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Adequate information exists to characterise the mutagenicity of Rosin Adducts and Rosin Adducts Salts. These are formed when rosin reacts with maleic anhydride. The available data includes includes results of tests conducted using Rosin, fumarated; Fatty acids, C14-18 and C16-18-unsatd., maleated Rosin, maleated; Tall oil, oligomeric reaction products with maleic anhydride and rosin, calcium magnesium zinc salts; and Rosin, fumarated, reaction products with formaldehyde which are summarised briefly below.

Bacterial cell mutation

In a bacterial reverse point mutation Ames test with Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2uvrA conducted according to guideline protocols, Rosin, fumarated was negative in increasing the number of revertant colonies when tested at 17, 50, 167, 500, 1667, and 5000 ug/plate with and without metabolic activation system from liver of rats treated with Aroclor 1254 (Inveresk, 2001). Concurrent positive (N-Ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine, 2 -nitrofluorene, and sodium azide), and negative (solvent) controls were used.

In a bacterial reverse point mutation Ames test with Salmonella typhimurium TA98, TA100, TA1535, and TA1537 conducted according to guideline protocols, Rosin, fumarated was negative in increasing the number of revertant colonies when tested at 50, 158, 500, 1580, 500 ug/mL3 with and without metabolic activation (Life Science Research, 1991d).

In a key bacterial reverse mutation assay (AMES), the mutagenic potential of the test material (Fatty acids, C14-18 and C16-18-unsatd., maleated) was tested using S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and E. coli strain WP2 in the presence and absence of metabolic activation (±S9).

Under the conditions of this assay, the test material was considered non-mutagenic if neither a dose-related increase in the number of revertants, nor a reproducible, biologically relevant positive response was evident in any of the dose groups, with or without metabolic activation. Precipitate / slight precipitate was observed in Experiment 2 in all tested bacterial strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2) at 5000 µg/plate concentration with and without metabolic activation. The positive and negative controls were observed to be valid for this assay and genotoxicity was not observed at any concentration either in the presence or absence of metabolic activation.Based on the results above, fatty acids, C14-18 and C16-18-unsatd., maleated is not considered to be mutagenic in this bacterial reverse mutation assay.

In a reverse gene mutation assay with Rosin maleated, a negative response was obtained in S typhimurium strains TA1535, TA1537, TA98, and TA100 exposed to concentrations of 50, 158, 500, 1580, 5000 mcg in the presence and absence of mammalian metabolic activation using the plate-incorporation method (Life Science Research, 1991c). Rosin, maleated was tested up to limit concentrations, and cytotoxicity was not reported. Based on the test conditions used in this study, rosin, maleated was not found to be mutagenic with or without metabolic activation regardless of strain or dose. The positive controls induced the appropriate responses in the corresponding strains.

A study conducted with Tall oil, oligomeric reaction products with maleic anhydride and rosin, calcium magnesium zinc salts evaluated Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrAusing both the Ames plate incorporation and pre-incubation methods at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors) (Harlan Laboratories Ltd, 2010a). The method conforms to OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines. The dose range was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to Experiment 1. An additional dose level (15 µg/plate) was selected in Experiment 2 in order to allow for potential toxicity following the change in test methodology. The vehicle (tetrahydrofuran) 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. No toxicologically 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. A small increase in revertant colony frequency for tester strain TA98 was noted, in the presence of S9, at 150 µg/plate in Experiment 2 only. However, this increase was non-reproducible in two separate experiments and the individual counts at 150 µg/plate were within the in-house historical control range for the strain. The increase was, therefore, considered to be of no biological consequence. Therefore, the test material was considered to be non-mutagenic under the conditions of this test.

A study conducted with Rosin, fumarated, reaction products with formaldehyde evaluated Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- 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) (Harlan Laboratories Ltd, 2010b). The method conforms to OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines. The dose levels used ranged between 1.5 and 5000 µg/plate, depending on bacterial tester strain type and exposures in the absence or presence of S9-mix. Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test material. 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.

Mammalian chromosomal aberrations

In a mammalian cell gene mutation assay, Chinese hamster ovary cells cultured in vitro were exposed to Rosin, fumarated in DMSO (Inveresk, 2002). The following concentrations were tested: 10, 20, and 40 ug/mL, +S9, for Test 1; 39, 78, and 156 ug/mL, -S9, for Test 1; 30, 40 and 50 ug/mL, +S9, for Test 2; 80, 95, and 110 ug/mL, -S9, for Test 2 at the 24-hour harvest; 40, 80, and 120 ug/mL, -S9, for Test 2 at 48-hour harvest. Rosin, fumarated did not show clastogenic effects under the conditions of the study, both in the presence and absence of activation system.

In a key in vitro mammalian cell gene mutation test, the mutagenic potential of the test material (Fatty acids, C14-18 and C16-18-unsatd., maleated) was tested using mouse lymphoma L5178Y cells in the presence and absence of metabolic activation (±S9). The metabolic activation system used was liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone (80 mg/kg/day over 3 consecutive days). Final concentration of liver homogenate in the test system was 2%. Dimethyl Sulfoxide (DMSO) was utilized as the vehicle in this study.

A preliminary toxicity testing was carried out at 3 h exposure with (+S9) and without (–S9) metabolic activation and at 24 h exposure without metabolic activation (–S9) at the following concentrations: 39.06, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. Three mutation experiments were subsequently carried out at the following test concentrations:

Experiment 1

3 h exposure (–S9): 12.5, 25, 50, 75, 100, 112.5, 125, 137.5, 150 and 200 μg/mL

3 h exposure (+S9): 12.5, 25, 50, 100, 150, 200, 250, 300 and 400 μg/mL

Experiment 2

24 h exposure (–S9): 6.25, 12.5, 25, 50, 75, 100, 112.5, 125, 137.5, 150 and 200 μg/mL

3 h exposure (+S9): 12.5, 25, 50, 100, 120, 140, 160, 180, 200 and 250 μg/mL

Experiment 3

24 h exposure (–S9): 25, 50, 75, 100, 110, 120, 130, 140, 150, 160 and 170 μg/mL

The experiments were conducted in duplicate in the presence and absence of metabolic activation.

Application of the test material WS400104 did not result in an increase in the number of mutant colonies in any of the three mutation experiments either in the presence or absence of metabolic activation at all exposure durations. Based on the results above, fatty acids, C14-18 and C16-18-unsatd., maleated is not considered to be mutagenic in this bacterial reverse mutation assay.

Mammalian cell mutation

A study conducted with Rosin, fumarated evaluated the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (Harlan Laboratories Ltd, 2010c). The method used meets the requirements of the OECD Guideline for Testing of Chemicals No. 476 and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. 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 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 4hour exposure group in the presence of metabolic activation (1% S9) and a 24hour exposure group in the absence of metabolic activation. The dose range for the first experiment was 2.5 to 80 µg/ml in the absence of metabolic activation, and 5 to 160 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 5 to 80 µg/ml in the absence of metabolic activation, and 5 to 120 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 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 a key in vitro mammalian chromosome aberration test, the mutagenic potential of the test material (Fatty acids, C14-18 and C16-18-unsatd., maleated) was tested using Chinese hamster lung fibroblast (V79) cells in the presence and absence of metabolic activation (±S9). The metabolic activation system used was liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone. Dimethyl Sulfoxide (DMSO) was utilized as the vehicle in this study.

Three assays were conducted at the following test concentrations:

Assay 1

3 h exposure (–S9): 2000, 1000, 750, 500, 375, 250, 125, 62.5 and 31.25 μg/mL

3 h exposure (+S9): 5000, 3750, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL

Assay 2

20 h exposure (–S9): 1000, 500, 375, 312.5, 250, 187.5, 125, 62.5 and 31.25 μg/mL

3 h exposure (+S9): 2500, 2187.5, 1875, 1562.5, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL

Assay 3

3 h exposure (+S9): 2000, 1500, 1000, 875, 750, 625, 500, 375, 250, 125, 62.5 and 31.25 μg/mL.

No repeatable dose-dependent induction of chromosome aberrations in Chinese hamster V79 cells was observed following treatment with Fatty acids, C14-18 and C16-18-unsatd., maleated in the presence or absence of metabolic activation.

Therefore, Fatty acids, C14-18 and C16-18-unsatd., maleated are considered not clastogenic in this test system.


Justification for selection of genetic toxicity endpoint
based on data available for the main constituents and/or related materials.

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 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).