Manganese dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-03 to 2009-10-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

manganese chloride

IUPAC Name:

manganese chloride

Constituent 2

Reference substance name:

Manganese dichloride

EC Number:

231-869-6

EC Name:

Manganese dichloride

Cas Number:

7773-01-5

Molecular formula:

Cl2Mn

IUPAC Name:

manganese(2+) dichloride

Details on test material:

- Name of test material : Manganese chloride, MnCl2
- Molecular formula : MnCl2
- Substance type: Light pink solid flakes
- Physical state: Solid
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Please see table 1 under section Any other information on materials and methods incl. tables for dosing regime

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: R0 medium

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

In the absence of metabolic activation. 400 µg/mL and 150 µg/mL for the 4 hour and 24 hour exposures respectively.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of activation. 2 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 or 24 hours
- Generation time : 12 hours
- Selection time (if incubation with a selection agent): 2 days

SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine (TFT) selective medium
STAIN : MTT

NUMBER OF REPLICATIONS:Each dose level was performed in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Plate scoring: Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgement. Large colonies were defined as those that covered approximately 1/4 to 3/4 of the surface of the well and were generally no more than one or two cells thick. Generally all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies normally are more than two cells thick. 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates to visualise the mutant colonies. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour.

% Relative Suspension Growth (%RSG), Day 2 Viability (%V), Relative Total Growth (RTG) and Mutation Frequency (MF) were all calculated to assess the mutagenic potential. Please refer to section: Any other information on materials and methods incl. tables under the appropriate headings for full details.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency over the concurrent vehicle mutant frequency value.

Statistics:

For a response to be considered positive, the induced mutation frequency value must exceed the set Global Evaluation Factor (GEF) value at 126 x 10^-6 for the microwell method. Any test material dose level that exhibits a mutation frequency value that is greater than the corresponding vehicle control by the GEF is considered positive. If a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. When a test material induced modest reproducible increases in the mutation frequencies that did not exceed the GEF value, then scientific judgement was applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Small significant increases designated by the UKEMS statistical package were reviewed using the criteria set out above and disregarded at the discretion of the Study Director.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Test material-induced toxicity was noted at the highest dose level employed in the test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of the test material was observed at and above 10 µg/mL in the 4-hour exposure groups in the absence of metabolic activation and at and above 20 µg/mL in the 4-hour exposure group in the presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: All three exposure groups employed in the screening test exhibited a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. A precipitate of the test material was observed at and above 78.75 µg/mL in the 4-hour exposure group in the absence of metabolic activation, at and above 39.98 µg/mL in the 4-hour exposure group in the presence of metabolic activation, and at and above 19.69 µg/mL in the 24-hour exposure group in the absence of metabolic activation. In the mutagenicity experiments the maximum dose level was limited by test-material-induced toxicity.

Any other information on results incl. tables

Table 2: Results from the preliminary toxicity test

Dose (µg/mL)	%RSG (-S9) 4-Hour Exposure	%RSG (=S9) 4-Hour Exposure	%RSG (-S9) 24-Hour Exposure
0	100	100	100
4.92	102	89	33
9.84	96	100	11
19.69	89	89	1
39.38	72	75	0
78.75	2	57	0
157.5	9	1	0
315	1	0	0
630	0	0	0
1260	0	0	0

 

Table 3: Summary of results for main experiment, 4 hour exposure

Treatment (µg/mL)	4-Hours –S9			Treatment (µg/mL)	4-Hours +S9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	81.37	0	100	1.00	74.20
2.5†	97			20	91	1.04	64.08
5	95	1.06	73.26	40	68	0.86	78.58
10	91	0.94	96.72	60	43	0.41	116.75
20	101	1.24	90.28	80	37	0.32	96.12
40	44	0.46	104.53	100	32	0.22	104.65
60	19	0.08	128.81	120	26	0.15	104.12
80	17	0.13	91.23	140	25	0.18	100.39
120†	14			160‡	13	0.04	42.87
Linear trend			NS	Linear trend
EMS				CP
400	71	0.62	656.51	2	55	0.22	1662.80
† Not plated for viability or 4-TFT resistance MF§ 5-TFT resistant mutants/10^6viable cells 2 days after treatment NS Not significant ‡ Treatment excluded from test statistics due to toxicity

 

Table 4: Summary of results for main experiment, 24 hour exposure

Treatment (µg/mL)	4-Hours –S9
	%RSG	RTG	MF§
0	100	1.00	103.16
0.31	97	0.99	91.33
0.63	105	0.97	79.60
1.25	95	1.07	50.22
2.5	76	0.81	100.92
5	41	0.38	173.75*
7.5	14	0.11	372.63*
10†	7
15†	4
Linear trend			***
EMS
150	54	0.32	1211.25
† Not plated for viability or 4-TFT resistance * p < 0.05 *** p < 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative With and without metabolic activation

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.

Executive summary:

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 material was observed at and above 10 µg/mL in the 4-hour exposure groups in the absence of metabolic activation and at and above 20 µg/mL in the 4-hour exposure group in the presence of metabolic activation. Toxicity in the cloned cultures was observed at doses at 120 and 160 µg/mL without and with S9 activation, respectively. Based on the results of the preliminary toxicity test, the doses chosen for treatment of the extended treatment assay ranged from 0.31 to 15 µg/mL for non-activated cultures with a 24-hour exposure. Toxicity in the cloned cultures was observed at doses of 10 and 15 µg/mL.

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

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.