Reaction mass of copper oxide and manganese dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 8, 2006 to Nov. 25, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 JUL 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

29 DEC 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Manganese dioxide

EC Number:

215-202-6

EC Name:

Manganese dioxide

Cas Number:

1313-13-9

Molecular formula:

MnO2

IUPAC Name:

Manganese dioxide

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Supplier: Sigma-Aldrich, Inc.
- CAS-No.: 1313-13-9
- lot/batch No.of test material: 16022KD
- Purity test date: September 2005
- Purity: 99.9%
- Titration: 60.3% (with KMnO4)
- Trace Metals in ppm: Na 20.9, Ca 17.3, Mg 6.0, Zn 5.9, Cr 3.3, Fe 2.2, Cu 0.2
- Appearance: Dark grey powder

VEHICLE
- Chemical name: Sodium carboxylmethyl cellulose (CMC)
- CAS No.: 9004-32-4
- Lot No.: AF2405
- Solvent manufacture: test substance was dissolved in Sterilization distilled water and autoclaved

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: insoluble in distilled water, DMSO and other solvents, suspended in CMC
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test substance was accurately weighed and suspended in 0.5% CMC to give each required concentration immediately before use


FORM AS APPLIED IN THE TEST (if different from that of starting material): Test substance suspended in 0.5% CMC

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix (rat liver, postmitochondrial supernatant, inducing agent Aroclor 1254 from MOLTOX™ Lot No: 2038)

Test concentrations with justification for top dose:

Dose-Range-Finding Test

When the test item at seven concentrations (1.2 ~ 5000 µg/plate) was treated to Salmonella typhimurium TA100, TA98, and Escherichia coli WP2uvrA, the exposure of the test substance at the concentrations of 1250 and 5000 µg/plate without S9 mix caused slight reduction in the number of revertant colonies. The number of revertant colonies did not decrease at any doses with S9 mix. The precipitation was observed on the agar plate at 1250 and 5000 µg/plate. Thus, a maximal dose for the mutation test was determined at 1250 µg/plate in the absence and presence of S9 mix.

Doses for the main study were selected as follows:
- all S. typhimurium strains and E.coli strain in the absence and presence of S9 mix:
39, 78.1, 156.3, 312.5, 625, 1250 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: Solubility test showed that the test item was insoluble in distilled water, DMSO, tetrahydrofuran, glycerol formal, dimethyl formamide, cyclohexane, tetrahydrofurfuryl alcohol, 1-methyl-2-pyrrolidinone, acetone, and cell culture solution, but could be suspended in 0.5% CMC
- historical data of vehicle control was provided

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% CMC (100 µL/plate)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

furylfuramide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- 0.1 mL of test item (or vehicle or positive control), 0.1 mL of bacterial culture, and 0.5 mL of S9 mix (or phosphate buffer, 0.2 M, pH 7.4) were added to a test tube

DURATION
- Preincubation period: for 30 min at 37°C
- then 2 mL of molten top agar containing trace histidin or tryptophan was added to each tube and the mixture was overlaid onto sterile plates of Vogel-Bonner Minimal gucose agar
- Exposure duration: 48 h at 37°C

SELECTION AGENT (mutation assays): top agar (0.6% Difco Bacto-agar and 0.55 sodium chloride with 10 mL of 0.5 mM histidine and 0.5 mM biotin or 0.5 mM tryptophan solution added to each 100 µL of top agar)

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

Slight reduction in the number of revertant colonies caused by exposure of the test substance at the concentrations of 1250 and 5000 µg/plate without S9 mix. Therefore 1250 µg/plate was determined to be a maximal concentration for S. typhimurium strains and E.coli strain in the absence and presence of S9 mix.

Evaluation criteria:

Data were presented using tables and figures as individual plate counts, the mean number of revertant colonies per plate and standard deviation.
Determination of a positive result:
- under S9- or + condition, the increase of reverse mutation colony of a strain in a dose-dependent manner, detection of repeated increase at doses more than one concentration, or having a significant linear relation
- biological relevance of the results was considered
- increase in revertant colonies over two times in bacterial strains TA 98, TA100 and WP2uvrA, and over three times in strains TA1535 and TA1537 than those in negative controls

Besides the above cases, it was judged negative.

Statistics:

Statistical significancy -analysis was not carried out.

Results and discussion

Test results

Test results 1

Key result

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

Test results 2

Key result

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

Test results 3

Key result

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

Test results 4

Key result

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

Test results 5

Key result

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

Additional information on results:

CONTROLS
- all positive controls induced marked increases in frequency of revertant colonies
- all negative controls were found to be in an acceptable range

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at 1250 µg/plate dose regardless of S9 mix


CONFIRMATION TEST:
- test item at any concentrations of 31.3 ~ 1000 µg/plate with and without S9 mix did not induce an increase in frequency of revertant colonies dose-related
- precipitation of test item was not observed at any dose

Any other information on results incl. tables

Table 1. Manganese dioxide: Summary of revertant colony numbers obtained per plate with/ without S9 mix

S9 mix (10%)	Dose (µg/plate)	Number of revertant colonies/plate
		Base replacement type			Frame shift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	NC	135 ±  4	21 ± 2	52 ± 3	55 ± 5	17 ± 1
	39	145 ± 10	21 ± 4	51 ± 4	57 ± 4	17 ± 1
	78.1	138 ±  6	20 ± 4	51 ± 3	57 ± 6	16 ± 3
	156.3	146 ±  9	22 ± 1	50 ± 2	58 ± 5	17 ± 2
	312.5	140 ±  9	20 ± 5	48 ± 2	59 ± 7	15 ± 2
	625	140 ± 10	20 ± 5	48 ± 3	54 ± 2	15 ± 1
	1250#	140 ± 12	18 ± 3	46 ± 8	54 ± 6	14 ± 2
+	NC	147 ±  6	20 ± 2	67 ± 3	70 ± 7	20 ± 5
	39	154 ± 11	20 ± 2	61 ± 6	70 ± 5	20 ± 3
	78.1	154 ±  6	19 ± 2	65 ± 6	74 ± 4	20 ± 2
	156.3	154 ±  7	21 ± 3	63 ± 3	73 ± 4	20 ± 1
	312.5	156 ± 12	21 ± 3	65 ± 5	70 ± 4	18 ± 2
	625	155 ± 14	20 ± 2	66 ± 3	68 ± 3	18 ± 2
	1250#	144 ± 14	18 ± 5	64 ± 4	66 ± 2	16 ± 2
-	PC	473 ± 46	698 ± 52	153 ± 20	309 ± 27	588 ± 47
+		311 ± 45	199 ±  5	320 ± 19	281 ± 53	105 ±  4

Data are presented as mean ± SD (N=3)

NC: Negative Control (0.5% CMC, 100 µL/plate), PC: Positive Control

#: Test item precipitates remained during incubation

Applicant's summary and conclusion

Conclusions:

In a guideline study according to OECD TG 471 under GLP conditions, the test item showed no mutagenic activity in preincubation experiments with and without metabolic activation (S9 mix rat liver postmitochondrial supernatant).

Executive summary:

In a guideline study according to OECD TG 471 under GLP conditions, mutagenic activity of the test item 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 was considered to be non-genotoxic under the present test conditions.