Overbased reaction products of distilled tall oil and magnesium hydroxide’

Endpoint summary

Additional information

In vitro Genetic toxicity in Bacteria

In an in vitro AMES test (Thompson, P. W., 2010), Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^-were treated with suspensions of the test material (fatty acid C18-(unsaturated) lithium salts) in acetone, using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number. However, the toxicity of the test material to the tester strains was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate. A greasy precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies. 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 or exposure method. The test material was considered to be non-mutagenic under the conditions of this test.

Bacterial mutation assays conducted on the potassium, calcium, calcium zinc and magnesium salts included in this category were also negative.

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 of E. coli were exposed to Resin acids and rosin acids, magnesium salts in ethanol at concentrations of 0.76 to 61 µg/plate in the presence and absence of a mammalian metabolic activation system using both the plate incorporation method and the pre-incubation method (Vivotecnia Research S.L., 2010b). Cytotoxicity was observed at the maximum dose level in some of the strains of bacteria. No experiment with the test substance showed ratios above 2.5 as compared to the negative control, either with or without S9 metabolic activation. No dose response was observed for any of the tested bacterial strains. All vehicle and positive controls induced the appropriate responses in the corresponding strains. Based on the test conditions used in this study, Resin acids and rosin acids, magnesium salts was found to be neither mutagenic nor pro-mutagenic.

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 of E. coli were exposed to Resin acids and rosin acids, calcium salts suspended in corn oil at concentrations of 62-5000 µg/plate in the presence and absence of a mammalian metabolic activation system using both the plate incorporation method and the pre-incubation method (Vivotecnia Research S.L., 2010c). No cytotoxicity was observed at any dose level up to the limit concentration of 5000 µg/plate. No experiment with the test substance showed ratios (R) above 2.5 as compared to the negative control, either with or without S9 metabolic activation. No dose response was observed for any of the tested bacterial strains. All vehicle and positive controls induced the appropriate responses in the corresponding strains. Based on the test conditions used in this study, Resin acids and rosin acids, calcium salts was found to be neither mutagenic nor pro-mutagenic.

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 of E. coli were exposed to Resin acids and rosin acids, calcium zinc salts in corn oil at concentrations of 62-5000 µg/plate in the presence and absence of a mammalian metabolic activation system using both the plate incorporation method and the pre-incubation method (Vivotecnia Research S.L, 2010a). No cytotoxicity was observed at any dose level up to the limit concentration of 5000 µg/plate. No experiment with the test substance showed ratios (R) above 2.5 as compared to the negative control, either with or without S9 metabolic activation. No dose response was observed for any of the tested bacterial strains. All vehicle and positive controls induced the appropriate responses in the corresponding strains. Based on the test conditions used in this study, Resin acids and rosin acids, calcium zinc salts was found to be neither mutagenic nor pro-mutagenic.

The mutagenic/genotoxic potential of Resin acids and rosin acids, hydrogenated, potassium salts has been characterized in a well conducted bacterial reverse mutagenicity test conducted according to OECD Guideline 471 (SafePharm Laboratories Limited, 2004). There was no increase in mutation frequency in any strain of S. typhimurium or E. coli at concentrations up to 5000 µg/plate in the presence or absence of metabolic activation. The study was run using the plate incorporation method. In this study, vehicle, negative and positive controls induced the appropriate responses.

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 of E. coli were exposed to Gum Rosin (Rosin) in ethanol at concentrations of 62-5000 µg/plate in the presence and absence of a mammalian metabolic activation system using both the plate incorporation method and the pre-incubation method (Vivotecnia Research S.L., 2010d). No cytotoxicity was observed at any dose level up to the limit concentration of 5000 µg/plate. No experiment with the test substance showed ratios (R) above 2.5 as compared to the negative control, either with or without S9 metabolic activation. No dose response was observed for any of the tested bacterial strains. All vehicle and positive controls induced the appropriate responses in the corresponding strains. Based on the test conditions used in this study, Gum Rosin was found to be neither mutagenic nor pro-mutagenic.

In vitroMammalian Cell Gene Mutation

In aL5178Y mouse lymphoma test (Flanders, L., 2010), two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to ten dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and for Experiment 1 was 2.5 to 80 µg/ml in the absence of metabolic activation, and 10 to 120 µg/ml in the presence of metabolic activation. The test material dose range for Experiment 2 was 10 to 80 µg/ml in the absence of metabolic activation, and 5 to 70 µg/ml in the presence of metabolic activation. The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of test material was observed in Experiment 1, at and above 20 µg/ml in the absence of metabolic activation, and at and above 80 µg/ml in the presence of metabolic activation. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

The mutagenic potential of Rosin has also been evaluated in a mouse lymphoma assay using the L5178Y mouse lymphoma cell line (Harlan Laboratories Ltd, 2010b). The method used met the requirements of the OECD (476) and EU Method B17. Two independent experiments were performed, with the maximum dose level limited by test material induced toxicity (Experiment 1: 2.5 to 40 µg/mL in the absence of metabolic activation, 10 to 80 µg/mL in the presence of metabolic activation. Experiment 2: 2.5 to 45 µg/mL in the absence of metabolic activation, 10 to 55 µg/mL in the presence of metabolic activation). Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

In vitroChromosome Aberration

In an In vitro study (Durward and Jenkinson, 2006),duplicate cultures of human lymphocytes, treated with the test material (Fatty acids C18-(unsaturated) lithium salts), were evaluated for chromosome aberrations at three or four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. 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. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

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 did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. The test material was toxic and exhibited a very steep dose-response curve. The dose range used included a dose level that induced approximately 50% mitotic inhibition or was the maximum sub-toxic dose level. The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Rosin dissolved in THF has been evaluated for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro (Harlan Cytotest Cell Research GmbH, 2010). The test was run using two independent experiments, with two parallel cultures analysed per study. Per culture, 100 metaphase plates were scored for structural chromosomal aberrations. The highest applied concentration in this study (3500.0 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. In Experiment 1 in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. However, in the presence of S9 mix, the highest applied concentration showed clear cytotoxic effects, but was not evaluable for cytogenetic damage. In Experiment 2 in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. An appropriate response (statistically significant increases (p < 0.05) in cells with structural chromosomal aberrations) was obtained with the positive controls.

Short description of key information:
Not mutagenic or clastogenic in bacterial and/or mammalian cells in vitro.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 orUN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).