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Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; the compound therefore represents a more bioavailable form of manganese. The below data are therefore considered to represent the worst case scenario in terms of exposure to manganese.

 

IN VITRO

- Bacterial Reverse Mutation Assay (e.g. Ames test)

The mutagenic potential of the test material, manganese dichloride, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.13/14.

During the study test strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and a tester strain of Escherichia coli (WP2uvrA) were exposed to the test material both in the presence and the absence of metabolic activation. Vehicle and positive controls were run concurrently. Two separate experiments were conducted, in the first five concentrations of test substance (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. In the second experiment the test materials and vehicle control were dosed using a pre-incubation method.

The test material was found to cause no visible reduction in growth of the bacterial background lawn at any dose and was therefore tested up to the maximum dose level of 5000 µg/plate A particulate precipitate was at 1500 µg/plate and above. This was considered not to prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for and of the bacterial strains, with any dose of the test material, with or without metabolic activation. In the TA100 revertant colony, a small but statistically significant increase was observed on the 1500 µg/plate in Experiment 2 (increase of less than 1.5 times). However the increase was within the range specified by the Standard Test Method, and proved non-reproducible over two separate experiments. This was concluded to have no biological or toxicological relevance. All of the positive control substances induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Therefore, under the conditions of the study the test material was concluded to be non-mutagenic.

 

- In Vitro Mammalian Chromosome Aberration Test

The potential of the test material, manganese dichloride, to induce structural chromosomal aberrations in human lymphocyte cells in vitro was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 473.

During the study duplicate cultures of human lymphocyte cells, treated with test material, were evaluated for chromosome aberrations over at least three dose levels. Vehicle and positive controls were run concurrently. Three treatment conditions were used for the study, as follows: firstly, cultures were exposed for 4 hours with a 20 hour expression time, both in the presence and absence of metabolic activation (S9 mix), secondly cultures were continuously exposed for 24 hours in the absence of metabolic activation. The frequencies of chromosome aberrations in both vehicle and positive controls were within the expected range and verified the sensitivity of the assay and the efficacy of the S9-mix.

The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups, in the absence or presence of a liver enzyme metabolising system, or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

- In Vitro Mammalian Cell Gene Mutation

The mutagenic potential of the test material, manganese dichloride, was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 476.

Based on the results from the preliminary toxicity test, the doses selected for treatment of the initial mutagenesis assay ranged from 2.5 to 120 µg/mL and 20 to 160 µg/mL for the S9 non-activated and activated cultures, respectively. Precipitate of the test material was observed at and above 10 µg/mL in the 4-hour exposure groups in the absence of metabolic activation and at and above 20 µg/mL in the 4-hour exposure group in the presence of metabolic activation. Toxicity in the cloned cultures was observed at doses at 120 and 160 µg/mL without and with S9 activation, respectively.

Based on the results of the preliminary toxicity test, the doses chosen for treatment of the extended treatment assay ranged from 0.31 to 15 µg/mL for non-activated cultures with a 24-hour exposure. Toxicity in the cloned cultures was observed at doses of 10 and 15 µg/mL.

Overall, the test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

 

IN VIVO

- In Vivo Micronucleus Assay

The potential of the test material, manganese dichloride, to induce chromosomal damage in vivo was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 476.

The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess the toxicity of the test material and set dose levels for the definitive study consisted of a toxicity study followed supplemental toxicity study. The second phase was the definitive micronucleus study.

In the range finding phase, male and female mice were treated with Mn2 + at 1000, 500, 250 and 125 mg/kg bw. However, based on the high levels of toxicity observed on the first day of treatment, the study was discontinued and restarted using doses of 175, 200 and 225 mg/kg bw. At the 1-hour post-treatment observation, the female mice in the 225 mg/kg group exhibited a hunched posture, decreased movement and piloerection. These mice were immediately euthanised. All other animals were considered normal throughout the study. Therefore, based on the range-finding study, the selected doses for the definitive study were 25, 50, 100 and 200 mg/kg manganese. Female mice only were dosed in the definitive test.

In the definitive micronucleus study there were no significant increases in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in the female mice at any treatment level.

The results of the assay indicate that under the conditions of the study, a administration of the test material at doses up to 200 mg/kg bw did not induce a significant increase in micronucleated polychromatic erythrocytes in female mice. Therefore, the test material was considered to be negative in this mouse micronucleus assay.

 

All studies with manganese dichloride were conducted under GLP conditions and in accordance with standardised guidelines. Since the studies were conducted on a more bioavailable form of manganese, rather than on the registration substance itself, they have been assigned a reliability score of 2 in line with the criteria of Klimisch (1997).

 


Justification for selection of genetic toxicity endpoint
Multiple studies have been provided to address the different endpoint of genetic toxicity, each addressing different types of genetic toxicity. Since all the studies showed negative results, a single study could not be selected as key over the others.

Short description of key information:
IN VITRO
- Bacterial Reverse Mutation Assay (e.g. Ames test) with MnCl2
Negative (TA98, TA100, TA1535, TA1537, WP2uvrA), MnCl2, OECD 471, Thompson & Bowles (2009)

- In Vitro Mammalian Chromosome Aberration Test with MnCl2
Negative (human lymphocyte cells), MnCl2, OECD 473, Morris & Durward (2009)

- In Vitro Mammalian Cell Gene Mutation with MnCl2
Negative (L5178Y cells), MnCl2, OECD 476, Flanders (2009)

IN VIVO
- In Vivo Micronucleus Assay with MnCl2
Negative (mouse erythrocytes), MnCl2, OECD 474, Streicker (2009)

The negative results obtained in the referenced studies are considered worst-case since the test material used in these studies (manganese chloride) is more readily available due to its much higher solubility compared to the registered substance (manganese dioxide).

A comprehensive literature review (Jenkinson P (2009), Manganese and its inorganic compounds: 2. Genotoxicity Aspects; attached) showed no evidence of genetic toxicity for the registered substance, manganese dioxide. To support this, in-vitro and in-vivo genetic toxicity studies conducted on a more bioavailable substance (manganese chloride) were negative; therefore, according to REACH Annex XI section 1.2, further studies with the registered substance are not necessary.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to mutagenicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to mutagenicity.