Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Justification for grouping of substances and read-across

The short chain methyl esters category (SCAE Me) covers fatty acid esters of methanol. The category contains both mono-constituent substances, with fatty acid C-chain lengths ranging from C6 to C18 and UVCB substances, composed of single methyl esters in variable proportions.

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate, environmental and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, 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 the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Sections 7.1 and 13) and within Chapter 5.1 of the CSR.

Table: Endpoint genetic toxicity

CAS

Genetic Toxicity in-vitro

Gene mutation in bacteria

Genetic Toxicity in vitro

Chromosome aberration in mammalian cells

Genetic Toxicity in vitro

Gene mutation in mammalian cells

106-70-7 (a)

Experimental result: not mutagenic

RA: 111-82-0 and 67762-38-3

RA: 111-82-0 and 124-10-7

111-11-5

RA: 106-70-7

RA: 111-82-0 and 67762-38-3

RA: 111-82-0 and 124-10-7

110-42-9

RA:

68937-83-7

RA: 111-82-0 and 67762-38-3

RA: 111-82-0 and 124-10-7

111-82-0

Experimental result: not mutagenic

Experimental result: not clastogenic in human lymphocytes

Experimental result: not mutagenic in mouse lymphoma cells

124-10-7 (b)

--

--

Experimental result: not mutagenic in mouse lymphoma cells

112-39-0

Experimental result: not mutagenic

RA: 111-82-0 and 67762-38-3

RA: 111-82-0 and 124-10-7

112-61-8

Experimental result: not mutagenic

RA: 111-82-0

RA: 111-82-0 and 124-10-7

85566-26-3

RA:

106-70-7 and 111-82-0

RA: 111-82-0

RA: 111-82-0

308065-15-8

RA: 111-82-0 and 544-35-4

RA: 111-82-0, 544-35-4 and 67762-38-3

RA: 111-82-0, 124-10-7 and 544-35-4

68937-83-7

Experimental result: not mutagenic

--

--

1234694-02-0

RA: 111-82-0 and 544-35-4

RA: 111-82-0, 544-35-4 and 67762-38-3

RA: 111-82-0, 124-10-7 and 544-35-4

85586-21-6

RA: 112-61-8, 111-82-0 and 544-35-4

RA: 111-82-0 and 544-35-4 and 67762-38-3

RA: 111-82-0, 124-10-7 and 544-35-4

67762-38-3

Experimental result: not mutagenic

Experimental result: not clastogenic in human lymphocytes

--

544-35-4 (c)

Experimental result: not mutagenic

Experimental result: not clastogenic in human lymphocytes

Experimental result: not mutagenic in mouse lymphoma cells

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

(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 “--“.

(c)   Surrogate substances are either chemicals forming part of a related category of structurally similar fatty acid esters or precursors/breakdown products of category members (i.e. alcohol and fatty acid moieties). Available data on these substances are used for assessment of (eco )toxicological properties by read-across on the same basis of structural similarity and/or mechanistic reasoning as described below for the present category.

 

Discussion

In vitro gene mutation study in bacteria

 

CAS 111-82-0 

Two studies investigating the in vitro gene mutation in bacteria of methyl laurate (CAS 111-82-0) are available.

An Ames test was performed according to OECD Guideline 471 with methyl laurate (CAS 111-82-0) dissolved in acetone in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and with E. coli WP2uvr A (National Institute of Health Sciences 2000). Test substance concentrations of 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/plate (TA 100 and TA 1537); 6.25, 12.5, 25, 50, 100 and 200 µg/plate (TA 1535 and TA 98); 313, 625, 1250, 2500 and 5000 µg/plate (E. coli WP2uvr A) without metabolic activation and 12.5, 25, 50, 100, 200 and 400 µg/plate (TA 1537); 25, 50,100, 200, 400 and 800 (TA 100 and TA 1535); 50, 100, 200, 400, 800 and 1600 (TA 98); 313, 625, 1250, 2500 and 5000 µg/plate (E. coli WP2 uvr A) with metabolic activation were tested in two independent experiments. Cytotoxic effects were observed in the absence of a metabolic activator at 25 µg/plate (TA 100 and TA 1537), 50 µg/plate (TA 1535 and TA 98) and in the presence of a metabolic activator (Phenobarbital- and 5,6-benzoflavone-induced rat liver S9) at concentrations of 150 µg/plate (TA 1537), 400 µg/plate (TA 100), 500 µg/plate (TA 1535) and 800 µg/plate (TA 98). 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, methyl laurate did not induce gene mutations in all tested strains under the given test conditions.

 

Another Bacterial Reverse Mutation Assay was performed with methyl laurate (CAS 111-82-0) according to OECD Guideline 471 (Banduhn, 1992). Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with methyl laurate diluted in acetone using theplate incorporation method. Two independent experiments were performed with identical dose levels of 0, 8, 40, 200, 1000 and 5000 µg/plate. Both experiments were performed in triplicates with and without the addition of a rat liver homogenate metabolising system (S9-mix).

Cytotoxic effects were observed at concentration of 5000 μg test substance per plate. 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, methyl laurate did not induce gene mutations in five tested Salmonella strains under the given test conditions.

 

CAS 112-39-0

Methyl palmitate (CAS 112-39-0) was tested for mutagenicity in S. tyhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 according to OECD Guideline 471 (Banduhn, 1992). Test substance concentrations of 0, 8, 40, 200, 1000 and 5000 µg/plate in acetone were tested in triplicates in two independent experiments using the plate incorporation method with and without the addition of a rat liver homogenate metabolising system (S9-mix).

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, methyl palmitate did not induce gene mutations in five tested Salmonella strains under the given test conditions. 

CAS 68937-83-7

The bacterial mutagenicity of fatty acids, C6-10, methyl esters (CAS 68937-83-7) was tested according to OECD Guideline 471 in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (Banduhn, 1988). Test substance concentrations of 0, 8, 40, 200, 1000 and 5000 µg/plate were used in the first experiment. In the second experiment 1.25, 5, 20, 80 and 360 µg/plate were used without S9-mix and 3.1, 12.5, 50, 200 and 800µg/plate were used with S9-mix. The test substance was dissolved in Tween 80/water.

Cytotoxicity occurred in the presence and absence of metabolic activation at test substance concentrations above 200 μg/plate. 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, C6-10, Me esters did not induce gene mutations in five tested Salmonella strains under the given test conditions.

 

CAS 106-70-7

A Bacterial Reverse Mutation Assay was performed with methyl hexanoate (CAS 106-70-7) according to OECD Guideline 471 in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (Banduhn, 1992). Test substance concentrations of 0, 8, 40, 200, 1000 and 5000 µg/plate in DMSO were tested in triplicates in two independent experiments using the plate incorporation method with and without the addition of a rat liver homogenate metabolising system (S9-mix).

Cytotoxic effects were observed at concentration of 5000 μg test substance per plate. 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, methyl hexanoate did not induce gene mutations in five tested Salmonella strains under the given test conditions.

 

CAS 112-61-8

Another Ames test was performed with methyl stearate (CAS 112-61-8) according to OECD Guideline 471 in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (Banduhn, 1992). Test substance concentrations of 0, 8, 40, 200, 1000 and 5000 µg/plate in acetone were tested in triplicates in two independent experiments using the plate incorporation method with and without the addition of a rat liver homogenate metabolising system (S9-mix).

Cytotoxic effects were observed at concentration of 5000 μg test substance per plate. 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, methyl stearate did not induce gene mutations in five tested Salmonella strains under the given test conditions.

  

CAS 67762-38-3

The substance Fatty acids, C16-18 and C18-unsatd., methyl esters (CAS 67762-38-3) was tested according to OECD Guideline 471 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 (Haddouk, 1999). Test substance concentrations of 0, 62.5, 125, 500 and 1000 µg/plate in a first experiment and concentrations of 0, 312.5, 625, 1250, 2500 and 5000 µg/plate in ethanol were used in two independent experiments.

Cytotoxic effects were not 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, Fatty acids, C16-18 and C18-unsatd., Me esters did not induce gene mutations in five tested Salmonella strains under the given test conditions.

 

CAS 544-35-4

Another Ames test was performed with 9,12-Octadecadienoic acid (Z,Z)-, ethyl ester (CAS 544-35-4) according to OECD guideline 471 with the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the E. coli strain WPA2uvr A (BASF, 2010). The bacterial tester strains were treated with 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate of the test substance in absence and presence of metabolic activation by rat liver S9-mix. The test substance slightly precipitates at concentrations ≥ 1000 µg/plate but induces no cytotoxic or genotoxic effects at any concentration neither in the presence nor in the absence of metabolic activation. Based on the study results,

9,12-Octadecadienoic acid (Z,Z)-, ethyl ester did not induce gene mutations in four tested Salmonella and one tested E. coli strains.

 

In vitro cytogenicity in mammalian cells

CAS 111-82-0 

An in vitro mammalian chromosome aberration test was performed with methyl laurate (CAS 111-82-0) in primary human lymphocytes according to OECD Guideline 473 (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 test substance concentrations of 33, 100 and 200 µg/mL in ethanol were used for 3 hours of exposure with and without metabolic activation. In the second experiment 100, 120 and 140 µg/mL were used for 24 hours exposure followed by 24 hours expression time and 30, 120 and 140 µg/mL for 48 hours exposure following 48 hours expression time without S9. 200 µg/mL was chosen as maximum dose due to limited solubility. Mitomycin C and cyclophosphamide were used as positive control substances. 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. 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.

 

A second, supporting in vitro mammalian chromosome aberration test was performed with methyl laurate (CAS 111-82-0) in chinese hamster cells similar to OECD Guideline 473 (National Institute of Health Sciences, 2000). The test material was evaluated for generation of chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment test substance concentrations of 0, 15, 30 and 60 µg/mL in acetone were used for 24 and 48 hours of exposure without metabolic activation. In the second experiment 0, 0.53, 1.1 and 2.1 mg/mL were used for 6 hours exposure followed by 12 hours expression time without metabolic activation and 0, 0.025, 0.05 and 0.1 mg/mL for 6 hours exposure following 12 hours expression time with metabolic activation. Mitomycin C and cyclophosphamide were used as positive control substances. 200 cells per dose were analysed for chromosome aberrations. All vehicle controls had frequencies of cells with aberrations within the expected range. 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 the two separate experiments. The test material was therefore considered to be non-clastogenic to chinese hamster cells in vitro.

 

CAS 67762-38-3

An in vitro mammalian chromosome aberration test was performed with Fatty acids, C16-18 and C18-unsatd., Me esters (CAS 67762-38-3) in primary human lymphocytes according to OECD Guideline 473 (Haddouk, 2000). Two independent experiments (with 3 or 20 and 44 hours of exposure) were performed in the absence and presence of S9-mix. In the first experiment test substance concentrations of 18.96, 37.93, 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml and in the second experiment concentrations of 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml with and without metabolic activation were used. The test substance did not induce any significant increase in the frequency of cells with chromosome aberrations in both experiments and at both harvest times, with and without S9 mix.

CAS 544-35-4

The ability of ethyl linoleate (CAS 544-35-4) to induce chromosome aberrations in cultured peripheral human lymphocytes was tested according to OECD guideline 473 (Verbaan, 2010) in two independent experiments. Test substance concentrations of up to 800 µg/mL dissolved in DMSO were tested in the presence and absence of metabolic activation. At concentrations of 333 µg/mL and higher precipitation of test substance occurred. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Ethyl linoleate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed.

In vitro gene mutation in mammalian cells

CAS 111-82-0 

An in vitro mammalian cell gene mutation assay was performed with methyl laurate (CAS 111-82-0) according to OECD guideline 476 in L5178Y mouse lymphoma cells (Verspeek, 2010). In the first experiment, methyl laurate was tested up to concentrations of 95 and 200 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. Methyl laurate was tested up to cytotoxic levels of 85 and 91% in the absence and presence of S9-mix, respectively. In the second experiment, methyl laurate was tested up to concentrations of 70 and 220 μg/ml in the absence and presence 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. Methyl laurate was tested up to a cytotoxic level of 90% in the absence of S9-mix and up to 88% in the presence of S9-mix. 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 12- and 8.4-fold for MMS in the absence of S9-mix, and by 17- and 15-fold for CP in the presence of S9-mix. In the absence of S9-mix, methyl laurate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, methyl laurate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that methyl laurate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

CAS 124-10-7

The mutagenic activity of methyl myristate (CAS 124-10-7) was evaluated in an in vitro mammalian cell gene mutation test with mouse lymphocytes (Baxter and Fish, 1981). After a test substance incubation time of 48 hours, 3H-thymidine were added to the cultures for 24 hours. The authors stated that methyl tetradecanoate had no proliferative effect. However, if the test substance was co-incubated with lectin (PHA) in mouse lymphocytes, a comitogenic effect was observed.

 

CAS 544-35-4

The mutagenic activity of ethyl linoleate (CAS 544-35-4) was evaluated in an in vitro mammalian cell gene mutation test according to OECD guideline 476 with L5178Y mouse lymphoma cells (Verspeek-Rip, 2010). Two independent experiments (with 3 or 24 hours of exposure) were performed in the absence and presence of S9-mix with test substance concentrations up to 300 μg/mL dissolved in dimethyl sulfoxide. At this dose level cytotoxicity occurred in the presence and absence of metabolic activation. No significant increase in mutation frequency occurred in any of the test conditions, indicating that ethyl linoleate is not mutagenic in the mammalian cells in vitro.

 

In summary, based on a weight of evidence approach, all reliable studies consistently showed no treatment-related effects on genetic toxicity.

 

Conclusions:

In summary, several studies investigating the genetic mutation in bacteria in-vitro are available within the SCAE Me category, all providing 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 structurally related fatty acid short-chain alcohol esters.

All available data investigating the genetic toxicity indicate that members of the category Fatty acid methyl esters have no genotoxic potential and classification according to EU classification criteria for genetic toxicity is not required.

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


Short description of key information:
In neither of the available studies on substances of the SCAE Me category were any mutagenic or clastogenic effects in bacteria or mammalian cells observed.

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 group concept is applied to the members of the SCAE Me category, data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the group concept, 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.