Reaction mass of tetramethylammonium hypochlorite and tetramethylammonium chloride

Endpoint summary

Additional information

In Vitro Genetic Toxicity

Gene mutation in bacteria (Ames test)

Ames study by Hurtado, S. (2014) is considered reliable without restrictions as the study was performed in compliance with GLP and according to OECD guideline 471. A preliminary range-finding assay was performed using four strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and one strain of E. Coli (WPuvrA) up to a maximum dose of 5.0 mg/plate to determine the optimal non-toxic test dose. Precipitates were not observed in any strain either with or without metabolic activation. Cytotoxicity (i.e.,reduction in the background lawn) was not observed in any strain with or without metabolic activation. The criteria for a negative response were met for all tester strains with and without metabolic activation.

The concentrations tested in the confirmatory assay were 100, 250, 500, 1000, 2500 and 5000 µg/plate using the preincubation method. Precipitates were not observed in any strain either with or without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn) was not observed in any strain either with or without metabolic activation. The criteria for a negative response were met for all tester strains with and without metabolic activation.

This test result is used as a key value in the hazard assessment.

In vitro cytogenicity in mammalian cells

In vitro chromosome aberration test by Wells, M. (2014) is considered reliable without restrictions as the study was performed in compliance with GLP and according to OECD guideline 473.

The objective of this study was to evaluate the potential of the test article to induce chromosome aberrations in Chinese hamster ovary (CHO‑WBL) cells with and without an exogenous metabolic activation system and with short- and/or long-term incubations. Chromosome alterations result from an inability of cells to repair breaks in chromosomes that may be caused by exposure to chemicals or other exogenous agents and are often associated with cancer. Therefore, a chromosomal aberration assay is used as an appropriate test for detecting potential clastogens and carcinogens. CHO‑WBL cells have been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals.

Halo Salt concentrations tested in the range-finding assay ranged from 9.77 to 5000 μg/mL, up to the highest concentration recommended by OECD guidance. Precipitates and changes in cell morphology (i.e. rounded cells) were not observed in any treatment at the end of test item treatment.

Based on the results of the range-finding assay, concentrations used in the aberration assay ranged from 37.5 to 5000 μg/mL. Precipitates were observed at ≥1250 μg/mL in the 3-hour treatment with metabolic activation at the end of test item treatment. Changes in cell morphology (i.e. rounded cells) were not observed in any treatment at the end of test item treatment.

In the 3 -hour and 20-hour treatments without metabolic activation, the 3 highest concentrations tested (1500, 3000, and 5000 μg/mL) were selected for evaluation of chromosome aberrations. In the 3-hour treatment with metabolic activation, the 3 highest concentrations up to and including the lowest insoluble concentration (800, 1000, and 1250 μg/mL) were selected for evaluation of aberrations.

No statistically significant test item related increases were observed in the percent of cells with aberrations. Additionally, no statistically significant increases in polyploidy or endoreduplication were observed. The aberrations in vehicle and positive control cultures were comparable to acceptable historical control ranges with the following exception. The mean percentage of aberrant cells in the vehicle control for the 20-hour treatment without metabolic activation (3%) was slightly higher than the historical control range (0-2%). Due to the limited number of studies contained in the historical control, this was considered to be comparable and acceptable.

The Halo Salt was considered negative for inducing structural aberrations in CHO-WBL cells with and without metabolic activation under the conditions of this test system.

In Vivo Genetic Toxicity

Read-across mouse bone marrow micronucleus assay (Hayashi, 1988) is used to evaluate the in vivo genotoxicity of the Halo Salt. Male mice were treated with a single intraperitoneal dose of a sodium hypochlorite at dose levels of 0, 312.5, 625, 1250 and 2500 mg/kg bw. In the second experiment four doses of 300 mg /kg bw spaced by 24 hours were similarly administered. Based on the behaviour and the composition of the Halo Salt there is a mechanistic reasoning to use read-across data from sodium hypochlorite (see read-across justification in IUCLID section 13).

Bone marrow cells were harvested at 24 after the last treatment. Femoral bone marrow cells were flushed out with foetal bovine serum and smeared on clean glass slides. One thousand polychromatic erythrocytes per mouse were scored and the number of micronucleated polychromatic erythrocytes (MNCPEs) was recorded. The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

No significant increase in the frequency of micronucleated cells (MNCPEs) was noted at any dose levels tested in single dose trial. Also four doses experiment did not show increased MNCPE level. In addition, the proportion of polychromatic erythrocytes (PCEs) was not affected by the treatment.

Justification for selection of genetic toxicity endpoint
No study was selected, since the conclusion is based on the following assays conducted for the target substance: Bacterial reverse mutation assay (Ames test), In vitro mammalian chromosome aberration test, and in vivo micronucleus assay conducted for read-across substance.

Short description of key information:
Ames test (OECD 471): Non-mutagenic with or without metabolic activation (Halo Salt).
Mammalian in vitro cytogenicity test (OECD 473): Negative with or without metabolic activation (Halo Salt).
Mammalian in vivo micronucleus study (similar to OECD 474): Negative based on the micronucleus assay (read-across substance).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on Ames test and in vitro cytogenicity study conducted for the target substance and in vivo micronucleus study conducted for read-across substance the Halo Salt will not be classified for germ cell mutagenicity in accordance with the criteria of CLP Regulation 1272/2008 and according to the EU directive 67/548/EEC.