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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read across from structurally similar substance, MnCl2

IN VITRO

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

The mutagenic potential of the test substance, 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 substance 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 substances and vehicle control were dosed using a pre-incubation method.

The test substance 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 substance, 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 substance was concluded to be non-mutagenic.

 

- In Vitro Mammalian Chromosome Aberration Test

The potential of the test substance, 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 substance, 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 substance 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 substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

- In Vitro Mammalian Cell Gene Mutation

The mutagenic potential of the test substance, 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 substance 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.

 

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

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

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


Endpoint Conclusion: No adverse effect observed (negative)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read across from structurally similar substance, MnCl2

IN VIVO

- In Vivo Micronucleus Assay

The potential of the test substance, 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 474.

The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess the toxicity of the test substance 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 substance-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 substance at doses up to 200 mg/kg bw did not induce a significant increase in micronucleated polychromatic erythrocytes in female mice. Therefore, the test substance was considered to be negative in this mouse micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

IN VITRO

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

The mutagenic potential of the test substance, 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 substance 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 substances and vehicle control were dosed using a pre-incubation method.

The test substance 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 substance, 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 substance was concluded to be non-mutagenic.

 

- In Vitro Mammalian Chromosome Aberration Test

The potential of the test substance, 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 substance, 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 substance 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 substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

- In Vitro Mammalian Cell Gene Mutation

The mutagenic potential of the test substance, 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 substance 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 substance, 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 substance 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 substance-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 substance at doses up to 200 mg/kg bw did not induce a significant increase in micronucleated polychromatic erythrocytes in female mice. Therefore, the test substance was considered to be negative in this mouse micronucleus assay.

All of the aforementioned studies were conducted under GLP conditions and in accordance with standardised guidelines; since the studies were conducted with the read across substance, manganese dichloride, rather than with the registered substance itself, they have been assigned a reliability score of 2 in line with the criteria of Klimisch (1997).

 

It was considered necessary to complete the aforementioned in vitro and in vivo studies to help judge the value of the available literature data and to definitively determine whether or not inorganic manganese should be regarded mutagenic, or not. The guideline studies were conducted with manganese dichloride since this represents a soluble manganese compound leading to high levels of metal ion release. Furthermore, for reasons of consistency, and to help draw comparisons and conclusions, since the majority of previous studies have used manganese dichloride.

Further justification for read-across from MnCl2 to MnSO4

The justification for read-across from the negative in-vitro results for MnCl2 to MnSO4 is based on the fact that in an in-vitro system, in the case of both substances, the aqueous solution surrounding the test cells will consist simply of Mn2+ cations with the anions not likely to play any part in the toxicity. Mn2+ cations in this case are likely to enter the cells in equal amounts in both cases.

The justification for read-across from the negative in-vivo result for MnCl2 to MnSO4 is based on the fact that for in-vivo systems there appears to be some indication that MnCl2 is absorbed more readily than MnSO4 (ref. Bales et al, IUCLID section 7.1, and the higher acute toxicity for MnCl2 compared to MnSO4, IUCLID section 7.2.1), therefore a negative result for MnCl2 is highly likely to produce a negative result for MnSO4 in the same test, assuming reasonably, that neither the chloride or sulphate anions exhibit a genotoxic effect.

Findings from Previous Studies - As Reported in Literature

The available mutagenicity tests with manganese sulphate show a mixed spectrum of results for all assay types, bacterial, mammalian cell and in vivo. However, in general it is clear that many of the studies were performed many years ago, before certain criteria on acceptable upper limits for testing were published and agreed. Many of the studies that gave positive results were difficult or impossible to evaluate because they did not provide concurrent toxicity data. Such toxicity data are absolutely necessary in order to judge the validity of the mutation data and the likely absence or presence of artefactual false positive results. In vitro genotoxicity assays, in particular those using mammalian cells, are known to be prone to the induction of false positive responses by extreme culture conditions, such as excessive toxicity, osmolarity, pH, or some other non-genotoxic mechanism. Other studies in which positive results were noted were either unreliable, used highly modified procedures, or were poorly reported. The papers are generally more than 20 years old, in many cases they are more than 30 years old. Consequently, very few of the studies they report have been performed in laboratories operating to OECD good laboratory practice (GLP) standards. Furthermore, many of the experiments are poorly reported and often lack the appropriate controls and rigour that one may expect to seee in a modern report, particularly one done for regulatory purposes. In many cases the source, purity and impurity profile of the test material were not provided. Consequently, the findings from the majority of the publications should be viewed with caution.

Although the published data suggest that manganese may induce mutagenic effects in genotoxicity test systems, it is clear that positive results were only obtained when extreme concentrations were used that exceed the upper threshold levels recommended by the latest test guidelines. Since it was not possible to reach a reliable conclusion on the genetic toxicity of manganese sulphate using published data, further testing was conducted. As outlined above, studies which were conducted with manganese chloride, under GLP conditions and in accordance with standardised guidelines, produced negative results. The ambiguous, and questionable findings from published data can therefore be disregarded and the findings from guideline studies taken forward for risk assessment.

Justification for selection of genetic toxicity endpoint

Four endpoints are regarded as key studies. Multiple studies have been provided to address the different endpoints of genetic toxicity, each assessing different types of genetic toxicity. All of the key studies were guideline studies which were conducted under GLP conditions, and all showed negative results.

Short description of key information:

IN VITRO

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

Negative (TA98, TA100, TA1535, TA1537, WP2uvrA), MnCl2, OECD 471, Thompson & Bowles (2009) MnCl2

- In Vitro Mammalian Chromosome Aberration Test

Negative (human lymphocyte cells), MnCl2, OECD 473, Morris & Durward (2009) MnCl2

- In Vitro Mammalian Cell Gene Mutation

Negative (L5178Y cells), MnCl2, OECD 476, Flanders (2009) MnCl2

IN VIVO

- In Vivo Micronucleus Assay

Negative (mouse erythrocytes), MnCl2, OECD 474, Streicker (2009)  MnCl2

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is required for MnSO4 based on findings of studies conducted with the read across substance, MnCl2, which included three negative in-vitro results (Thompson and Bowles (2009), Morris and Durward (2009) and Flanders (2009)) and a negative result from the in vivo test performed (Streicker(2009)).