolic activation (Sterling-Winthrop Research Institute, 1981, as cited in US EPA HPVIS, 2010). In the first experiment, cells were exposed to concentrations of 56, 624, 1249, 2498, 3997, 4996 µg/mL according to the method described by Clive and Spector (1975). In two further experiments, cells were treated with concentrations of 2573, 3431, 4288, 5146, 6861 µg/mL and 4977, 5806, 6636, 7465, 8295 µg/mL, respectively. According to the information reported, the test substance did not produce repeatable increases in mutant frequency at the TK locus in L5178Y mouse lymphoma cells under the conditions of S9 microsomal activation and adjustment of the assay mixture to a neutral pH range (pH 7.0 to pH 7.4) in any of the experiments performed. Concentrations from 156-8295 µg/mL (with pH adjustment) induced, at best, moderate toxicity. Sporadic increases in mutant frequency were observed, but could not be confirmed in replicate treatments and/or at higher concentrations of the test substance. The positive and solvent controls showed the expected results. In conclusion, the test substance did not induce gene mutation in mouse-lymphoma L5178Y cells in the presence and absence of metabolic activation.

Conclusions for genetic toxicity in vitro

There are no data available on the genetic toxicity of Glycerides, mixed C8-C10 and succinyl. The substance is composed of mixed esters of glycerol with octanoic (C8), decanoic (C10) and succinic acid, and the ester bond is anticipated to undergo enzymatic hydrolysis. Therefore, hazard assessment is conducted by means of read-across and in a weight of evidence approach with available data for esters of glycerol with C8 and/or C10 acids as well as for representative products of glycerides hydrolysis (glycerol and heptanoic acid) and succinic acid (and its sodium salts). Available data on analogue dicarboxylic acids (fumaric and glutaric acid) is taken into account for assessment of endpoints for which no data on succinic acid (and/or its sodium salts) are available.

All available in vitro studies on the induction of gene mutations in bacteria and mammalian cells as well as on the induction of chromosome aberrations yielded negative results. Overall, based on read-across the available data provide sufficient weight of evidence leading to the conclusion that the substance Glycerides, mixed C8-C10 and succinyl is not mutagenic and not clastogenic in vitro.

Justification for selection of genetic toxicity endpoint

No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:

Based on read-across from glycerides, representative hydrolysis products (glycerol and heptanoic acid) and dicarboxylic acids (succinic acid and its sodium salts, fumaric and glutaric acid) and in a weight of evidence approach, no hazard was identified.

In none of these studies mutagenicity in bacteria could be observed.

In none of these studies clastogenic effects in mammalian cells could be observed.

In none of these studies mutagenicity in mammalian cells could be observed.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on read-across from structural analogues and surrogates, the available data on the in vitro genetic toxicity of Glycerides, mixed C8-C10 and succinyl do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.