Ethylene distearate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA with and without metabolic activation (OECD 471, GLP, WoE).
Negative results in mammalian chromosomal aberration test with Chinese hamster lung fibroblasts (V79) and Chinese hamster lung cells (CHL/IU) 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).
Negative results in mammalian erythrocyte micronucleus test in vivo (OECD 474, GLP).

 

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category 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.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

 

 

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 glycol esters category 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.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

 

Genetic toxicity (mutagenicity) in bacteria in-vitro

CAS 627-83-8

Two independent in vitro gene mutation studies in bacteria are available.

A bacterial gene mutation assay (Ames test) was performed with ethylene distearate following OECD guideline 471 and in compliance with GLP (Wnorowski, 1991). The plate incorporation procedure was performed with Salmonella typhimurium strains TA 100, TA 1535, TA 1537 and TA 98 in the absence and presence of metabolic activation (Aroclor 1254-induced rat liver S9-mix). Two independent experiments were conducted each in triplicates at concentrations from 8 to 5000 µg/plate (vehicle: DMSO). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results, however instead of the solvent control DMSO, water was included as negative control substance. Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

 

More recently, a gene mutation test in bacteria was sponsored by the Ministry of Health, Japan (Tokyo Laboratory, BoZo Research Center, 2012). The study was conducted following OECD guideline 471 and according to "OECD Principles of Good Laboratory Practice". Briefly, Salmonella typhimurium strains TA 100, TA 1535, TA 1537 and TA 98 and E. coli WP2uvrA were treated with the test substance in the absence and presence of metabolic activation (Phenobarbital and 5,6-benzoflavone induced rat liver S9-mix). Two independent experiments were conducted in triplicates using the plate incorporation method. The applied concentrations were selected based on the results of a dose-range finding pre-test. Precipitation of the test substance determined the highest concentration tested. In the presence of metabolic activation, concentrations ranging from 19.5 - 313 µg/plate (vehicle: Tetrahydrofuran (THF)) were applied. In the absence of S9 mix, bacteria were treated with 4.88 - 78.1 µg/plate (vehicle: THF). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and vehicle controls are considered valid. Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

Overall, based on the available studies, the test substance is not considered to exhibited mutagenic properties in bacteria.

 

CAS 68583-51-7

Two bacterial gene mutation assays were performed with Decanoic acid, mixed diesters with octanoic acid and propylene glycol following OECD guideline 471 and in compliance with GLP (Banduhn, 1991; Ebert, 1995). The strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested in two independent experiments according to the plate incorporation procedure at concentrations from 8 to 5000 µg/plate (vehicle: suspension medium Tween 80/bidest. water) with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). No increase in the number of revertant colonies was noted in any strain of bacteria tested, with and without a metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results and were therefore considered as valid. Under the conditions of this study, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested (Banduhn, 1991). A further experiment in the tester strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 confirmed the above described results (Ebert, 1995). In concentrations from 50 to 5000 µg/plate with and without a metabolic activation system, no cytotoxicity was apparent and no increase in the number of revertant colonies was observed in any of the strains tested.

Thus, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

 

CAS 84988-75-0

A study investigating the genetic toxicity in vitro of Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol is available. The study was conducted according to OECD guideline 471 under GLP conditions (Banduhn, 1991). In two independent experiments, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested according to the plate incorporation procedure. Concentrations from 8 to 5000 µg/plate were investigated with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results and were therefore considered as valid. Thus, under the conditions of this study, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.

 

CAS 624-03-3

No studies are available investigating the gene mutation properties of ethane-1,2-diyl palmitate in bacteria. However, there are available data on the category members ethylene distearate (CAS 627-83-8) and Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1). The studies of the category members were considered for assessment and read-across was conducted based on a category and weight of evidence approach.

The gene mutation properties of the category member Fatty acids, C16-18, esters with ethylene glycol were determined according to OECD guideline 471 under GLP conditions (Banduhn, 1991). The tester strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used. The main study was performed in triplicates each in two independent experiments according to the plate incorporation procedure at concentrations from 8 to 5000 µg/plate (vehicle: Tween 80; 1:1 (w/w) dilution with water) with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. Precipitation was noted at the highest test concentration of 5000 µg/plate. The positive and negative controls showed the expected results and were therefore considered as valid. Under the conditions of this study, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.

One bacterial gene mutation assay was performed with ethylene distearate following OECD guideline 471 and in compliance with GLP. The study of the category member showed no induction of gene mutations in the bacterial mutation test and is discussed under the respective CAS number.

All available data on the category members were consistently negative. Thus, the available data on the category members, do not provide evidence for gene mutation properties of ethane-1,2-diyl palmitate in bacteria.

 

Genetic toxicity (cytogenicity) in mammalian cells in-vitro

CAS 627-83-8

An in vitro mammalian chromosome aberration test was conducted with ethylene distearate in accordance with OECD guideline 473 under GLP conditions (Tokyo Laboratory, 2012). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster lung (CHL/IU) cells, incubated for a short-term (6 h) treatment period with and without metabolic activation and for a continuous (24 and 48 h) treatment period without metabolic activation. The metabolic activation system was S9-mix from rats treated with Phenobarbital and 5,6-Benzoflavone. Concentrations of 1250, 2500 and 5000 µg/mL (6 h treatment, with and without S9) and 1480, 2220, 3330 and 5000 µg/mL (24 and 48 h treatment, without S9) of the test substance in the vehicle 0.5% (w/v) carboxymethyl cellulose sodium aqueous solution were applied. Demecolcin was added approximately 2 hours before the end of the culturing period to arrest the cells in metaphase. At least 200 metaphases were scored for each of the tested concentrations.

Precipitation in the test media was observed at all tested concentrations at the start and end of the treatment period. In the 6 h treatment with and without S9, no cytotoxicity was observed, with the 50% growth inhibition concentration determined to be > 5000 µg/mL. In the 24 h continuous treatment, the 50% growth inhibition concentration was also > 5000 µg/mL, but on microscopic examination, evidence of cytotoxicity was apparent at 2500 and 5000 µg/mL. In the 48 h continuous treatment, the 50% growth inhibition concentration was 3372 µg/mL and microscopic evidence of cytotoxicity was observed from 1250 µg/mL.

For all treatment methods, the test article was judged to be negative for cytogenicity since the incidence of cells with structural chromosome aberrations and/or polyploidy was lower than 5%. For all treatment methods, the incidence of cells with structural chromosome aberrations and/or polyploidy in the negative control group was lower than 5% and thus considered negative for cytogenicity. In contrast, structural chromosome aberrations were markedly increased in the positive control groups. Therefore, it was judged that the test system functioned correctly.

In conclusion, under the conditions of the study the test material did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation in Chinese Hamster lung (CHL/IU) cells.

 

CAS 853947-59-8

An in vitro mammalian chromosome aberration test was conducted with C8-10, 1,3-Butandiolester in accordance with OECD guideline 473 under GLP conditions (Dechert, 1997). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster lung fibroblasts (V79) cells, incubated for 18 and 28 h with and without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 10-100 µg/mL (18 h incubation) and 80 and 100 µg/mL (28 h incubation) of the test substance in the vehicle ethanol were applied. The solubility limit of the test substance in the vehicle ethanol in the culture medium was determined to be 100 µg/mL. In the first experiment without metabolic activation, the negative controls exhibited a mitotic index of 2.0% only and the experiment was therefore repeated. Thereafter, the negative as well as the positive controls showed the expected results and were within the range of historical control data. The frequency of polyploidy cells with and without metabolic activation was within the expected range (< 10%). In the experiments both with and without metabolic activation, a systematic influence of the test substance was observed, which led to a reduction in the mitotic index. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed.

Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in Chinese hamster lung fibroblasts in vitro.

The available data on the category members were consistently negative. Thus, the available data on the category members, do not provide evidence for cytogenicty of ethane-1,2-diyl palmitate in mammalian cells.

Genetic toxicity (mutagenicity) in mammalian cells in-vitro

Within the Glycol Ester category, one study investigating the gene mutation properties in mammalian cells is available. Therefore, the study of the category member Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1) was considered for assessment and read-across was conducted based on a category approach.

CAS 91031-31-1

The in vitro mammalian cell gene mutation study of Fatty acids, C16-18, esters with ethylene glycol was carried out according to OECD guideline 476 under GLP conditions (Verspeek-Rip, 2010). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/β-naphtoflavone-induced rat liver S9). In the first experiment, cells were exposed for 3 h to test substance at concentrations of 0.1-333 µg/mL (in DMSO) with and without metabolic activation. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 3-175 µg/mL and with metabolic activation (3 h; 12% S9-mix) from 0.1-333 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data. No cytotoxicity was observed up to the precipitating concentration of 100 µg/mL and up to 333 µg/mL, respectively. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, Fatty acids, C16-18, esters with ethylene glycol did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

 

Genetic toxicity in vivo

Within the Glycol Ester category, one study investigating the cytogenetic properties of Fatty acids, C18 and C18 unsatd., epoxidized, ester with ethylene glycol (CAS 151661-88-0) in vivo is available. Therefore, the study was considered for assessment and read-across was conducted based on a category approach.

CAS 151661-88-0

The in vivo micronucleus assay of Fatty acids, C18 and C18 unsatd., epoxidized, ester with ethylene glycol was carried out according to OECD guideline 474 under GLP conditions (Banduhn, 1990). Based on the results of a preliminary dose range finding study, the test substance diluted in arachis oil was administered at 3000, 4000 and 5000 mg/kg bw as single oral doses to groups of 6 male and female CFW1 mice, observed for 24, 48 and 72 h post-dose. A concurrent negative control with the vehicle alone and a positive control group given cyclophosphamide was included in the study. No mortality and no signs at clinical examinations were reported. The test substance did not induce a statistically significant increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of the animals. The negative and positive controls showed the expected results. Therefore, under the conditions of the study, Fatty acids, C18 and C18 unsatd., epoxidized, ester with ethylene glycol did not induce chromosomal mutations in the bone marrow of mice.

 

Additional data

Three bacterial gene mutation studies are available for the category members C8-C10-1,3-Butandiolester (CAS 853947-59-8), Stearic acid, monoester with propane-1,2-diol (CAS 1323-39-3) and Fatty acids, C18 and C18 unsatd. epoxidized, ester with ethylene glycol (CAS 151661-88-0) all showing negative results.

 

Conclusion for genetic toxicity

In summary, all available studies within the Glycol Ester category investigating the genetic mutation in bacteria in-vitro provide negative results. Furthermore, no cytogenicity in mammalian cells in-vitro, no mutagenicity in mammalian cells in-vitro and no Genetic toxicity in-vivo was observed with members of the Glycol Ester category.

Therefore, no properties for genetic toxicity were observed within the Glycol Ester group for any member.

References

Agency for Toxic Substances and Disease Registry (ATSDR) (1997): Toxicological Profile for Propylene Glycol. US Department of Health and Human Services. Atlanta, US.

Agency for Toxic Substances and Disease Registry (ATSDR) (2010): Toxicological Profile for Ethylene Glycol. US Department of Health and Human Services. Atlanta, US.

Gubicza, L., Kabiri-Badr, A., Keoves, E., Belafi-Bako, K. (2000): Large-scale enzymatic production of natural flavour esters in organic solvent with continuous water removal. Journal of Biotechnology 84(2): 193-196.

Heymann, E. (1980): Carboxylesterases and amidases. In: Jakoby, W.B., Bend, J.R. & Caldwell, J., eds., Enzymatic Basis of Detoxication, 2nd Ed., New York: Academic Press, pp. 291-323.Gubicza, L. et al. (2000). Large-scale enzymatic production of natural flavour esters in organic solvent with continuous water removal. Journal of Biotechnology 84(2): 193-196.

International Programme on Chemical Safety (IPCS) (2001): Ethylene Glycol. Poisons Information Monograph. PIM 227.

Lilja, J. et al. (2005). Esterification of propanoic acid with ethanol, 1-propanol and butanol over a heterogeneous fiber catalyst. Chemical Engineering Journal, 115(1-2): 1-12.

Liu, Y. et al. (2006). A comparison of the esterification of acetic acid with methanol using heterogeneous versus homogeneous acid catalysis. Journal of Catalysis 242: 278-286.

Miller, O.N., Bazzano, G. (1965): Propanediol metabolism and its relation to lactic acid -metabolism. Annals of the New York Academy of Sciences 119, 957-973.

Radzi, S.M. et al. (2005). High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica. Electronic Journal of Biotechnology 8: 292-298.

Ritchie, A.D. (1927): Lactic acid in fish and crustacean muscle. Journal of Experimental Biology 4, 327-332.

Stryer, L. (1994): Biochemie. 2nd revised reprint, Heidelberg; Berlin; Oxford: Spektrum Akad. Verlag.

Tocher, D.R. (2003): Metabolism and Functions of Lipids and Fatty Acids in Teleost Fish. Reviews in Fisheries Science 11(2), 107-184.

WHO (2002): Ethylene Glycol: Human Health Aspects. Concise International Chemical Assessment Document 45.

Zhao, Z. (2000). Synthesis of butyl propionate using novel aluminophosphate molecular sieve as catalyst.Journal of Molecular Catalysis 154(1-2): 131-135.

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 group concept is applied to the members of the Glycol Ester Category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labeled on this basis.

Therefore, based on the group concept, all 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.