Isobutyl acetate

Endpoint summary

Additional information

Genetic toxicity in vitro

 

Various studies have been identified to assess the genetic toxicity potential of isobutyl acetate and supporting substances like isobutanol and n-butalyacetate. In all studies, no evidence was found for mutagenic/genotoxic potential. (For details on read-across hypothesis and justification please see read-across report in section 13).

 

Reverse gene mutation assay in bacteria

This endpoints requirements are covered by using valid bacterial reverse gene mutation assays with supporting substances isobutanol and n-butyl acetate as well as QSAR predictions from the QSAR Toolbox.

Mutagenic activity of n-butyl acetate (purity: 99.6%) and iso butanol (purity: 99.7%) was investigated in a bacterial reverse gene mutation assay according to Ames (1975, similar/equivalent to OECD TG 471). The tests were performed using Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA1538 and TA100 as well as Escherichia coli strain WP2uvrA with (polychlorinated biphenyl (KC 500) induced rat liver S9 mix) and without metabolic activation at various substance concentrations up to 5000 µg/plate using the preincubation method. Both test items did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagens showed distinct positive mutagenic effects (Shimizu et al., 1985; RL2).

A QSAR prediction with OECD QSAR Toolbox v3.1 was performed. A category comprising isobutylacetate (target substance), isobutanol (source) and n-butyl acetate (source) was formed (category verfication via profiling reults obtained in QSAR Toolbox, for details cf. Toolbox generated report). Based on the results for these substances a negative test result in bacterial reverse mutation assay with E. coli WP2uvrA was predicted for isobutuyl acetate (Toolbox prediction, 2013, RL2).

Studies using isobutyl acetate lack testing with required 5th strain (either Salmonella typhymurium TA 102 or Escherichia coli WP2uvrA

to identify certain oxidizing mutagens, cross-linking agents, or hydrazines). Thus even tough these tests are of high quality (well performed studies performed to former versions of OECD TG 471, Hoechst Celanese study GLP conform) they are insufficient to cover the endpoints' requirements (RL3). In these two studies five strains of S. typhimurium were exposed to isobutyl acetate at various concentrations either up to concentrations of 5000 or even 10,000 µg/plate in the presence and absence of mammalian metabolic activation. There was no evidence of induced mutant colonies over background. Isobutyl acetate was negative under test conditions (Huels AG, 1988, RL3; Hoechst-Celanese (GLP), 1989, RL3). These resulst supporth the findings from studies of the WoE approach.

 

Cytogenicity study in mammalian cells

In a valid mammalian cell cytogenetics assay (Chromosome Aberration test according to OECD TG 473) performed under GLP, V79 Chinese hamster lung cell (CHL) cultures were exposed to isobutyl acetate (purity: pure active substance) at 10 different concentrations between 25 and 5000 µg/L with and without metabolic activation. Three of the tested concentrations (600, 3000 and 5000 µg/L) were scored for chromosomal aberration induction.

 There was no evidence of the induction of chromosomal aberrations over background. Isobutyl acetate is shown to be not clastogenic in vitro (Huels AG, 1996, RL2).

 

Gene mutation study in mammalian cells

For isobutyl acetate, no study concerning mutagenicity in mammalian cells could be identified.

As substitute, data for isobutanol will be used (read-across from supporting substance).

 

In a mammalian cell gene mutation assay (forward mutation assay, HPRT gene, performed according to OECD TG 476), V79 Chinese hamster lung fibroblast cells cultured in vitro were exposed to 2-methylpropan-1-ol (isobutanol, commercial grade, Riedel de Haen) at concentrations up to the highest non toxic concentration of 107 mM (7930 µg/mL) in the presence and absence of mammalian metabolic activation (S9-mix of Aroclor 1254 induced rat liver).

 The highest test concentration exceeds the maximum concentration according to the OECD test guideline 476. The positive controls (0.5 mM methyl methansulfonate) did induce the appropriate response. There was no evidence of induced mutant colonies over background. 2 -Methylpropan-1 -ol is not mutagenic in this assay (Kreja 2002, RL2).

 

Genetic toxicity in vivo

 

For isobutyl acetate, no study concerning genetic toxicity in vivo could be identified. As substitute, data for isobutanol will be used (read-across from supporting substance).

The genetic toxicity in vivo of isobutanol has been evaluated in one valid study of high reliability (GLP study) which is used as key study.

 In this mouse bone marrow micronucleus test (MNT according to OECD TG 474 and GLP conform), 5 NMRI-mice per sex/dose were treated orally with isobutanol (purity: 99.87%) at doses of 0, 500, 1000, and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post treatment. The test substance was administered orally dissolved in olive oil in single doses.

 There were signs of toxicity during the study. Isobutanol was tested at adequate doses. The positive controls induced the appropriate responses.

 There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any dose and any treatment time. Under the experimental conditions of the test, isobutanol had no chromosome damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (CMA/BASF, 2000, RL2).

  

In summary, it is concluded that isobutyl acetate lacks a potential for genetic toxicity. The information basis underlying this conclusion comprises valid studies conducted in-vitro and in-vivo on isobutyl acetate, or on closely related substances isobutanol (a metabolite that is rapidly formed, cf. section 7.1) and n-butyl acetate (cf. section 7.1) as tabulated below.

 

 

Test substance	Source	Test method	Result	Reliabilty
Bacterial cell test systems
Isobutyl acetate	Hoechst-Celanese (1989)	Bacterial cells Ames test; 5 strains of S. typhimurium; w/wo metabolic activation	negative	3
Isobutyl acetate	Huels AG (1988)	Bacterial cells Ames test; 5 strains of S. typhimurium; w/wo metabolic activation	negative	3
Isobutanol	Shimizu (1985)	Bacterial cells Ames test; 5 strains of S. typhimurium and E. Coli WP2 uvrA w/wo metabolic activation	negative	2
n-butyl acetate	Shimizu (1985)	Bacterial cells Ames test; 5 strains of S. typhimurium and E. Coli WP2 uvrA w/wo metabolic activation	negative	2
Isobutyl acetate	Lazar (2013	In silico prediction; OECD Toolbox v 3.1	negative	2
Mammalian cell test systems in-vitro and in-vivo
Isobutanol	Kreja and Seidel (2002)	Mammalian cell mutagenicity in-vitro; V79 cells; w/wo metabolic activation	negative	2
Isobutanol	CMA/BASF (2000)	Mammalian cells in vivo; OECD 474 EU method B.12; erythrocyte micro-nucleus test	negative	2
Isobutyl acetate	Huels AG (1996)	Chromosome aberration; OECD 473 EU method B.10 Mammalian cells (V79); w/wo metabolic activation	negative	1

 

Justification for selection of genetic toxicity endpoint
No study was selected since, all available data (covering the data requirements) were negative.

Short description of key information:
Genetic toxicity in vitro
No valid bacterial reverse gene mutation assays were located for isobutyl acetate (available testes were missing the required 5th strain, however both available AMES studies negative with and without metabolic activation). Results from supporting substances isobutanol and n-butylacetate as well as a QSAR prediction were used in a weight-of-evidence approach instead. None of the tests (with and without metabolic activation) or the QSAR prediction revealed any evidence for mutagenic potential in bacteria.
In addition, isobutyl acetate did not show genotoxic potential in vitro in mammalian cells (CHL) with and without metabolic activation in a Chromosomal Aberration test.
In a gene mutation study in mammalian cells (HPRT mutation test) with and without metabolic acitivation using isobutanol as supporting substance, no evidence of mutagenic potential was observed.

Genetic toxicity in vivo
No data for isobutyl acetate could be located. Data for isobutanol are used as supporting substance.
In a valid mouse micronucleus test, isobutanol did not increase the rate of micronuclei in polychromatic and normochromatic erythrocytes at the three doses tested.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative results obtained in all tests for genotoxicity performed with isobutyl acetate in vitro and with supporting susbtances isobutanol (metabolite) and n-butyl acetate (structural analogue) in vivo and in vitro it is concluded that isobutyl acetate has not to be classified for genotoxicity according to Regulation (EC) No 1272/2008.