Alcohols, C11-15-secondary, ethoxylated

Endpoint summary

Additional information

Gene mutation in bacteria

In this in vitro assessment of the mutagenic potential of Softanol 30, realised according to OECD guideline 471 and in compliance with GLP, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to Softanol 30 diluted in dimethyl sulphoxide (DMSO) (HLS 2010, PLZ0021). DMSO was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of Softanol 30 up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to Softanol 30.

No evidence of mutagenic activity was seen at any concentration of Softanol 30 in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

It is concluded that Softanol 30 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

 

 

Chromosomal aberration in vitro

A study was performed to assess the ability of Softanol 30 to induce chromosomal aberrations in human lymphocytes cultured in vitro, in a protocol realised according to OECD guideline 473 and in compliance with GLP (HLS 2010, PLZ0022).

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also included.

Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

In order to determine the toxicity of Softanol 30 to cultured human lymphocytes, the mitotic index was assessed for all cultures treated with the test substance and the solvent control, ethanol. A final concentration of 2004 μg/mL, dosed at 1%v/v, was used as the maximum concentration in order to test up to the maximum concentration that did not cause a change in osmolality of greater than 50 mOsm/kg. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test

In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 30, 40 and 50 μg/mL.

In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 60, 80 and 100 μg/mL.

Second test

In the absence of S9 mix - 21 hour continuous treatment: 5, 18 and 20 μg/mL.

In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 140, 155 and 160 μg/mL.

In both the absence and presence of S9 mix, Softanol 30 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test.

A quantitative analysis for polyploidy was also determined quantitatively for all cultures used in the analysis for chromosomal aberrations in the presence of S9 mix (5%v/v). A statistically significant increase in the proportion of polyploid cells was observed, when compared to the solvent control. The increase was within the laboratory historical control and therefore considered to be of questionable biological relevance.

All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. It is concluded that Softanol 30 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

 

Gene mutation in mammalian cells

Softanol 30 was tested for mutagenic potential in an in vitro mammalian cell mutation assay, in a protocol realised according to the OECD 476 guideline and in compliance with GLP (HLS 2010, PLZ0023).

The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Softanol 30 was found to be soluble at 334 mg/mL in ethanol (1M). A final concentration of 1670 μg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the maximum concentration that did not cause a change in osmolality of greater than 50 mOsm/kg.

Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to Softanol 30 at concentrations from 3.26 to 1670 μg/mL, relative suspension growth (RSG) was reduced from 111 to 0% and from 112 to 0% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 106 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3 hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presences of S9 mix were 45 and 70 μg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 11 and 16% respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 40 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed at concentrations up to 40 μg/mL, where RTG was reduced to 15%.

In all tests the concurrent vehicle and positive control were within acceptable ranges. It was concluded that Softanol 30 did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Short description of key information:
A gene mutation on bacteria (HLS 2010, PLZ0021), a chromosomal aberration test on human lymphocytes (HLS 2010, PLZ0022) and a gene mutation test on mammalian cells gave negative result (HLS 2010, PLZ0023).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

A gene mutation test on bacteria, a chromosomal aberration test on human lymphocytes and a gene mutation test on mammalian cells gave negative result. Therefore, Softanol 30 is not classified for mutagenicity.