Manganese di(acetate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vitro: - Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimurium TA 102: negative without metabolic activation (equivalent to OECD 471) - Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 97, TA 98, TA 100: negative with and without metabolic activation (equivalent to OECD 471) - DNA damage and/or repair (bacterial SOS/umu test): E. coli PQ37 uvrB-: negative without metabolic activation (no guideline available) - DNA damage and/or repair (FADU: Fluorescence Analysis of DNA Unwinding): lymphocytes (human white blood cells, WBC): negative without metabolic activation (no guideline available) - DNA damage and/or repair (Bacillus subtilis recombination assay): Bacillus subtilis H17 (Rec+, arg-, trp-), Bacillus subtilis M45 (Rec-, arg-, trp-): negative without metabolic activation (only slightly positive induction of DNA repair, no distinct effect, no guideline available) Genetic toxicity in vivo: - Gene mutation (somatic mutation assay in Drosophila): Drosophila melanogaster (males), cross C(1)DX, y w f females x sc z w+ f (zeste) males: negative (no guideline available) - Chromosome aberration (Chromosome aberration and micronucleus assay): negative (no substance-related effects, Mice were dosed daily orally with an aqueous solution of the test substance at dose levels of 1/5, 1/15 and 1/30 of the preliminary determined lethal dose for up to 21 days)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No guideline was available, test was performed on the read-across substance MnSO4 and the chromosome aberration scoring system bears some deficiencies. Nevertheless, the present study covers both the stipulated alternatives for mammalian cell cytogenicity, i.e. chromosomal aberration and micronucleus test. Furthermore, it is an in vivo study which is more valuable than an in vitro study and it also includes the testing of repeated applications up to 21 days. So it can be concluded that this study is sufficient to cover and detect all possible cytogenicity effects of the compound and can therefore considered to be reliable with restrictions.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice were dosed daily orally with an aqueous solution of the test substance at dose levels of 1/5, 1/15 and 1/30 of the preliminary determined lethal dose for up to 21 days.
For Chromosome aberration (CA) testing, animals were killed after 7, 14 or 21 days of treatment. 1,5 h prior to sacrifice the animals were injected 0.04% colchicin solution, bone marrow cytogenetic preparations were made, 60 well scattered metaphase plates were scanned for chromosome analysis.
For the micronucleus test (MNA), each animal of the single dose groups received the same dose orally twice with an interval of 24 h. Animals were killed 6 h after the second exposure and scoring slides were prepared. 1000 polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were examined per animal. The endpoint was percentage of micronuclei in PCEs/NCEs.

GLP compliance:

not specified

Type of assay:

other: Chromosome aberration and micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 25 - 30 g

Route of administration:

oral: gavage

Vehicle:

Vehicle/solvent used: distilled water

Details on exposure:

no further details available

Duration of treatment / exposure:

Chromosome aberation (CA) testing: Daily for 7, 14 or 21 days
Micronucleus assay (MNA): 30 h (24 between the two dosage times and 6 h between second dosage and sacrifice)

Frequency of treatment:

daily

Post exposure period:

not applicable

Remarks:

Doses / Concentrations:
1/5, 1/50, 1/30 of lethal concentration
Basis:
nominal conc.
MnSO4

Remarks:

Doses / Concentrations:
61, 20.5, 10.25 mg/100g bw/day
Basis:
nominal conc.
MnSO4

Remarks:

Doses / Concentrations:
610, 205, 102.5 mg/kg bw/day
Basis:
nominal conc.
MnSO4

No. of animals per sex per dose:

5 for each timepoint

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

CA: Bone marrow cells
MNA: polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: percentage (i.e. 1/5, 1/15, 1/30) of lethal dose as determined in preliminary experiments

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no further details available

DETAILS OF SLIDE PREPARATION:
- CA: Bone marrow cytogenetic preparations were made following the method of Preston (Preston, R.J., B.J. Dean, S. Galloway, H. Holden, A.F. McFee and M. Shelby (1987) Mammalian in vivo cytogenetic assays, Analysis of chromosome aberrations in bone marrow cells, Mutation Res., 180, 157-165.)
-MNA: slides were prepared according to Schmid's method (Schmid, W. (1976) The micronucleus test, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Plenum, New York, pp. 31-53.) modified by Das and Kar (Das, R.K., and R.N. Kar (1980) Sodium citrate solution as a substitute for fetal calf serum in micronucleus preparation, Stain Technol., 55, 43-45.).

METHOD OF ANALYSIS:
- CA: 60 well scattered metaphase plates were scanned for chromosome analysis (Sharma, A.K., and A. Sharma (1980) Chromosome Techniques: Theory and Practice, 3rd edn., Butterworth, London.)
-MNA: 1000 polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were examined per animal. The end point was percentage of micronuclei in PCEs/NCEs.

Statistics:

Values were determined as mean ± standard deviation.
Significance was determined at alpha = 0.05 following the Cochran-Armitage trend test.

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Remarks:

Doses was chosen lower than preliminary determined LD50

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: percentage (1/5, 1/15, 1/30) of preliminary determined LD50

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): The chromosomal aberrations screened were chromatid gaps, breaks, fragments, isochromatid gaps, chromatid exchanges and double minutes
- Induction of micronuclei (for Micronucleus assay): determined
- Ratio of PCE/NCE (for Micronucleus assay):determined
- Statistical evaluation: values were determined as mean ± standard deviation, significance was determined according to Cochrane-Armitage trend test

Table 1: Total chromosomal aberrations (CA) and break per cell (B/C) in bone marrow of mice after treatment with MnSO4

Dose in mg/kg bw	Interval (days)	CA (mean_+ SD)	B/C
0	7	8.46 ± 0.83	1.14 ± 0.05
	14	9.28 ± 0.36	0.64 ± 0.06
	21	12.15 ± 0.39	0.61 ± 0.05
102.5	7	19.53 ± 1.45	4.98 ± 0.83
	14	19.25 ± 0.57	5.18 ± 0.67
	21	24.49 ±2.73	5.23 ± 0.33
205	7	38.05 ± 1.84	12.15 ± 1.41
	14	44.13 ± 2.22	12.98 ± 3.12
	21	46.41 ± 0.60	10.73 ± 0.94
610	7	49.93 ± 4.04	15.46 ± 1.45
	14	51.94 ± 5.28	15.19 ± 0.48
	21	53.39 ± 3.39	14.48 ± 0.33

 

Table 2: Micronucleus (MN) formation in mouse bone marrow erythrocytes following MnSO4 treatment

Dose in mg/kg bw	MN % in polychromatic erythrocytes (mean ± SD)	MN % in normochromatic erythrocytes (mean ± SD)	Total MN %
0	0.19±0.35	0.04±0.41	0.23
102.5	0.46±0.49	0.08±: 0.29	0.54
205	0.62±0.52	0.07±0.42	0.69
610	1.34±0.29	0.08 ± 0.29	1.43

 

Table 3: Statistical analysis of CA, B/C and MN following treatment with MnSO4

Endpoint	Interval (days)	Trend test P value
CA	7	< 0.0002*
	14	< 0.0002*
	21	< 0.0002*
B/C	7	< 0.0002*
	14	< 0.0002*
	21	< 0.0002*
MN	PCE-MN	0.0003*
	Total MN	0.4247

*Trend test showing statistically significant value at alpha = 0.005.

Following 7 days of exposure to the lowest dose of MnSO4, 102.5 mg/kg body weight, the frequency of breaks per cell was lowest while the highest dose (610 mg/kg) induced the highest frequency of breaks per cell.

Following exposure to MnSO4, the frequency of total chromosomal abnormalities increased at a rate directly proportional to the concentration of chemical used.

Compared to controls, all 3 doses of the salt increased the frequencies of micronucleated PCEs and NCEs. This increase was, however, statistically not significant.

Conclusions:

Interpretation of results (migrated information): negative
In the in vivo experiments reported here, even the lowest dose of MnSO4 induced a significantly higher number of breaks than the control. One conclusion might be that this effect is possibly due to the direct action of available Mn2+ ions on the chromosomal material leading to specific breaks and rejoining. Manganese is an intracellular oligoelement which is highly concentrated by the mitochondria and within the nucleus. As a result, the frequency of chromosomal aberrations like chromatid gaps and breaks (including fragments), isochromatid gaps and chromatid exchanges increased significantly with increasing concentrations of MnSO4. The overall similarity of the number of breaks induced by the highest and the middle doses of MnSO4, unrelated to the duration of exposure, may indicate a saturation of sites available for action with cationic Mn2+. However, a dose-related increase in the effect was seen but not with increased exposure time, although this could be expected when a saturation of sites available was on hand. Additionally, there is a lack of clarity in the scoring system, in particular for chromosome damage that raises further doubt on the presence of positive genotoxic effects. This is further supported by the lack of clearly significantly increased frequencies of micronucleated poly- and normochromatic erythrocytes. It is most likely that this slight, insignificant increase in micronucleated cells is due to the systemic stress induced by the high doses (up to 20% of the LD50 value) of MnSO4. This present systemic stress due to toxic doses of any chemical is usually accompanied with an increase in e.g. cytokines and reactive oxygen species, whereas the latter are known inducers of genotoxic effects. Consequently, the observed minor increases in micronucleus frequency are irrelevant for the assessment of the intrinsic toxic properties of MnSO4. So it can be furthermore concluded, taking into account the non-prevalence of micronucleus-inductions and deficiencies in the scoring system of CAs and expected time-dependent effects, that even the overall effects on chromosomal structure can be neglected.
This study observed the effects on two endpoints correlated with chromosomal damage and includes furthermore the testing of repeated applications up to 21 days. So it can be concluded that this study is sufficient to cover and detect all possible toxic effects of the compound. Hence, it can considered to be sufficient to fulfill the data requirements under REACH for this specific endpoint, as it is a) as an in vivo study more reliable than an in vitro one, b) covers both stipulated alternatives and c) MnSO4 serves as a read-across substance for manganese acetate.
As an overall conclusion it can be stated that Manganese (II) acetate is not genotoxic regarding cytogenicity.

Executive summary:

In a combined Swiss albino mouse bone marrow chromosomal aberration (CA) and erythrocyte micronucleus (MNA) test, five males per dose and time point were treated orally with MnSO4 in distilled water at daily doses of 0, 102.5, 205 and 601 mg/kg bw/application. Bone marrow cells were harvested at 7, 14 or 21 days of treatment with daily (i.e. repeated) application for chromosome aberration analysis, erythrocytes were harvested 6 h after the second treatment (gavage twice with 24 h spacing) for the micronucleus test. Although there were signs of cytogenic toxicity, i.e. chromosomal aberrations, they were clearly not substance-related and can therefore be neglected. Also, there was no induction of micronuclei significantly over control. MnSO4 was tested up to relatively high doses based on the LD50 of MnSO4.

There was no significant increase in the frequency of micronucleated poly- and normochromatic erythrocytes and no directly substance-related effects in bone marrow after any treatment time induced by MnSO4, which consequently also applies to Manganese (II) acetate.

This study was classified as acceptable with restrictions and satisfies general scientific requirements to assess the in vivo cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

According to REACH Regulation 1907/2006 Annexes VII – IX, testing for genetic toxicity must include an in vitro gene mutation study in bacteria, an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study and an in vitro gene mutation study in mammalian cells; an in vivo study is only required if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII.Although bearing restrictions, such as being performed on read-across substances, the available studies on genetic toxicity are assessed with Klimisch 2 (reliable with restrictions). Furthermore, when using a Weight-of-Evidence approach for the assessment of the genetic toxicity of Manganese (II) acetate, all the required study types are available:

 

a) In vitro gene mutation study in bacteria: Two bacterial reverse mutation assays (Ames tests) are available, both equivalent to OECD 471, one on S. typhimurium TA 102 on MnSO4 without metabolic activation and one on S. typhimurium TA 1535, TA 1537, TA 97, TA 98, TA 100 on MnSO4 with and without metabolic activation. In both assays congruent negative results were obtained. This is supported by two assays on bacterial DNA damage and/or repair, i.e. bacterial SOS/umu test (E. coli PQ37 uvrB-, KMnO₄, MnCl₂*4H₂O, MnSO₄*H₂O) and Bacillus subtilis recombination assay (Bacillus subtilis H17/M45 (Rec+/-, arg-, trp),Mn(CH₃COO)₂, MnCl₂, Mn(NO₃)2, MnSO₄), which both revealed negative results without metabolic activation. Hence, this required study type is available, and all studies provide congruent negative results.

 

b) In vitro cytogenicity study in mammalian cells or in vitro micronucleus study: An in vitro study is not available. However, Regulation 1907/2006 Annex VIII states, that the study does not usually need to be conducted, if adequate data from an in vivo cytogenicity test are available. This is supported by the scientific consideration that in vivo data are generally more reliable than in in vitro data because they are more effectively able to mimic a substance’ effect in humans. A combined in vivo Swiss albino mouse bone marrow chromosomal aberration (CA) and erythrocyte micronucleus (MNA) test is available, which observed the effects on two endpoints correlated with chromosomal damage and includes furthermore the testing of repeated applications up to 21 days. So it can be concluded that this study is sufficient to cover and detect all possible toxic effects of the compound. There was no significant increase in the frequency of micronucleated poly- and normochromatic erythrocytes and no directly substance-related effects in bone marrow after any treatment time induced by MnSO4, which consequently also applies to Manganese (II) acetate. So, Manganese (II) acetate is not genotoxic regarding chromosomal damage in mammalian cells and in vivo.

 

c) In vitro gene mutation study in mammalian cells: To cover this endpoint, a study performed with a well established method to detect somatic mutations in Drosophila melanogaster. Although not performed on an mammalian cell system or organism, Drosophila provides a different enzyme composition and metabolic network than bacteria and are closer related to higher organisms (mammals) than bacteria are. There was no significant increase of somatic mutations over background. In combination with the results obtained in a FADU assay (Fluorescence Analysis of DNA Unwinding) on freshly isolated human lymphocytes, also this endpoint can be considered to be covered out of the following reasons. First, human lymphocytes are mammalian cells and are therefore one of the foreseen test systems to cover this endpoint. Second, the FADU assay detects single-stranded DNA and damaged DNA. DNA damage is, if not repaired, the precursor of DNA mutations. Also, besides during DNA replication and transcription, single-stranded DNA occurs during repair processes as a consequence of DNA damage and can consequently furthermore serve as a surrogate for mutations as a consequence of DNA damage and error-prone repair. Also in this assay, there was no evidence that DNA damage was induced and consequently, no mutations in mammalian cells can be expected to occur. In conclusion, also the endpoint “In vitro gene mutation study in mammalian cells” can be considered to be covered with negative results.

Additional Remark: Under certain experimental conditions, manganese compounds are mutagenic in bacteria. In addition, positive results have been reported in some mammalian cell assays conducted in vitro using the divalent manganese ion (Oberly et al.1982, Miyaki et al. 1979), but not manganese (II) acetate itself. The positive in vitro result arise from a secondary consequence of the substitution of magnesium ions by divalent manganese ions in DNA polymerases, which reduces the fidelity of DNA replication. No reliable relevant in vivo data are available. The reason for that fact could be, that under in vivo conditions e.g. under homeostatic conditions, the substitution will not take part, because the living cell can avoid or minimize the substitution and therefore the restriction of the DNA fidelity. Since this effect does not occur in vivo, these results are neglected for further risk assessment.

So, all relevant endpoints regarding genotoxicity are covered with negative results, no further in vivo studies are triggered and there are no datagaps identified.

Justification for selection of genetic toxicity endpoint
This study observed the effects on two endpoints correlated with chromosomal damage, i.e. chromosomal aberrations and induction of micronuclei, and includes furthermore the testing of repeated applications up to 21 days. So it can be concluded that this study is sufficient to cover and detect all possible toxic effects of the compound. Also, it is as an in vivo study more reliable than an in vitro one and MnSO4 serves as a read-across substance for manganese acetate.
As an overall conclusion, also taking into account all the other available genotoxicity studies, it can be stated that Manganese (II) acetate is not genotoxic regarding cytogenicity.

Justification for classification or non-classification

All required endpoints under "Genetic toxicity" are covered, all studies are assessed as reliable with restrictions and have a negative or no substance related outcome. Consequently, Manganese (II) acetate does not need to be classified as a mutagen, neither according to Regulation 1272/2008/EC nor Directive 67/548/EEC.