Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results in mammalian cell gene mutation test using mouse lymphoma cells, with and without metabolic activation (OECD 490, GLP, substance-specific data)

Negative results in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 102 and E.coli strain with and without metabolic activation (OECD 471, GLP, analogue approach).
Negative results in mammalian chromosomal aberration test with lymphocytes (OECD 473, GLP, analogue approach).
Negative results in mammalian cell gene mutation tests using mouse lymphoma cells, with and without metabolic activation (OECD 476, GLP, analogue approach).

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the polyol esters family will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative results in mammalian erythrocyte micronucleus test in vivo (OECD 474, GLP, analogue approach).

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the polyol esters family will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the polyol esters family will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

 

Justification for grouping of substances and read-across

Apart from one in vitro gene mutation assay in mammalian cells (Nyco, 2021), there are no data available for the genetic toxicity of the substance Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids (EC 453-490-7). 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 was 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 toxicological properties may be predicted from data for the reference substance(s) on the basis of structural similarity, the substances listed below are selected as reference substances for assessment of toxicological endpoints, for which information gaps are identified.

 

Overview for genetic toxicity

CAS/EC	Bacterial gene mutation	Chromosomal aberration	Mammalian gene mutation	In vivo
EC 453-490-7 (a)	RA: CAS 67762-52-1 RA: CAS 84418-63-3	RA: CAS 131459-39-7	negative in addition: RA: CAS 15834-04-5	RA: CAS 68424-31-7
CAS 84418-63-3 (b)	Negative	--	--	--
CAS 67762-52-1	Negative	--	--	--
CAS 68424-31-7	--	--	--	Negative
CAS 15834-04-5	--	--	Negative	--
CAS 131459-39-7	Negative	Negative	--	--

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

1. b) Substances that are either already registered under REACh or not subject to the REACh Phase-in registration deadline of 31 May 2013 are 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 and similar properties and/or activities. The available endpoint information is used to predict the same endpoints for Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids (EC 453-490-7).

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

 

In vitro gene mutation study in bacteria

Since no studies investigating the in vitro gene mutation in bacteria of Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids (EC 453-490-7) are available, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5, a read-across from the structurally related analogue substances Fatty acids, C5-9, hexaesters with dipentaerythritol (CAS# 67762-52-1), and Isononanoic acid, mixed esters with dipentaerythritol, heptanoic acid and pentaerythritol (CAS# 84418-63-3) was conducted.

 

CAS 67762-52-1 

 

A study investigating the in vitro gene mutation in bacteria of analogue substances Fatty acids, C5-9, hexaesters with dipentaerythritol (CAS# 67762-52-1) is available.

An Ames test was performed according to OECD Guideline 471 and GLP with the test substance dissolved in acetone in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and with E. coli WP2uvr A (Exxon Mobil, 1999). Test substance concentrations of 33.3, 100, 333, 1000, 3330, and 5000 µg/plate with and without metabolic activation. Cytotoxic effects were not observed in the absence and presence of a metabolic activator. No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C5-9, hexaesters with dipentaerythritol did not induce gene mutations in all tested strains under the given test conditions.

 

 

CAS 84418-63-3

 

Isononanoic acid, mixed esters with dipentaerythritol, heptanoic acid and pentaerythritol (CAS# 84418-63-3) was tested for mutagenicity in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 according to OECD Guideline 471 (Stearinerie, 2006). Test substance concentrations of 312.5, 625, 1250, 2500 and 5000 µg/plate in ethanol were tested in triplicates in two independent experiments. The plate incorporation method with and without the addition of a rat liver homogenate metabolising system (S9-mix) was used in experiment 1 and the preincubation method was used in experiment 2 with metabolic activation.

No cytotoxicity was observed. No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. Thus, the test substance acid did not induce gene mutations in five tested Salmonella strains under the given test conditions. 

 

In an additional study Isononanoic acid, mixed esters with dipentaerythritol, heptanoic acid and pentaerythritol (CAS# 84418-63-3) was tested for mutagenicity in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strains WP2 uvr A pKM 101 and WP2P (WP2 pKM101) according to OECD Guideline 471 (Croda, 1991). Test substances concentrations of 100, 200, 500, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

The plate incorporation method with and without the addition of a rat liver homogenate metabolising system (S9-mix) was used in experiment 1 and in experiment 2 without metabolising system and the preincubation method was used in experiment 2 witht metabolic activation. No cytotoxicity was observed. No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. Thus, the test substance acid did not induce gene mutations in five tested Salmonella and E. coli strains under the given test conditions. 

 

In vitro cytogenicity in mammalian cells

Since no studies investigating the in vitro cytogenicity in mammalian cells of Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids (EC 453-490-7) are available, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from the structurally related analogue substance 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS# 131459-39-7) was conducted.

 

CAS 131459-39-7 

 

An in vitro mammalian chromosome aberration test was performed with 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS# 131459-39-7) in primary human lymphocytes according to OECD Guideline 473 (Croda, 2000). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix).

In the first experiment test substance concentrations of 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL in acetone were used for 4 hours of exposure with and without metabolic activation. In the second experiment 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/mL were used for 24 hours exposure without S9 and 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for 4 hours with S9. Ethylmethanesulphate and cyclophosphamide were used as positive control substances. Evaluation of 200 cells from each culture for chromosomal aberrations revealed no increase in the frequency of chromosome aberrations at any dose level in comparison to the negative controls. The test material demonstrated showed no cytotoxicity. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

In vitro gene mutation in mammalian cells

 

EC 453-490-7 (former CAS 70983-72-1)

 

There is one recent study (Nyco, 2021) available investigating in vitro gene mutation in mammalian cells of EC 453-490-7 (former CAS 70983-72-1). This study was a mouse lymphoma assay in L5178Y cells, applying both short-term and long-term treatment without or with metabolic activation (2.5% (v/v) S9-mix), according to OECD 490 (2016) and GLP. The concentrations 128, 320, 800, 2000, 3500 and 5000 µg/mL were evaluated for short-term treatment conditions, and 320 - 5000 µg/mL for long-term treatment conditions. The short-term treatment was defined by an exposure time of 3 or 4 h (+S9 and -S9 mix, respectively), and the long-term treatment was defined by an exposure time of 24 h. Untreated negative and vehicle controls (absolute ethanol), as well as a positive control (either cyclophosphamide monohydrate for +S9 conditions, or methylmethanesulfonate for -S9 conditions) were run in parallel. 5-trifluorothymidine was used as a selective agent. After 10 - 12 days treatment with the selective agent, 2000 cells/well were evaluated. Mutant frequency served as a measure for gene mutation. Cytotoxicity was determined by relative suspension growth (RSG). There was no change in osmolality or pH after treatment with the test substance. The negative and positive controls fulfilled the assay acceptability criteria. In presence of the test material an increase of mutation frequency was measured at the highest doses tested in the absence and presence of the metabolic activation system for short- and long-term treatment, but the global evaluating factor (GEF) indicated that values fell below the GEF criteria for each of the experiments conducted. Therefore, it was concluded that the test substance did not induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells.

 

As additional data consideration, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from the structurally related analogue substances 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate (CAS# 15834-04-5) was conducted and reported below:

 

CAS 15834-04-5

An in vitro mammalian cell gene mutation assay was performed with 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate (CAS# 15834-04-5) according to OECD Guideline 476 in L5178Y mouse lymphoma cells (Croda, 2010). In the first experiment, the test item was tested up to concentrations of 100 μg/mL in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. In the second experiment, 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate was tested up to cytotoxic levels of 100 µg/mL and 250 µg/L in the presence and absence of 12% (v/v) S9-mix respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. In the test, no cytotoxicty was noticed. However, the test item was test up at or above the precipitating dose level of 100 µg/mL. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 13- and 11-fold for methylmethansulfonate (MMS) in the absence of S9-mix, and by 13- and 11-fold for cyclophosphamide (CP) in the presence of S9-mix. In the absence and presence of S9-mix, 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate did not induce a significant increase in the mutation frequency. In the absence presence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the second experiment. It is concluded that 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

Conclusion for genetic toxicity in vitro

 

Three studies are available to assess the mutagenic potential in bacteria for Fatty acids C5-10 esters with dipentaerythritol (CAS# 70983-72-1). Since, all studies are negative for reverse gene mutation, Fatty acids C5-10 esters with dipentaerythritol (CAS# 70983-72-1) is not considered to induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested. Cytogenicity and mammalian gene mutation data are available from structural related read-across substances as well as on the substance itself in case of one mammalian gene mutation test (mouse lymphoma assay). The study investigating cytogenicity using human lymphocytes with 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS# 131459-39-7) was negative for induction of chromosomal aberrations. Equally, no gene mutation effects were found in mammalian L5178Y cells treated with 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate (CAS# 15834-04-5) and with the registration substance, respectively, in neither of the two MLA assays. Therefore, according to data on the substance itself, as well as on structural based read-across from analogue substances, Fatty acids C5-10 esters with dipentaerythritol (CAS# 70983-72-1) is not considered to have cytogenetic or gene mutation potential in mammalian cells in vitro.

 

Genetic toxicity in vivo

Since no studies investigating the in vivo genetic toxicity of Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids (EC 453-490-7) are available, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5, a read-across to the structurally related analogue substancesFatty acids, C5-10, esters with pentaerythritol (CAS# No. 68424-31-7) was conducted.

An in vivo micronucleus assay with Fatty acids, C5-10, esters with pentaerythritol (CAS# 68424-31-7) was conducted according to OECD guideline 474 and GLP (Croda, 1992). The substance was found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application. A single intraperitoneal injection was given to groups of 5 male and 5 female CD-1 mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 hours after dosing.

The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be biologically significant compared to the concurrent control values. Therefore, no statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times. The substance is found not to be clastogenic in the mouse micronucleus test in vivo.

 

Conclusion for genetic toxicity in vivo

There is one in vivo micronucleus assay available conducted with the source substance Fatty acids, C5-10, esters with pentaerythritol (CAS# 68424-31-7). The outcome of the test was negative was negative and therefore, Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids (EC 453-490-7) was considered not to have clastogenic potential in vivo.

 

Justification for classification or non-classification

Based on substance-specific data and read-across from structurally similar substances, the available data on genetic toxicity 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.