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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item, n-butyl-3 -hydroxybutyrate was assessed for its potential to induce mutations in the bacterial reverse mutation (AMES) assay. In addition, the related compound isopropyl-3 -hydroxybutyrate was used to assess its potential to induce mutations in the mouse lymphoma thymidine kinase assay and for its potential to induce chromosome aberrations.

The AMES Assay was conducted to evaluate the potential of the test substance, n-butyl-3- hydroxybutyrate, to induce reverse mutations in histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in S. typhimurium and E. coli, respectively. An initial range finding study was conducted to determine toxicity and select test concentrations for the definitive assay. The test substance was dissolved in Dimethyl Sulfoxide (DMSO) and tested at 5, 1.6, 0.5, 0.18, 0.06 and 0.02 IJL per plate using TA98 strain in the absence of metabolic activation. The results of the range finding assay showed that the test substance was not cytotoxic at 5, 1.6, 0.5, 0.18, 0.06 and 0.02 IJL per plate. The definitive assay was conducted using 5, 1.6, 0.5, 0.18, 0.06 and 0.02 IJL per plate of the test substance. The definitive assay was conducted with four strains of S. typhimurium and one strain of E. coli in the presence or absence of an exogenous mammalian metabolic activation system using plate incorporation method of exposure. The results of the definitive assay showed that the test substance did not increase the frequency of revertants at any of the test concentrations in any of the strains tested in the presence and absence of metabolic activation. The positive controls exhibited a significant increase in the frequency of revertants as compared to the negative controls in the presence and absence of metabolic activation validating functioning of the assay. A confirmatory assay was performed in order to verify the results of the definitive reverse mutation assay using preincubation method of exposure. The results of the confirmatory assay were consistent with the definitive assay. Under the test conditions, the test substance is non mutagenic in the test species.

The mouse lymphoma thymidine kinase locus utilized the cell line L5178Y The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 9.627 mM (with metabolic activation) and I 0.0 mM (without metabolic activation) were selected as the highest concentrations. In experiment II 10.0 mM (with and without metabolic activation) was selected as the highest concentration. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay. In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories [13, 14, 15]) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

A chromosome aberration assay was carried out in order to investigate a possible potential of Isopropyl-3-hydroxybutyrate for its ability to induce structural chromosome aberrations in Human Lymphocytes. The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation (experiment I) and 4 h with and 24 h without metabolic activation (experiment II). Two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated: Experiment I: Without and with metabolic activation, 4 h treatment, 24 h preparation interval: 2.5, 5.0 and 10.0 mM Experiment II: Without metabolic activation, 24 h treatment, 24 h preparation interval: 2.5, 5.0 and 10.0 mM With metabolic activation, 4 h treatment, 24 h preparation interval: 4.0, 8.0 and 10.0 mM No precipitation of the test item was noted without and with metabolic activation in all dose groups evaluated in experiment I and II. No cytotoxic effects of the test item were noted without and with metabolic activation in all dose groups evaluated in experiment I and II. In experiment I and II no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. EMS (400 and 900 f.lg/mL) and CPA (5 f!g/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

 

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Isopropyl-3-hydroxybutyrate did not induce structural chromosomal aberrations in human lymphocyte cells. Therefore, Isopropyl-3-hydroxybutyrate is considered to be non-clastogenic in this chromosome aberration test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification