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Results are available to meet the standard information requirements for in vitro genotoxicity. Bacterial tests have been undertaken on Rosin (which, since it is in solution includes the unionised material and the ionised material, and therefore includes the monovalent cation salts of rosin), the monovalent cation (potassium) salt of Hydrogenated rosin and each of the (insoluble) divalent cation salts of Rosin. A mammalian chromosomal aberration test and a mammalian cell mutation assay were conducted on the lead substance, Rosin. All tests were followed OECD guidelines and were subject to GLP audit. The results in all cases were negative.

Bacterial cell mutation

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.

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

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, 2004a). 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 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., 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 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, 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., 2010c). 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 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.

Mammalian chromosomal aberrations

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, 2010a). 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.

Mammalian cell mutation

The mutagenic potential of Rosin has also been evaluated in a mouse lymphoma assay using the L5178Y mouse lymphoma cell line (Harlan Laboratories Ltd, 2010k). 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.


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).