Tetraesters of pentaerythritol with 2-ethylhexanoic acid and heptanoic acid and nonanoic acid

Endpoint summary

Additional information

Justification for analogue read-across

Data on the in vitro genetic toxicity in bacterial and mammalian cells of Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid (EC 806-879-4) are not available. The genetic toxicity assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 67762-53-2

The in vitro mutagenic potential of Fatty acids, C5-9, tetraesters with pentaerythritol was assessed in a reverse mutation assay according to OECD Guideline 471 and under GLP conditions (Mecchi, 1999). The plate incorporation method was applied, using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvrA. The tester strains were incubated with test substance dissolved in ethanol at concentrations of 33.3 to 5000 µg/plate, with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9-mix). Vehicle and positive controls were included and shown to be valid. No cytotoxicity was observed. Slight precipitation was observed from 100 µg/plate without metabolic activation, and from 1000 µg/plate with metabolic activation. The test substance did not induce an increase in reversions in the S. typhimurium strains, with or without metabolic activation and is considered to be not mutagenic under the conditions of this study.

CAS 78-16-0

A bacterial reverse mutation assay (Ames test) was performed according to a protocol similar to OECD guideline 471 and under GLP conditions, using 2-ethyl-2-[[(1-oxoheptyl)oxy]methyl]propane-1,3-diyl bisheptanoate (Wagner, 1997). The plate incorporation method was applied, exposing Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA1538 and E. coli WP2 uvrA to test substance concentrations of 10, 33, 100, 333 and 1000 µg/plate without metabolic activation, and of 33, 100, 333, 1000 and 5000 µg/plate with metabolic activation (Arochlor 1254 induced rat liver S9 mix). The positive and vehicle controls included for each tester strain were shown to be valid and were in the range of historical control data. However, only 2-aminoanthracene was used in the positive control with metabolic activation. No increase in the frequency of revertant colonies compared to concurrent vehicle controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed. Precipitation was observed at concentrations of 1000 µg/plate and above in experiment 1 and at 300 µg/plate and above in experiment 2, with metabolic activation, and from 333 µg/plate without metabolic activation in both experiments. No increase in the mean number of revertants per plate was observed when compared to controls. The test substance was considered to be not mutagenic under the conditions of this study.

CAS 7299-99-2

A bacterial reverse mutation assay (Ames test) was performed according to a protocol similar to OECD guideline 471 and under GLP conditions, using Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester .The preincubation method was applied, exposing Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli WP2 uvrA to concentrations of 8.19 – 5000 µg /plate (range-finding study) and 78.1 - 5000 µg /plate (main study), with and without metabolic activation. No cytotoxicity was observed. Precipitation was noted at concentrations of 800 µg /plate and above in the range-finding study, and at 1250 µg /plate and above in the main study, with and without metabolic activation. A repeat experiment with strain TA 1535 was performed as the first experiment was contaminated. The positive and negative controls included for each tester strain were shown to be valid. No increase in the mean number of revertants per plate was observed when compared to controls. The test substance was considered to be not mutagenic under the conditions of this study.

CAS 11138-60-6

The potential mutagenicity of Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol was assessed in a bacterial reverse mutation assay (Ames test) according to OECD guideline 471 and under GLP conditions (Bailey, 1996). The test substance was diluted in ethanol and test substance concentrations of 0, 10, 33, 100, 333 and 1000 µg/plate were tested in triplicate on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E.coli WP2 uvr A, with and without metabolic activation. No cytotoxicity was observed, although precipitation was noted at concentrations from 100 µg/plate. No increase in the mean number of revertants per plate was observed when compared to controls. The positive and negative controls included for each tester strain were shown to be valid. Based on the study results, the test substance was considered non-mutagenic in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 7299-99-2

The cytogenetic potential of Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester was assessed in an in vitro mammalian chromosome aberration test in Chinese hamster lung (CHL/IU) cells, performed according to OECD guideline 473 and under GLP conditions (Masumori, 2005). Duplicate cultures of CHL cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation. In the range-finding test, CHL cells were incubated with a total of 10 test substance concentrations in the range of 9.77 - 5000 µg/mL for 6 hours with a 24-hour fixation time and for 24 hours with a 24-hour fixation time, in the absence of a metabolic activation system. The first experiment was also performed with cells exposed to a concentration range of 9.77 - 5000 µg/mL for 6 hours with a 24-hour fixation time in the presence of metabolic activation. In the main test, cells were incubated with 1250, 2500 and 5000 µg/mL for 6 hours followed by a 24-hour expression time, and for 24 hours with a 24-hour fixation time without metabolic activation. In the presence of metabolic activation cells were exposed to 1250, 2500 and 5000 µg/mL for 6 hours followed by a 24-hour fixation time. No cytotoxicity was observed. In the pre-test, visible precipitation was observed at the end of exposure period at concentrations ≥ 625 µg/mL. However, no inhibition of cell growth was detected in any concentrations with and without S9 mix after short-term and continuous exposure. The vehicle (solvent) and positive controls were shown to be valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation.

CAS 11138-60-6

An in vitro mammalian chromosome aberration test was performed with Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol in Chinese hamster ovary cells (CHO) according to OECD guideline 473 and under GLP conditions (Gudi, 1996). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (Arochlor 1254-induced rat liver S9-mix). Cells were exposed to test substance concentrations of 625, 1250, 2500 and 5000 µg/mL for 4 and 20 hours without and for 4 hours with metabolic activation. The fixation time was 20 hours for all exposure times, with and without metabolic activation. Cytotoxicity was observed at the highest concentration tested with and without metabolic activation. The vehicle (solvent) controls induced aberration frequencies within the expected range. The positive controls were shown to be valid. Evaluation of 200 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 15834-04-5

An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and under GLP conditions was performed with 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). In the first experiment, the cells were treated for 3 hours with 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL test substance in the presence or absence of metabolic activation (8% (v/v)). In the second experiment, concentrations of 0.03 - 100 µg/mL test substance were applied with metabolic activation (12%, v/v) for 3 hours, while cells were exposed to concentrations of 0.1 - 250 µg/mL test substance without metabolic activation for 24 hours. The expression time was 48 hours. The cells were then incubated for 11-12 days to determine the mutation frequency. Precipitation was observed at concentrations ≥ 100 µg/mL. Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No cytotoxicity was observed. The positive and negative controls were valid and the results were in the range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was 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.

 

Genetic toxicity in mammalian cells in vivo

CAS 68424-31-7

An in vivo mammalian erythrocyte micronucleus test was performed according to OECD guideline 474 and under GLP conditions, using Fatty acids, C5-10, esters with pentraerythritol (Griffiths and Mackay, 1992). 5 mice/sex/dose were administered 5000 mg/kg bw of the test substance via intraperitoneal injection and sacrificed after 24 and 48 hours respectively. Bone marrow cells from the femur were extracted, and slides with bone marrow smears were prepared and stained with polychrome methylene blue and eosin. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the control values were seen in either sex at either of the sampling times. No increase in the percentage of polychromatic erythrocytes was observed in the females, compared with the control group. A small, but significant decrease was noted in treated male mice. This small decrease is considered not to be biologically significant compared with the concurrent control values. No toxicity was observed at the limit dose of 5000 mg/kg bw. The positive control substance (cyclophosphamide) induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes and a reduction in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls, showing the positive control was valid.

Overall conclusion for genetic toxicity

There are no studies available on the in vitro and in vivo genetic toxicity of the target substance Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid.Therefore analogue read-across from source substances was applied fromin vitro studies on bacterial and mammalian cells,and from in vivo studies, using six source substances. The results of the available in vitro and in vivo studies on source substances were negative. Based on the available data, and following the analogue approach Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid is not expected to be mutagenic and clastogenic in vitro and in vivo.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from structural analogues. 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 substances and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Genetic toxicity in vitro:
Ames test (OECD 471, WoE): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102 and TA 1538 and in E. coli WP2 uvrA pKM 101
Chromosome aberration (OECD 473, WoE): negative in Chinese hamster lung cells and Chinese hamster ovary cells with and without metabolic activation
Gene mutation in mammalian cells (OECD 476, WoE): negative in mouse lymphoma L5178Y cells with and without metabolic activation
Genetic toxicity in vivo:
Micronucleus test (OECD 474, WoE): negative in mouse bone marrow cells with and without metabolic activation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid (EC 806-879-4), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.