Isobutyl laurate

Endpoint summary

Additional information

Justification for grouping of substances and read-across

There are only limited data available on genetic toxicity of isobutyl laurate (CAS 37811-72-6). 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 substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of genetic toxicity

CAS	Chemical name	Molecular weight [g/mol]	Gene mutation in vitro (Ames)	Chromosome aberration in vitro	Gene mutation in vitro (MLA)
37811-72-6 (a)	Isobutyl laurate	256.42	WoE: RA: CAS 110-27-0 RA: CAS 163961-32-8	WoE: RA: CAS 10233-13-3 RA: CAS 163961-32-8	WoE: RA: CAS 10233-13-3 RA: CAS 163961-32-8
110-27-0 (b)	Isopropyl myristate	270.45	Experimental result: negative	--	--
10233-13-3 (b)	Isopropyl laurate	242.40	--	Experimental result: negative	Experimental result: negative
163961-32-8 (b)	Fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters	312.53-340.58	Experimental result: negative	Experimental result: negative	Experimental result: negative

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances 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 similar properties and/or activities. The available endpoint information is used to predict the same endpoints for isobutyl laurate (CAS 37811-72-6). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

No data on genetic toxicity is available with isobutyl laurate (CAS 37811-72-6). Therefore, read across from the structurally analogue substances isopropyl myristate (CAS 110-27-0), isopropyl laurate (CAS 10233-13-3) and fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters (CAS 163961-32-8) was applied.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 110-27-0

In the first study, isopropyl myristate (CAS 110-27-0) was investigated for mutagenicity to bacteria (Ames test) according to OECD TG 471 and in compliance with GLP (Verspeek-Rip, 2014). The Salmonella typhimurium strains TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2 uvrA were exposed to concentrations ranging from 52 to 5000 µg/plate in ethanol with and without the addition of metabolic activation system (S9-mix at concentrations of 5% and 10% v/v) in three independent assays. The experiments were conducted according to the plate incorporation methodology. Cytotoxicity was observed in tester strain TA 100 in the presence of S9-mix, where a moderate to extreme reduction of the revertant colonies was observed at test substance concentrations of 512 µg/plate and above. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the other tester strains in the absence and presence of S9-mix. In tester strain TA 98 a 3.3-fold increases in the number of revertant colonies compared to the solvent control in the absence of S9-mix was observed. Since this increase was observed at a precipitating dose level and was not reproducible in two other experiments, this increase was not considered to be toxicologically relevant. Appropriate solvent and positive controls were included into the test and gave the expected results. Based on these results, the test item was considered to be not mutagenic to bacteria under the conditions of the test. 

CAS 163961-32-8

A reliable bacterial gene mutation study (Ames test) performed according to OECD TG 471 and in compliance with GLP with fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters (CAS 163961-32-8) is available (Bowles, 2002). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment I+II) procedure in the absence and presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix). Two independent experiments were conducted in three repetitions at each concentration from 50 to 5000 µg/plate (experiment I and II). No cytotoxicity was observed in a preliminary toxicity test performed with tester strain TA 100 after treatment with the test item up to 5000 µg/plate in the absence and presence of metabolic activation. Precipitation was recorded at a concentration of 5000 µg/plate. Appropriate solvent (acetone) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. Therefore, the test material was considered to be non-mutagenic under the conditions of the test.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 10233-13-3

An in vitro mammalian chromosome aberration test was performed with isopropyl laurate (CAS 10233-13-3) in primary human lymphocytes according to OECD TG 473 and GLP (Buskens, 2010). 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 cells were exposed for 3 hours to test substance concentrations of 10, 33 and 100 µg/mL in ethanol with and without metabolic activation. In the second experiment cells were exposed for 24 hours to 66, 150 and 250 µg/mL followed by 24 hours expression time. Additionally, 3, 125 and 150 µg/mL were the concentrations used for 48 hours exposure followed by 48 hours expression time. Both incubations were done without metabolic activation. 250 µg/mL was chosen as maximum dose due to limited solubility. Mitomycin C and cyclophosphamide were used as positive control substances. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Positive control materials induced statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level in comparison to the negative controls. The test material demonstrated only modest cytotoxicity. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

CAS 163961-32-8

A reliable in vitro chromosome aberration test in human lymphocytes was performed with fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters (CAS 163961-32-8) according to OECD TG 473 and in compliance with GLP (Durward, 2004). Human peripheral lymphocytes were cultured and treated with the test material or vehicle (arachis oil) in the absence or presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix) in duplicates at concentrations of 625, 1250 and 2500 µg/mL for 4 h (with and without S9-mix) and 312.5, 468.75 and 625 µg/mL for 24 h (without S9-mix). Fixation and staining of the cells were performed 24 hours after start of exposure with the test material. Cytotoxicity was assessed by determination of the mitotic index. Appropriate solvent and positive controls were included in the test and gave the expected results. Neither precipitation nor excessive cytotoxicity was recorded at any concentration during the course of the study. The test substance did not cause a statistically significant, dose-related increase in chromosome aberrations under the tested conditions. Based on the results of the study the test material is considered not to be clastogenic in this chromosome aberration test in vitro.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 10233-13-3

An in vitro Mammalian Cell Gene Mutation Assay according to OECD TG 476 and in compliance with GLP was performed with isopropyl laurate (CAS 10233-13-3) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). The cells were treated for 3 and 24 hours with 8% (v/v) and without S9-mix and with 12% (v/v) and without S9-mix, respectively. The test substance was tested up to precipitation, the following concentrations were tested: 0.01, 0.03, 0.1, 0.3, 1, 3, 5 and 10 μg/mL. Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9-mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with isopropyl laurate either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the isopropyl laurate treated cultures were comparable to the numbers of small and large colonies of the solvent controls. It was concluded that isopropyl laurate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

CAS 163961-32-8

An in vitro Mammalian Cell Gene Mutation Test was performed with fatty acids, C16-18 and C18-unsaturated, branched and linear, butyl esters (CAS 163961-32-8) in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) according to OECD TG 476 and under GLP (Flanders, 2007). The cells were treated with the test substance in duplicate, together with vehicle (acetone) and positive controls. 4 hour exposures were used both with and without activation in experiment l. In experiment II, the exposure time without activation was increased to 24 hours. A confirmatory third experiment was performed due to a statistically significant response being observed in the lower / mid-dose range in the presence of metabolic activation in experiment II that had not been seen in experiment I. The dose range of test material in the first and second experiment was 156.25 to 5000 μg/mL following the results of a preliminary toxicity test without evidence of toxicity. The confirmatory experiment III was performed using the dose range of 39.06 to 1250 μg/mL. A precipitate of test material was observed at and above 78.13 μg/mL. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant increases in the mutant frequency at any dose level in any of the exposure groups, which included dose levels up to and including the maximum recommended dose level of 5000 μg/mL. Thus, the test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Taken together, the available data on genetic toxicity from structural analogue substances do not indicate any mutagenic and clastogenic potential. Therefore, according to EU classification criteria, the target substance isobutyl laurate (CAS 37811-72-6) is not to be classified.

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.

Short description of key information:
Genetic toxicity in vitro:
Bacterial reverse mutation assay (WoE, Ames test / OECD 471): negative (RA CAS 110-27-0 and RA CAS 163961-32-8)
In vitro chromosome aberration test in human lymphocytes (WoE, CA / OECD 473): negative (RA CAS 10233-13-3 and RA CAS 163961-32-8)
In vitro gene mutation assay in mouse lymphoma cells (WoE, MLA / OECD 476): negative (RA CAS 10233-13-3 and RA CAS 163961-32-8)

Endpoint Conclusion: No adverse effect observed (negative)

References:

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within the CSR.

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 isobutyl laurate (CAS 37811-72-6), 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.