Reaction mass of 2-methylbutyl acetate and pentyl acetate

Endpoint summary

Additional information

Some genetic studies were available with the reaction mass of 2-methylbutyl acetate and pentyl acetate available. Data were also adopted by read-across from isopentyl acetate (= isoamyl acetate; CAS: 123-92-2), an isomer of the reactant pentyl acetate (= amyl acetate; CAS: 628-63-7). Due to the structural similarities, the same results can be expected for the reaction mass of 2-methylbutyl acetate and pentyl acetate.The same principle apply for 3-Methylbutanol-1 (isoamylalkohol) used for as surrogate for in vivo genetic toxicity, which is an isomer of 2 -methylbutanol (one of the metabolic product of the reaction mass of 2 -methylbutyl acetate and pentyl acetate).

In vitro

- Bacteria gene mutation

In an Ames Test conducted equivalent or similar to the OECD Guideline 471 (Bacterial Reverse Mutation Assay using TA98, TA100, TA1535, TA1537 and TA1538), the test substance was tested in triplicate of 5 doses (0.01, 0.03, 0.1, 0.3 and 1.0 µl/plate, with and without metabolic activation, chosen because dose levels > 1 µl/plate inhibited all growth of the background lawn, while the 0.3 µl/plate allowed some sparse growth of the background lawn) chosen from a preliminary study to span a range which included moderately toxic to relatively nontoxic concentrations (Union Carbide Corporation, 1983). Concurrent solvent (ethanol) and positive (4-nitro-o-phenylenediamine, sodium azide, 9-aminoacridine or 2-aminoanthracene) controls were run in each test. No evidence of mutagenicity was observed at any of the dose levels evaluated, either by evidence of a dose-response relationship or a doubling of the number of colonies over the solvent control value.

An additional Ames test was performed using E.coli uvrA (BASF 2015), since the TA strains used in the previous Ames test do not detect certain oxidising mutagens, cross-linking agents and hydrazines. The test substance was tested in triplicate in doses up to 5300µg/plate with and without metabolic activation (rat S9). No cytotoxicity was observed in the standard plate test, but background growth was reduced in the pre-incubation assay at the two highest concentrations (2650 and 5300µg/plate). The test substance did not induce an increase in mutant colonies in any of concentration tested.

 

CAS No. 123-92-2

Isoamyl acetate was not mutagenic in a preincubation Ames test (according to OECD 471) with and without metabolic activation (tested up to10 mg/platein Salmonella typhimuriumTA 97, TA 98, TA 100, TA 1535, TA 1537; metabolic activation: liver S-9 mix from Aroclor 1254-induced Sprague-Dawley rats and Syrian hamsters; Zeiger, 1992). Cytotoxicity (reduction of the background lawn) was observedin part at the highest concentration(s) (depending on strain and activation system). In further tests isoamyl acetate was also not mutagenic with (Ishidate, 1984) with and without metabolic activation, respectively (McCarroll, 1985)

- Gene mutation in mammalian cells

In a HPRT Test conducted equivalent or similar to the OECD Guidelineto the OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test), Chinese hamster Ovary (CHO) cells were treated with 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20 or 0.40% test substance (vol/vol; for 24 hours) without S9 activation mix, or with 0, 0.04, 0.06, 0.08, 0.10, 0.20 and 0.30% (for 24 hours) in the presence S9 activation (Union Carbide Corporation, 1983). The dose levels were chosen based on the results of a preliminary test, where concentration of 1.0% produced complete lyses of CHO cells, and concentration of 0.5% allowed only 4.5% of the culture growth attained by the control cultures. The surviving fraction was determined at 24 to 28 hours after the start of the exposure to the test agents and the mutant fraction was determined after a 7 to 10 day period to allow "expression" of the mutant phenotype. Only the data from the top five concentrations which allowed sufficient cell survival for assessment of mutagenicity. Dimethylnitrosamine (DMN;CAS #62-75-9) and ethylmethanesulfonate (CAS #62 -50 -0) were used as positive control agents to assure the sensitivity of the test system for detecting metabolic activation dependent and independent mutagens, respectively. Cell culture medium was used as the negative control. Absolute ethyl alcohol (ETOH; CAS #64-17-5) was used as the solvent for dilutions and appropriate aliquots were tested as the solvent control.

The test substance did not produce repeatable, significant mutagenic effects in the CHO test system. Tests were performed with and without the incorporation of an S9 metabolic activation system over a range of concentrations which included doses which produced a marked cytotoxic effect. No dose-related or repeatable significant effects of the test chemical were observed in any of these tests. Therefore, the test substance was not mutagenic to CHO cells under the conditions of this assay.

 

CAS No. 123-92-2

Isoamyl acetate (not further specified) was tested in a TK-Assay with mouse lymphoma cells (with and without metabolism, 0.2 - 1 mg/ml). Also this test was negative (McCarroll, 1985).

 

- Cytogenicity in mammalian cells

In the mouse lymphoma test conducted equivalent or similar to the OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test), cultured lymphocyte cells obtained from Sprague-Dawley derived CD ISG (outbred Crl:CD(SD)) male rats were treated with test substance concentrations in DMSO of 20.5, 40.9, 81.9, 163.8, 327.5, 655 and 1310 µg/ml for 4 hour test without S9, 10.2, 20.5, 40.9, 81.9, 163.8, 327.5, 655 and 1310 µg/ml for 24 hour test without S9 or 20.5, 40.9, 81.9, 163.8, 327.5, 655 and 1310 µg/ml for 4 hour test with S9 (Union Carbide Corporation, 2006). The cell division was inhibited with colcemid (1 μg/culture) added 2-3 hours prior to harvesting, and the cells were then swollen by hypotonic treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa. Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. 100 metaphases per replicate were examined from coded slides for structural abnormalities (the number of cells examined for structural abnormalities was reduced to 50 metaphases per replicate when approx. 20% or higher numbers of aberrations were observed.

Based upon the mitotic indices, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 327.5, 655, and 1310 μg/ml in the absence and presence of S9 activation and cultures treated for 24 hours with 0 (solvent control), 163.8, 327.5, and 655 μg/ml were selected for determining the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in either the presence or absence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells in all assays. Based upon these results, the test material, primary amyl acetate-mixed isomers, was considered to be non-genotoxic in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

 

In an Union Carbide Corporation (1983) test conducted equivalent or similar to the OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells), Chinese hamster Ovary (CHO) cells were treated with 0, 0.06, 0.08 and 0.10 % v/v (for 5 hours without S9) or 0, 0.06, 0.10 and 0.20% v/v 2 hr (for 2 hours with S9) test substance dissolved in DMSO (toxicity of the test chemical was determined prior to assessment of mutagenic potential to select a range of doses which produce a maximum of 50 to 60% cell killing). The respective solvent is tested as a negative control at the maximum concentration used to add the test agent. Dimethylnitrosamine (DMN) and ethyl methanesulfonate are used as positive control mutagens for tests with or without the addition of an S9 metabolic activation system, respectively. Colcemid (0.1 µg/ml) or Colchicine (0.2 ug/ml) was added to culture flasks 1 to 2 hrs prior to harvesting to arrest cells in mitosis. Cells were then fixed with Carnoy's fixative (3:l methanol acetic acid) and chromosome spreads prepared from cells suspended in a small volume of fixative. Chromosomes are stained for SCE's by treatment with 5.0 µg/ml of Hoechst 33258 dye for 20 min, irradiated with high intensity sun lamp, and the chromosomes were stained in Gurr's giemsa (diluted 1:25 with water), rinsed in water and dried before application of coverslips. The number of chromosomes and the number of SCE's in a minimum of 25 cells/duplicate culture were recorded for each dose level, whereas doses which markedly inhibit cell division and/or SCE differentiation were recorded as cytotoxic (and SCE's not counted). The mean number of SCE/cell and SCE/chromosome were then calculated and recorded.

The test substance did not reproducibly affect the frequency of SCE over the range of concentrations tested with or without addition of an active S9 metabolic activation system. No dose-related effects of exposure on the SCE frequency were evident and the test agent did not produce significant genetic toxicity in the present in vitro assay.

 

CAS No. 123-92-2

A chromosomal aberration test in Chinese hamster fibroblasts without metabolic activation (test concentrations: 0.5, 1, 2 mg/ml, exposure period: 48 hours) was also negative (Ishidate, 1984). 

 

In vivo

CAS No. 123 -51 -3

The genetic toxicity in vivo was analyzed in a micronucleus assay performed according to OECD guideline 474 (BG-Chemie, 1999). Five male and five female NMRI mice received a single dose of 1500 mg/kg bw 3-methylbutan-1-ol in 0.25% aqueous Methocel K4M premium by gavage. Although the dose of 1500 mg/kg bw was severely toxic in a preceding range finding test, no mortality occurred. 24 or 48 hours after administration, the animals were sacrificed and bone marrow was prepared for analysis. As result, 3-methylbutan-1-ol did not produce a significant, exposure-related increase in the incidence of micronucleated polychromatic erythrocytes in male or female animals. Therefore, the test substance can be regarded as not mutagenic in vivo.

Short description of key information:
1) In Vitro tests
- Gene mutation in bacteria: negative; Ames test Union Carbide Corporation, 1983; BASF 2015, CAS No. 123-92-2: Zeiger, 1992);
- Cytogenicity in mammalian cells: negative; Chromosomal aberration test, without metabolic activation (The Dow Chemical, 2006; CAS No. 123-92-2; Ishidate 1984);
- Gene mutation in mammalian cells: negative; Mouse Lymphoma TK Assay (CAS No. 123-92-2: McCarroll, 1985), HPRT (Union Carbide Corporation, 1983);
- Sister chromatid exchange assay in mammalian cells: negative (Union Carbide Corporation, 1983).
2) in vivo tests
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test): negative (CAS No. 123-51-3; BG-Chemie, 1999)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the above results of the test substance or its surrogates, there is no indication of genetic toxicity potential according to the current regulation.

• GHS classification (REGULATION (EC) No 1272/2008 (CLP)):no classification required