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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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.

Data source

Reference
Reference Type:
publication
Title:
Comparison of clastogenicity of inorganic Mn administered in cationic and anionic forms in vivo
Author:
Joardar M, Sharma A
Year:
1990
Bibliographic source:
Mutation Research, 240 (1990) 159-163, Elsevier

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese sulphate
EC Number:
232-089-9
EC Name:
Manganese sulphate
Cas Number:
7785-87-7
Molecular formula:
H2O4S.Mn
IUPAC Name:
manganese (2+) sulfate
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Manganese sulphate
- Molecular formula (if other than submission substance): MnSO4*H2O
- Substance type: inorganic salt
- Physical state: solid
- Other: Supplier: Sarabhai Chemicals, Bombay, India

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.

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