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Additional information

In Vitro data

The in vitro genotoxicity of tertiary butyl alcohol has been investigated in four Ames tests, a mammalian cell gene mutation assay (mouse lymphoma assay), a chromosome aberration study, a sister chromatid exchange study and a comet assay. The majority of studies were non-guideline, but were conducted using methods considered equivalent to guideline.

The majority of the Ames tests were negative. A positive result was reported in strain TA 102 in the presence of metabolic activation in the published Williams-Hill et al (1999) study. In this study, the number of revertants increased in a dose dependent manner up to approximately 2.75 mg/plate. At this dose level, the number of revertants had almost doubled. At higher doses the number of revertants decreased dose dependently. Although the authors concluded tertiary butyl alcohol was mutagenic, no criteria as to what constituted a positive result were included in the paper and no information on toxicity was provided, which is imperative given the very high dose level employed. In follow up, the mutagenic potential of tertiary butyl alcohol in strain TA102 was investigated in a study conducted at two independent laboratories (McGregor et al (2005)). The first laboratory used a pre-incubation method to restrict evaporation, whereas the second used the plate incorporation procedure (as was used in the Williams-Hill study). Both studies were conducted in accordance with OECD 471 in GLP-accredited laboratories. No significant increase in the numbers of mutations per plate was observed in either laboratory. Given the increase reported in the Williams-Hill study was less than 2-fold and the results of the follow-up studies showed no significant increase in mutations, tertiary butyl alcohol is not considered mutagenic in bacteria.

The mutagenic potential of tertiary butyl alcohol was investigated in mammalian cells in a mouse lymphoma assay (McGregor et al (1988)). In this study, a small dose-related increase was observed in one study conducted in the absence of S9; however, the increase was only 1.6-fold at the top dose (5000 ug/plate) and, as such, is not considered to constitute a positive result. The results of both trials conducted in the presence of metabolic activation were clearly negative. Overall, tertiary butyl alcohol does not appear to be mutagenic in mammalian cells.

The results of a chromosome aberration study (non-guideline) and a sister chromatid exchange study have been included in NTP summary of tertiary butyl alcohol. A weak positive result was observed in one test of the SCE assay; however, as this result was not reproducible, the overall result of the study is considered negative (in accordance with the criteria outlined in OECD guideline 476). There were a number of deficiencies in the in vitro chromosome aberration study; however, a statistically significant increase in the number of cells with aberrations was observed at the top dose in one trial of the chromosome aberration study (6 % of cells had aberrations compared to none in the controls) in the presence of S9. The increase is small compared to the positive control, was not dose-related, and may have been exacerbated by the absence of aberrations in control cells. On this basis the biological significance of this increase is doubtful. This conclusion is supported by the negative response observed in the in vivo micronucleus study (see discussion below). Severe toxicity in the repeat trial meant that only 13 metaphases could be scored at the top dose.

A dose-related positive response was reported in an in vitro comet assay following incubation of tertiary butyl alcohol with HL-60 cells for one hour (Tang, 1997). No OECD guideline is available for the in vitro comet assay and at the time the methodology is unlikely to have been well developed. There are a number of issues with the reporting and interpretation of the data that reduce confidence in the result. Firstly, the results are presented as % DNA damage; however, it is not clear what this relates to. No information on the % DNA present in the tail or tail length or the number of cells undergoing apoptosis has been reported (a minimum requirement for acceptance of an in vivo comet assay by EFSA: http://www.efsa.europa.eu/en/efsajournal/doc/ 2977.pdf). Furthermore, the results of three separate experiments have been grouped together and no information on the variability of the data is available to determine whether this approach was justified. Finally, there are concerns with the assessment of cytotoxicity (estimated by measuring % LDH released). The authors concluded that tertiary butyl alcohol was not cytotoxic based on a similar % of LDH released in treated cells (3.62 – 4.49 %) and controls (3.29 %). However, similar levels of LDH release were also reported for other substances in the same paper, but in these cases (due to a comparison to the internal control) the extent (2.79 - 4.15 %) was considered indicative of cytotoxicity, raising doubts over the use of LDH as a measure of cytotoxicity. Overall, the results of this study are not considered reliable.

In Vivo Data

One study investigating the potential for tertiary butyl alcohol to cause cytogenetic damage to peripheral blood cells of mice following 13 week exposure is available. No increase in micronucleus formation was observed following oral administration of very high doses of tertiary butyl alcohol. No change in the P/N ratio was observed; however, detection of tertiary butyl alcohol in the blood (see section 5.1) suggests the bone marrow will have been exposed. In addition, deaths in the high dose group suggest the maximum tolerated dose was exceeded.

Summary

There are a number of studies available investigating the mutagenic potential of tertiary butyl alcohol in vitro and in vivo. Although a positive response was reported in the Ames test for strain TA102, this result was not replicated in two tests conducted in two independent GLP-accredited laboratories and, therefore, tertiary butyl alcohol is not considered mutagenic in bacteria. The results of the other in vitro studies did not provide any convincing evidence that tertiary butyl alcohol was mutagenic. The results of an in vivo micronucleus study indicate tertiary butyl alcohol is not clastogenic or aneugenic.

Overall, tertiary butyl alcohol is not considered genotoxic.

Short description of key information:

Several in vitro and in vivo tests are available and the total weight of evidence available indicates that tertiary butyl alcohol is not expected to induce heritable mutations in the germ cells of humans.  

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The mutagenic profile of tertiary butyl alcohol has been investigated in vitro and in vivo. Although a positive response was reported in the Ames test strain for TA102, the result was not replicated in tests conducted in two independent GLP accredited laboratories. Tertiary butyl alcohol is not considered mutagenic in bacteria. The results of the other in vitro studies and the in vivo study did not provide any convincing evidence that tertiary butyl alcohol is genotoxic. Overall, the genotoxic potential of tertiary butyl alcohol has been adequately investigated and tertiary butyl alcohol is not considered genotoxic. No classification is warranted.