Tetramethylammonium hydrogen phthalate

Endpoint summary

Additional information

TMHP (tetramethylammonium hydrogen phthalate) was tested for mutagenic properties in a reverse mutation assay (Ames test) according to OECD guideline 471. Test strains Salmonella typhimurium TA1535, TA100, TA1537, TA1538 and TA 98 were included, and TMAP was tested up to and including 5000µg/plate, both in the presence an the absence of metabolic activation (S9). Reliable negative and positive controls were included. No toxicity was observed. TMAP did not significantlyincrease the number of revertant colonies in any of the tested bacterial strains either with or without metabolic activation. As a result, TMHP is considered to be non-mutagenic under the conditions of the test. However, as no E coli strain was tested, no conclusion can be given on the potential of TMHP for induction of DNA cross linking.

TMHP was tested in human lymphocytes for induction of chromosome aberrations according to OECD guideline 473. The tested doses were 625, 1250 and 2500 µg/ml TMHP in the absence of metabolic activation and 312.5, 625, 1250 and 2500 µg/ml TMHP in the presence of metabolic activation. Reliable negative and positive controls were included. TMHP did not induce a significant increase in the frequency of cells with chromosome aberrations or polyploid cells in either the presence or absence of a liver enzyme metabolising system, while cytotoxicity was observed. TMHP is therefore considered to be non-clastogenic to human lymphocytes in vitro.

To fulfill the data requirements, the test results from Ames tests with the analogues TMAC, TMAH and phthalic acid/ phthalic anhydride were included:

An AMES test was performed with TMAC according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to 5000 ug TMAC/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that TMAC is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

An AMES test with TMAH was conducted according to OECD guideline 471 and GLP principles. Considerable growth inhibition was observed in all strains treated at the highest concentrations of TMAH. However, no increase in the number of revertant colonies was observed in either strain (S. typhimurium TA98, TA100, TA1535, TA1537, or E.coli WP2 uvrA) treated at any concentrations of TMAH with or without metabolic activation (S9 mix). These results have led to the conclusion that TMAH is negative for mutagenicity in the bacterial reverse mutation assay (Ames test) regardless of metabolic activation.  

An Ames Salmonella mutation test with phthalic acid was performed with according to the plate incorporation procedure described by Maron and Ames (1983). Five Salmonella typhimurium strains were used: TA98, TA100, TA102; TA1535, and TA1537. This assay was performed with and without metabolic activation. Five concentrations of the test substance were examined: 0, 20, 100, 500, 2500 and 12500 µM using triplicate plates per dose. A significant increase in the number of revertants was observed in the presence of the positive control compounds. Negative and strain-specific positive control values were within historical lab range, demonstrating that the test conditions were effective and that the metabolic activation system functioned properly. Phthalic acid did not produce mutagenic activity in any of the five bacterial strains tested under any of the activation conditions examined.

Furthermore, the test substance Phthalic Anhydride was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster up to concentrations of 10 mM. The study was conducted according to the OECD guideline and according to GLP principles. The test substance was found to be not mutagenic in this test, with or without metabolic activation.

A mouse lymphoma assay was conducted with TMAH up to limit concentrations (10 mM) according to OECD 476 guideline and GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. No cytotoxicity, nor precipitation was observed. In the absence of S9-mix, TMAH 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 8% v/v S9-mix, TMAH did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with 12% v/v S9 for metabolic activation. It is concluded that TMAH is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

The rationale to Read-Across these data to TMHP is attached in section 13.

Justification for selection of genetic toxicity endpoint
No single study was selected, since several relevant in vitro studies were performed. The conclusion was based on a weight-of-evidence approach.

Short description of key information:
An Ames test (no E coli is included, Klimisch 2) and a chromosome aberration test in human lymphocytes (Klimisch 1) are available, both performed with TMHP. The potential mutagenicity of the phtalate part of the test substance is addressed with an AMES test with phthalic acid and an in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells with Phthalic Anhydride. The potential mutagenicity of the tetramethylammonium part is addressed with AMES tests with tetramethylammonium chloride (TMAC) and hydroxide (TMAH), and a mammalian cell gene mutation test with L5178Y mouse lymphoma cells with TMAH (all studies Klimisch 2).The rationale for Read-Across of these data on substance analogues is attached in section 13.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, tetramethylammonium hydrogen phtalate is not classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008.