Condensation products of oil shale alkylresorcinols and formaldehyde

Endpoint summary

Additional information

In vitro bacterial reverse mutation assay

The genetic toxicity of the test material was examined in an AMES bacterial reverse mutation assay using Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100, without and with S9 activation.

The method was designed to meet the requirements of the OECD 471, EU Method B.13/14 and the USA, EPA (TSCA) OPPTS harmonised guidelines. The test was conducted to GLP standard, in a certified laboratory.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9, initially from 1500 μg/plate.

The test material was considered to be non-mutagenic under the conditions of this test.

In vitro cytogenicity assay

The genetic toxicity of the test material was examined in an in-vitro chromosomal aberration test. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period.

The study was performed according to the OECD 473 and EU Method B.10 guidelines. The study was performed to GLP standard in a certified laboratory.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material was toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50 % mitotic inhibition.

The test material was considered to be non-clastogenic to human lymphocytes in vitro. Small but statistically significant increases in the frequency of polyploid cells were observed at two dose levels in the 4-hour without-S9 exposure group. No similar responses were observed in either of the 4-hour exposure groups with S9 or in the 24-hour exposure group without S9. It was considered that the polyploidy response had little or no biological significance.

In vitro mammalian cell gene mutation assay

The test material was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in Chinese hamster lung V79 cells plated into Petri dishes. The study consisted of a cytotoxicity Range-Finder Experiment followed by two main mutation experiments, each conducted in the absence and presence of metabolic activation by 20-methylcholanthrene induced rat liver post-mitochondrial fraction (S-9). An additional third mutation experiment was conducted with extra concentrations.

In Experiment 1 in the absence of S-9, no statistically significant increase in mean mutant frequency (MMF) was observed following treatment with the test substance at any concentration tested. Cytotoxicity (expressed in terms of % RPE at the end of treatment) was reduced to ca. 22 % at 60 µg/mL.

In the presence of S-9, exposure of V79 cells to concentrations of the test substance up to 120 µg/mL for 3h resulted in a negative response. No cytotoxic effects of test item were observed in the concentration range of 7.5 - 120 µg/mL.

In Experiment 2 in the absence of S-9, statistically significant increase in mean mutant frequency was observed following treatment with the test material at a concentration of 60 µg/mL only. At this level RPE was reduced to ca. 16 %. Whilst the increase in MMF was greater than 3- fold above that of the concurrent vehicle control, the increase was accompanied by marked cytotoxicity with no evidence of an increase in MMF at any of the other concentrations tested. In the presence of S-9, no statistically significant increase in mean mutant frequency was found following treatment with the test substance at any concentration tested. No cytotoxic effects of test item were observed in the tested concentration range of 7.5 - 120 µg/mL.

In additional Experiment 3 in the absence of S-9, no statistically significant increase in mean mutant frequency (MMF) was observed following treatment with the test material at any tested concentration. Cytotoxicity (expressed as % RPE) at the end of treatment was ca. 17 %, ca. 14 % and ca. 12 % at 60, 65 and 70 µg/mL, respectively.

In the presence of S-9, exposure of V79 cells to concentrations of the test material up to 160 µg/mL for 3h resulted in a negative response. Cytotoxicity (% RPE) was ca. 22 and ca. 13 % at concentrations 150 and 160 µg/mL, respectively.

It is concluded that the test material did not induce mutations at the hprt locus of V79 Chinese hamster lung cells when tested under the conditions employed in this study. These conditions included treatments with doses up to 70 µg/mL and 160 µg/mL in the absence and presence of a rat liver metabolic activation system (S-9) in three independent experiments. The maximum tested concentrations were limited by cytotoxicity.

Justification for selection of genetic toxicity endpoint
All three of the studies address different endpoint. All of them are considered to be key studies, as all of them are sufficiently reliable to adequately address the respective endpoints.

Short description of key information:
Thompson (2004): In vitro bacterial reverse mutation assay: Negative in the absence and presence of metabolic activation.
Wright and Jenkinson (2004): In vitro cytogenicity assay: Negative in the absence and presence of metabolic activation.
Bednáriková (2012): In vitro mammalian cell gene mutation assay: Negative in the absence and presence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

With respect to the genetic toxicity information available for this substance, no classification is required in accordance with Regulation (EC) No. 1272/2008 or Directive 67/548/EEC.