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Reverse mutation assay "Ames test" using Salmonella typhimurium and Escherichia coli:

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, using both the Ames plate incorporation and pre-incubation methods at 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 ranged between 0.15 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

The vehicle (sterile distilled water) 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.

In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 µg/plate in both the absence and presence of S9-mix. In the main test (pre-incubation), the test item induced a stronger toxic response with weakened bacterial background lawns initially noted from 15 µg/plate in the absence of S9-mix (TA1535 and TA1537) and 150 µg/plate in the presence of S9-mix (all strains). The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. The test item was tested up to the toxic limit. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.

The test item,Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, was considered to be non-mutagenic under the conditions of this test.

Chromosome aberrations in cultured peripheral human lymphocytes (Read-across result),

This result has been read-across from Fatty acids, C18 unsaturated, reaction products with diethylenetriamine (also referred to as Tall oil diethylenetriamine imidazoline). This read-across substance is a starting material in the manufacture ofFatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, which is a complex mixture of reaction products derived from the neutralisation of fatty acid-diethylenetriamine imidazoline (the read-across substance) with acetic acid. It is therefore considered that the results obtained from studies onFatty acids, C18 unsaturated, reaction products with diethylenetriamine are suitable to read-across to and for use in the evaluation ofFatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts.

Tall oil diethylenetriamine imidazoline was studied for its effect on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix), in two independent experiments.

The study was performed under GLP and according to the most recent OECD and EU guidelines.

In the first cytogenetic assay, Tall oil diethylenetriamine imidazoline was tested up to 22 and 35 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, Tall oil diethylenetriamine imidazoline was tested up to 12 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 10 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix Tall oil diethylenetriamine imidazoline was tested up to 50 µg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly

Tall oil diethylenetriamine imidazoline did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

It was noted that Tall oil diethylenetriamine imidazoline increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the presence of S9- mix in the second cytogenetic assay. This may indicate that Tall oil diethylenetriamine imidazoline has the potential to inhibit mitotic processes.

No effects of Tall oil diethylenetriamine imidazoline on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix in both cytogenetic assays.

Therefore, it is concluded that Tall oil diethylenetriamine imidazoline is not clastogenic in human lymphocytes.

It is therefore concluded that Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, would not be clastogenic in human lymphocytes.

Mouse Lymphoma Assay (Read-across result).

This result has been read-across from Fatty acids, C18 unsaturated, reaction products with diethylenetriamine (also referred to as Tall oil diethylenetriamine imidazoline). This read-across substance is a starting material in the manufacture of Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, which is a complex mixture of reaction products derived from the neutralisation of fatty acid-diethylenetriamine imidazoline (the read-across substance) with acetic acid. It is therefore considered that the results obtained from studies on Fatty acids, C18 unsaturated, reaction products with diethylenetriamine are suitable to read-across to and for use in the evaluation of Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts.

Tall oil diethylenetriamine imidazoline was evaluated for its possible induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix. The study was performed under GLP and according to the most recent OECD and EU guidelines.

In the first experiment, Tall oil diethylenetriamine imidazoline was tested up to concentrations of 6.5 and 60 µg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. Toxicity was observed at these dose levels in the absence and presence of S9-mix. Tall oil diethylenetriamine imidazoline was tested up to cytotoxic levels of 88 and 92% in the absence and presence of S9-mix, respectively.

In the second experiment, Tall oil diethylenetriamine imidazoline was tested up to concentrations of 8.8 and 70 µg/ml, in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Tall oil diethylenetriamine imidazoline was tested up to cytotoxic levels of 94 and 85% in the absence and presence of S9-mix, respectively.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 14-fold for MMS in the absence of S9-mix, and by 9.2- and 8.5-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Tall oil diethylenetriamine imidazoline did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Tall oil diethylenetriamine imidazoline did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Tall oil diethylenetriamine imidazoline is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions used in the study.

It is therefore concluded that Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, would not be mutagenic in a TK mutation test.

Discussion on Cytotoxicity

The level of cytotoxicity observed in the three studies was evaluated. The above discussion of the studies gives information regarding the cytotoxicity observed in the main mutagenicity experiments. The below details refer to the toxicity seen in the preliminary dose-range finding studies (preliminary toxicity tests) conducted in the studies.

Ames test:

In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 µg/plate in both the absence and presence of S9-mix.

Chromosome aberration:

Toxicity was observed at dose levels of 20 µg/ml and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 10 µg/ml and above in the absence of S9 for the continuous treatment of 24 hr and at dose levels of 20 µg/ml and above iin the absence of S9 for the continuous treatment of 48 hr

Mouse lymphoma assay:

Toxicity was observed at dose levels of 10 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 100 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 8 µg/mL in the absence of S9, 24 hours treatment.

The levels of toxicity of the substance Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts and the read-across substance, Tall oil diethylenetriamine imidazoline, are considered to be similar, with the read-across substance being potentially showing a slightly greater toxic effect. All studies were conducted upto toxicity limits and no genotoxicity was seen at these levels. It is considered that reading-across the results of the chromosome aberration and mouse lymphoma studies conducted on Tall oil diethylenetriamine imidazoline is valid.


Short description of key information:
An in-vitro reverse mutation assay 'Ames test' using S. typhimurium and E. coli has been conducted on the substance (Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts). The study gave a negative result.

A chromosome aberration study and mouse lymphoma assay have been conducted on a structural analogue substance, Fatty acids, C18 unsaturated, reaction products with diethylenetriamine (also referred to a Tall oil diethylenetriamine imidazoline). These two studies gave negative results and have been read-across to assess the mutagenicity of Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test item, Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, was considered to be non-mutagenic under the conditions of an reverse mutation assay "Ames test".

A read-across substance, Tall oil diethylenetriamine imidazoline, is not mutagenic in the Salmonella typhimurium reverse mutation assay

and in the Escherichia coli reverse mutation assay, is not clastogenic in human lymphocytes, and not mutagenic in the TK mutation test with L5178Y mouse lymphoma cells.

It can therefore be concluded that Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salt is not genotoxic.

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