Reaction mass of copper oxide and manganese dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data are available for the registration substance. However adequate and reliable studies performed with each of the two constituents of the registration substance are at hand.

For MnO2 an in vitro bacterial reverse mutation assay is available showing negative test results.

There are two studies available investigating the mutagenic effects in bacterial cells of copper chloride dihydrate, a structural analogue of one main constituent of Hopcalite. In none of the tested strains, i.e. Salmonella typhimurium strain TA100, TA98, TA102, TA1535, and TA1537 (the latter 4 strains were tested in the presence or absence of metabolic activation (S9 -mix)) a mutagenic activity was observed.

In order to asses gene mutations in mammalian cells for copper the results of an indicator test are provided. In this study, copper sulphate (1 mM for 3h; 250 mg/l) caused-single strand breaks in isolated rat hepatocytes as assessed by the alkaline elution unwinding assay., however cytotoxicity was demonstrated as well. These results in combination with the knowledge of a negative test result obtained in a proprietary study investigating the unscheduled DNA synthesis in the livers of orally dosed rats are sufficient (BPC, 2011; OECD, 2014).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

based on MnO2 as main constituent of the reaction mass

Conclusions:

MnO2, as main constituent of Hopcalite, did not show activity in a bacterial reverse mutation test.

Executive summary:

The study used as source investigated manganese dioxide. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference "assessment report".

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Remarks on result:

other:

Remarks:

Cytotoxicity assay was mainly done with the wild type.

Conclusions:

Copper chloride dihydrate, a structural analogue of one main constituent of Hopcalite, did not show activity in a bacterial reverse mutation test with Salmonella typhimurium strain TA100.

Executive summary:

The study used as source investigated copper chloride dihydrate. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference "assessment report".

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Copper chloride, a structural analogue of one main constituent of Hopcalite, did not show activity in a bacterial reverse mutation test in the presence or absence of metabolic activation (S9 -mix) in Salmonella typhimurium strain TA98, TA102, TA1535, and TA1537.

Executive summary:

The study used as source investigated copper chloride. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference "assessment report".

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

other: at 1 mM for 3h (250 mg/l) DNA-single strand breaks in isolated rat hepatocytes were induced. Cytotoxicity was observed at this concentration (only 3% cell viability, GOT release). no effect when a non-toxic dose (0.3 mM for 3h; 75 mg/l, 94% viability).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In this study, copper sulfate (1 mM for 3h; 250 mg/l) caused DNA-single strand breaks in isolated rat hepatocytes as assessed by the alkaline elution DNA unwinding assay (Sina et al, 1983). Cytotoxi city was demonstrated at this concentration by 3% cell viability, as assessed by glutamate-oxaloacetate transaminase release. The authors reported no effect when a non-toxic dose (0.3 mM for 3h; 75 mg/l) was investigated (94% viability).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No data are available for the registration substance. However adequate and reliable studies performed with each of the two constituents of the registration substance are at hand.

For MnO2 a micronucleus test (OECD TG 474) should be viewed as the in vivo test of choice for chromosomal aberrations.... (quote from OECD 475).

The repeated daily exposure to manganese dioxide for 28 days, showed in a micronucleus test a statistically significant increase in polychromatic erythrocytes at the highest dose level (i.e. 1000 mg/kg bw/day) compared to negative control. Therefore manganese dioxide was considered to be genotoxic in this study.

For MnO2 there is no gene mutation test in mammalian cells, but as given in ECHA guidance on information requirements and chemical safety assessment a COMET assay, which determines possible DNA damage (indicator assay) may be used as alternative. Thus the results obtained in a COMET assay with repeated dosing is used as key study. In this study the repeated daily exposure to manganese dioxide for 28 days, showed in an alkaline comet assay a statistically significant increase in % tail DNA damage at the highest dose level (1000 mg/kg bw/d) in comparison to control. Therefore manganese dioxide was considered to cause DNA damage in this study.

There is no reliable single study available to determine the cytogenetic activity of copper compounds. However there are three individual studies in which clastogenic effects of copper sulfate pentahydrate were investigated in the bone marrow or sperm cells of mice (Agarwal et al., 1990;Bhunya and Pati, 1987;Tinwell and Ashby, 1990). While in two studies a positive clastogenic effect was observed (however only at cytotoxic concentrations and including gaps), in the another study no increase of micronuclei formation in treated animals was observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

In a micronucleus test, MnO2, a main constituent of Hopcalite, revealed a significant increase of polychromatic erythrocytes compared with negative control at the highest dose level and was considered to be genotoxic.

Executive summary:

In an in vivo study comparable to the standardised guideline OECD 474, male and female Wistar rats (5 animals per dose) were administered MnO2 orally by gavage (0, 100, 500, and 1000 mg/kg bw) every day for 28 days and the presence of polychromatic erythrocytes (PCEs) were determinded. A statistically significant increase of PCEs was observed at the highest dose level compared to negative control at 24 h after treatment. Therefore MnO2 was considered to be genotoxic. Results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross-reference “assessment report”.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Remarks:

At highest dose rats showed dullness, irritation and moribund symptoms (no mortality), reduction in body weight gain and feed intake (but loss was not significant)

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

In an alkaline comet assay, manganese dioxide, a main constituent of Hopcalite, induced a significant increase in % tail DNA damage.

Executive summary:

The potential of manganese dioxide to induce DNA damage was investigated in an alkaline comet assay (no guideline available when conducted) in rats. Results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross-reference “assessment report”.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

the Test material (Copper sulfate pentahydrate) administered intraperitoneally to Swiss albino mice in vivo induced a significant increase in the frequency of chromosomal aberrations in bone marrow cells at all concentrations used (1.1-6.6 mg/kg b.w.), when compared to the negative control. Statistical analysis indicates that the degree of clastogenicity was directly related to the concentrations used and indirectly to the period of exposure. The effect was maximal at 6 h after treatment as compared with 12 and 24 h.

Executive summary:

The study used as source investigated copper sulfate pentahydrate.

In a cytogenetic test, Swiss albino male mice were administered a single ip dose of copper sulphate (0, 1.1, 1.65, 2.0, 3.3 or 6.6 mg/kg, representing 1/30, 1/20, 1/15, 1/10 and 1/5 of the LD50,respectively). Animals (6 per group) were sacrificed at 6, 12 and 24h after dosing. The principal classes of CAs reported were isochromatid breaks and chromatid gaps. When gaps were excluded, there was a statistically significant, dose-dependent increase in both the number of chromosomal aberrations per cell (CA/cell) and the percentage of cells with at least one chromsomal aberration (%DC) at all time points and doses investigated (p<0.01 at 6 and 12h; p<0.05 at 24h). Further analysis of the data demonstrated that both CA/cell and %DC (excluding gaps) were significantly higher at 6h compared to 12 and 24h at all doses investigated, indicating a relative early onset of clastogenesis. No evidence of cytotoxicity was presented in this study. In this study, ip injection of copper sulphate caused a dosedependent increase in chromosome aberrations in bone marrow cells from Swiss albino mice.

The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Sex:

not specified

Genotoxicity:

positive

Remarks:

ip-treated animals, a statistically significant, dose-dependent increase in percentage of cells with chromosomal aberrations was reported at the 24h sampling time

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not specified

Remarks on result:

other: the major type of aberrations was chromatid gaps in all cases and when gaps were excluded, results were similar to controls.

Sex:

not specified

Genotoxicity:

positive

Remarks:

statistically significant elevation in the frequency of chromosomal aberrations following c hronic subcutaneous (4.66%) dosing

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not specified

Remarks on result:

other: the major type of aberrations was chromatid gaps in all cases and when gaps were excluded, results were similar to controls.

Sex:

not specified

Genotoxicity:

positive

Remarks:

statistically significant elevation in the frequency of chromosomal aberrations following c hronic oral dosing (4.00%)

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not specified

Remarks on result:

other: the major type of aberrations was chromatid gaps in all cases and when gaps were excluded, results were similar to controls.

Sex:

not specified

Genotoxicity:

other: positive: statistically significant, dose-dependent increase in frequency of micronuclei

Remarks:

polychromatic erythrocytes (PCE; 0.2, 1.3, 1.8 and 2.06% for controls, 5, 10 and 20 mg/kg) and normochromatic erythrocytes (NCE; 0.15, 0.98, 1.41 and 1.76% for controls, 5, 10 and 20 mg/kg)

Toxicity:

yes

Remarks:

increased frequency of nuclei in lysis

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not specified

Conclusions:

In this study chromosomal aberrations in Swiss mice increased after acute or chronic intraperitoneal, as well as chronic oral or subcutaneous dosing. However the study results are thought to be of low reliability as the study was only performed with a low number of animals and no positive controls were included. Moreover the values are from pooled data, which further diminish the results substantially. Further, in all cases, chromosomal aberrations were predominantly chromatid gaps and when gaps are excluded, results were similar to negative controls.
The study also investigated micronuclei formation in bone marrow cells of ip treated mice. A statistically significant, dose-dependent increase in the frequency of micronuclei was observed in both polychromatic erythrocytes and normochromatic

Executive summary:

The study used as source investigated copper sulfate pentahydrate.

The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

marked depression in erythropoiesis was observed at both 13.2 and 19.8 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

historical controls of other vehicles

Positive controls validity:

valid

Conclusions:

This study investigated micronuclei formation in bone marrow cells of ip treated mice. No increase in the frequency of micronuclei was observed at any dose level. Cytotoxicity was observed in the mid and high dose group but not in the low dose group.

Executive summary:

The study used as source investigated copper sulfate pentahydrate.

The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

General mechanistics:

- Copper:

It is known mostly from in vitro studies that free copper (II) ions are able to redox cycle and catalyse the generation of reactive oxygen species (ROS). ROS are capable of damaging DNA, providing a mechanism by which copper may indirectly cause genetic damage in cells. Also the fidelity of DNA repair is affected by free copper (II) ions as e.g. DNA repair enzymes like 8-oxo-dGTPase are impaired.

However under physiological conditions the majority of copper is bound to proteins such as ceruloplasmin or albumin and thus the concentration of free copper is extremely low (as 10^-18M, that is less than one free copper atom per cell). Moreover cells under normal conditions have potent antioxidants, all of which protect against the secondary copper genotoxicity. Consequently, under normal physiological conditions it seems unlikely that copper-induced oxidative damage will occur (ECI, 2008 - VOLUNTARY RISK ASSESSMENT Report).

Studies investigating genotoxic effects in vivo and finding positive effects most often use unphysiological exposure routes such as IP, which circumvent the physiological copper homeostatic systems which are in place when Copper compounds are e.g. ingested.

- Manganese:

From a mechanistic point of view genotoxic activity observed in various assays with manganese dioxide can be explained rather by secondary mechanisms than by a direct genotoxic activity (see also OEDC QSAR Toolbox – no alert for genotoxic activity indicating direct activity). Secondary mechanisms which should be considered are generation of reactive oxygen species and exhaustion of antioxidant capacity of cells as well as the possible impairment of DNA repair. Such mechanisms are considered relevant also by others, e.g. by Greim, 1994 “The effects of manganese in its various target tissues are probably all a result of the fact that manganese is a reducing agent or an oxidizing agent, dependent on its oxidation state. Manganese(II) can capture superoxide radicals (O· 2 –) and so is a powerful antioxidant. In the higher oxidation states (III, IV) the manganese ion is an effective oxidizing agent. It catalyses the oxidation of biologically important substances such as catecholamines, unsaturated fatty acids and glutathione.” However oxidation of catecholamines is relevant for the most critical endpoint identified after manganese exposure – neurotoxicity – but not for genotoxicity.

Detailed discussion of studies reported here:

- Copper compounds:

Copper in vitro

There are two studies available investigating the mutagenic effects in bacterial cells of copper chloride dihydrate, a structural analogue of one main constituent of Hopcalite. In none of the tested strains, i.e. Salmonella typhimurium strain TA100, TA98, TA102, TA1535, and TA1537 (the latter 4 strains were tested in the presence or absence of metabolic activation (S9 -mix)) a mutagenic activity was observed.

Copper in vivo

In order to asses gene mutations in mammalian cells the results of an indicator test are provided.In this study, copper sulphate (1 mM for 3h; 250 mg/l) caused-single strand breaks in isolated rat hepatocytes as assessed by the alkaline elutionunwinding assay (Sinaet al,1983). Cytotoxicity was demonstrated at this concentration by 3% cell viability, as assessed by glutamate-oxaloacetate transaminase release. The authors reported no effect when a non-toxic dose (0.3 mM for 3h; 75 mg/l) was investigated (94% viability). These results in combination with the knowledge of a negative test result obtained in a proprietary study investigating the unscheduled DNA synthesis in the livers of orally dosed rats are sufficient to cover this endpoint(BPC, 2011;OECD, 2014).

There is no reliable single study available to determine the cytogenetic activity of copper compounds. However there are three individual studies in which clastogenic effects of copper sulfate pentahydrate were investigated in the bone marrow or sperm cells of mice(Agarwal et al., 1990;Bhunya and Pati, 1987;Tinwell and Ashby, 1990).

“In a cytogenetic test, Swiss albino male mice were administered a single ip dose of copper sulphate (0, 1.1, 1.65, 2.0, 3.3 or 6.6 mg/kg, representing 1/30, 1/20, 1/15, 1/10 and 1/5 of the LD50, respectively). Animals (6 per group) were sacrificed at 6, 12 and 24 h after dosing. The principal classes of CAs reported were isochromatid breaks and chromatid gaps. When gaps were excluded, there was a statistically significant, dose-dependent increase in both the number of chromosomal aberrations per cell (CA/cell) and the percentage of cells with at least one chromsomal aberration (%DC) at all time points and doses investigated (p<0.01 at 6 and 12h; p<0.05 at 24h).   Further analysis of the data demonstrated that both CA/cell and %DC (excluding gaps) were significantly higher at 6h compared to 12 and 24h at all doses investigated, indicating a relative early onset of clastogenesis. No evidence of cytotoxicity was presented in this study. In this study, ip injection of copper sulphate caused a dose-dependent increase in chromosome aberrations in bone marrow cells from Swiss albino mice (Agarwal et al., 1990).

In contrast to the results of Agarwalet al(1990), the study by Tinwell and Ashby (1990) reported a negative result for copper sulphate. Malemice (5 animals per group) were administered a single ip injection of copper sulphate (6.6, 13.2 or 19.8 mg/kg) and sacrificed after either 24 h (all doses) or 48h (6.6 mg/kg only). No toxicity was reported at the lowest dose level (6.6 mg/kg) during the course of the experiment. At the other two dose levels, the animals were reported as appearing subdued. In addition, a marked depression in erythropoiesis was observed at both 13.2 and 19.8 mg/kg, indicating cytotoxicity. The dose of 19.86 mg/kg (60% of LD₅₀) was estimated by the authors to be the maximum tolerated dose. In this study, intraperitoneal administration of copper sulphate failed to induce micronuclei in the bone marrow inmice in two separate assays at any of doses or time points investigated.

The findings of Tinwell and Ashby (1990) make the clastogenic effects seen by Agarwalet al(1990) difficult to explain. Although the study by Agarwalet al(1990) reported principally chromatid gaps and breaks, it also reported exchange figures (referred to as isochromatid gaps and breaks). These would have produced acentric fragments, some of which would be expected to yield micronuclei (Savageet al, 1976). The age and sex of the test animals were the same and as noted by Tinwell and Ashby (1990), as was the exposure route. However, the mouse strain was different and an explanation for these conflicting results may be a strain-specific bone marrow response for which there appears to be no precedent.

Bhunya and Pati, 1987: A cytogenetic test of inferior quality was reported by Bhunya and Pati (1987). The study deviated from Annex V guidelines by using less than the recommended number of animals (3animals in each treatment group and at each time-point; 10 animals are recommended in Annex V) and by pooling the data from these three animals before analysing for statistical significance. Further, no positive control data were presented in this study. The findings of this study are consequently considered to be unreliable. In the study, inbred Swiss mice were administered copper sulphate as a single ip injection at 5, 10 or 20 mg/kg (24h sampling time) or 20 mg/kg (6, 24 and 48h sampling time). Copper sulphate was also administered by subcutaneous injection and orally (20 mg/kg - 24h sampling time). In addition, as a repeated-dose treatment, animals were administered 5 daily ip injections of copper sulphate (5 mg/kg/day) and were killed 24h following the final injection. At the appropriate sampling times, bone marrow was analysed for the presence of chromosomal aberrations. 100 metaphases were scored for each animal and then results from three animals pooled. The total number of negative control animals with each route of administration was 6 (oral and sc) or 10 (ip), although it is not clear whether there was a negative control group at each sampling time in the ip-dosing study. In the ip-treated animals, a statistically significant, dose-dependent increase in percentage of cells with chromosomal aberrations was reported at the 24h sampling time (0.70% in controls; 4.0, 4.66 and 5.00% at 5, 10 and 20 mg/kg CuSO4, respectively). However, given that these values are from pooled data, the importance of this finding is diminished substantially. Further, in all cases, chromosomal aberrations were predominantly chromatid gaps and when gaps are excluded, results were similar to negative controls. The authors also reported a statistically significant elevation in the frequency of chromosomal aberrations following both subcutaneous (4.66%) and oral dosing (4.00%) with CuSO4, and following the chronic treatment regime (4.00%) compared to a composite control group (0.66%). Again, these values are from pooled data. As before, the major type of aberrations was chromatid gaps in all cases and when gaps were excluded, results were similar to controls. 

The same authors also investigated micronucleus formation. Animals (3 per group) were administered two daily ip injections of copper sulphate (5, 10, and 20 mg/kg) and sacrificed 6h following the second injection. A statistically significant, dose-dependent increase in the frequency of micronuclei was observed in both polychromatic erythrocytes (PCE; 0.2, 1.3, 1.8 and 2.06% for controls, 5, 10 and 20 mg/kg, respectively) and normochromatic erythrocytes (NCE; 0.15, 0.98, 1.41 and 1.76% for controls, 5, 10 and 20 mg/kg, respectively) was observed. However, a statistically significant increase in the frequency of nuclei in lysis compared to controls was also reported for all doses investigated, indicating that all the doses of copper sulphate used in this study were cytotoxic. This study deviated from Annex V guidelines by using less than the recommended number of animals, the use of a single sampling time and the absence of positive control data” (note – the discussion was taken from(ECI, 2008)).

- Manganese compounds:

Manganese dioxide In vitro

In a guideline study according to OECD TG 471 under GLP conditions, mutagenic activity of manganese dioxide was investigated in a preincubation method in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2uvrA with (rat liver postmitochondrial supernatant S9 mix) and without metabolic activation at concentrations of 0, 39, 78.1, 156.3, 312.5, 625 and 1250 μg/plate and 3 plates at each dose levels were used. Precepitation of the test substance was observed at 1250 µg/plate in all strains with and without metabolic activation. All positive controls induced marked increases in frequency of revertant colonies. All negative controls were found to be in an acceptable range. The test item showed no mutagenic activity.

Besides these key results in a publication by Dutta and colleagues(2006)genotoxic activity of manganese dioxide was also assessed in various in vitro tests. In a so called "Fluorimetric detection of Alkaline DNA Unwinding" (FADU) assay the characteristic of manganese dioxide to induce DNA strand breaks was investigated. It was observed that the percentage of DNA stand breaks induced in human polymorphonuclear leukocytes by 1,2 mmol MnO2/L after 1 hour of exposure was 79% in comparison to the negative control value of 24%. In the same publication clastogenic activity was assessed in human peripheral lymphocytes which were treated with manganese dioxide (0 and 1.2 mmol/L). The frequencies of micronuclei or chromosomal aberrations were assessed. Manganese dioxide showed a statistically significant increase in frequencies of micronuclei compared to controls. Manganese dioxide showed also a statistically significant increase in chromosomal aberrations compared to controls (%aberrantcells of 46.4 ± 3.5 compared to DMSO (0.1%) % aberrant cells: 3.5 ± 0.707). Thus a genotoxic effect of manganese dioxide was presented. Besides other methodological deficiencies of these assays published the major deficiency is that only one test concentration was investigated and therefore no concentration response relationship could be investigated.

Manganese dioxide In vivo

There are two publications by the same group describing examinations of various endpoints concerning genotoxic activity after acute(Singh et al., 2013a)or repeated dosing(Singh et al., 2013b)of rats.

The following tests were identified as key studies to fulfil the data requirements for assessment of cytogenetic effects and gene mutations in mammalian cells.

For MnO2 a micronucleus test (OECD TG 474) should be viewed as the in vivo test of choice for chromosomal aberrations.... (quote from OECD 475).

The repeated daily exposure to manganese dioxide for 28 days, showed in a micronucleus test a statistically significant increase in polychromatic erythrocytes at the highest dose level (i.e. 1000 mg/kg bw/day) compared to negative control. Therefore manganese dioxide was considered to be genotoxic in this study.

For MnO2 there is no gene mutation test in mammalian cells, but as given in ECHA guidance on information requirements and chemical safety assessment a COMET assay, which determines possible DNA damage (indicator assay) may be used as alternative. Thus the results obtained in a COMET assay with repeated dosing is used as key study. In this study the repeated daily exposure to manganese dioxide for 28 days, showed in an alkaline comet assay a statistically significant increase in % tail DNA damage at the highest dose level (1000 mg/kg bw/d) in comparison to control. Therefore manganese dioxide was considered to cause DNA damage in this study.

As supporting studies the results from the same 28 day repeated dosing regime, however assessment of chromosomal aberrations are provided. A statistically significant increase in chromosomal aberrations and percentage of aberrant cells in all animals at the highest dose level was observed. The mitotic index was not significantly decreased or differed by manganese dioxide compared to control groups. Due to the observed effects, manganese dioxide is considered to be genotoxic.

Moreover as indicated the same authors investigated genotoxic endpoints after acute exposure. To this end manganese dioxide was administered once to female Wistar rats (5 per dose) orally by gavage at various doses (0, 100, 500, and 1000 mg/kg bw) and different assessments were performed thereafter. In a micronucleus test comparable to OECD guideline 474, bone marrow cells extracted from the femurs were collected 24 and 48 h after treatment. For each animal three slides were prepared and thereof a total of 2000 polychromatic erythrocytes were scored for the presence of micronuclei. Both controls, negative and positive (cyclophosphamide), were valid. No statistically significant increase in polychromatic erythrocytes compared to negative control was seen.

In a bone marrow chromosome aberration assay comparable to OECD guideline 475, bone marrow cells extracted from the femurs and tibia were collected 18 and 24 h after treatment. For each animal three slides were prepared and assessed for the presence of CAs (500 metaphases investigated) and the mitotic index was determined after analysing more than 1000 cells. Both controls, negative vehicle and positive (cyclophosphamide), were valid. No statistically significant increase in aberrant cells or the mitotic index compared to control groups was observed.

And also single oral exposure of female rats to manganese dioxide did not induce significant DNA damage (% tail DNA damage). Furthermore, the cell viability was > 90%. In conclusion, manganese dioxide is not considered to cause DNA damage under the test conditions.

Overall there were mixed results obtained with Manganese dioxide in the different genotoxicity studies.

Negative results in the bacterial mutagenicity assay might only be due to inadequate bioavailability of the metal ion due to presence of chelating agents or other compounds competing with the active uptake transporter in the media, which hinder a genotoxic activity of the metal instead of absence of mutagenicity per se (see also discussion(Greim, 1994). In vivo genotoxicity studies yielded positive results indicating clastogenic/genotoxic activity when a repeated dosing regimen was applied (yielding higher systemic manganese concentrations) compared to negative test results obtained after single manganese dioxide exposure.

Additional information

Additional results:

- Copper:

In reviews like e.g. the Biocidal products assessment report for Copper (II) oxide (2011) and SIDS Initial Assessment Profile (2014) further proprietary studies are mentioned. These studies investigate the mutagenic potential of copper (II) sulfate pentahydrate. Negative test results were obtained in a bacterial reverse mutation assay (AMES Test; Salmonella typhimurium Strains TA98, TA100, TA1535, TA1537, TA102, tested up to cytotoxic concentrations; with and without metabolic activation) and also in an in vivo micronucleus assay in mice (dose 447 mg/kg bw). No unscheduled DNA synthesis was observed in the hepatocytes of orally dosed male rats (gavage dosing of 632.5 or 2000 mg/kg bw). These studies thus support the notion that copper is not mutagenic in these test systems used.

- Manganese dioxid:

Additional proprietary studies were cited in a summary provided by OCED (2007; not included in IUCLID). Here manganese dioxide elicited positive results in in vitro chromosomal aberration test [OECD TG 473] for CHL/IU cells and in in vivo mammalian erythrocyte micronucleus assay [OECD TG 474]. The available information suggests that manganese dioxide is genotoxic ( SIDS Initial Assessment Profile for SIAM 25 (17-18 October 2007). Manganese dioxide; online: http://webnet.oecd.org/HPV/UI/Search.aspx).

Justification for classification or non-classification

Free copper and manganese ions are able to induce the formation of reaction oxygen species and thus in turn may lead to secondary genotoxic effects. As both metals are essential metals and homeostasis is tightly regulated, under physiological conditions none of these effects will occur. No data are available for the registration substance. However adequate and reliable studies performed with each of the two constituents of the registration substance are at hand.

In someof these in vivo and in vitro tests positive test results were obtained, however this is most probably only due to unphysiological exposure conditions (e.g. intraperitoneal). Based on this mechanistic considerations and in light of also a lot of negative test results obtained in several reliable in vitro and in vivo studies the submission substance has not to be classified for genotoxic effects according to Regulation (EC) No 1272/2008.