Manganese oxalate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Manganese Oxalate, dihydrate

IUPAC Name:

Manganese Oxalate, dihydrate

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:

phenobarbital/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment I and Experiment II: 3; 10; 33; 100; 333; 1000; 2500; 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item Manganese oxalate, dihydrate was suspended in DMSO (MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, NaN3 (TA 1535, TA 100), 4-nitro-o-phenylene-diamine, 4-NOPD (TA 1537, TA 98), methyl methane sulfonate, MMS (WP2 uvrA)

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

Experimental Performance

For each strain and dose level including the controls, three plates were used.

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL Overlay agar

In the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.


Acceptability of the Assay

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation criteria:

Evaluation of Results

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Example:
TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the test tubes and on the incubated agar plates at 2500 and 5000 µg/plate. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA:
performed

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 Summary of Results Pre-Experiment and Experiment I

Study Name: 1360602	Study Code: Harlan CCR 1360602
Experiment: 1360602 VV Plate	Date Plated: 11/08/2010
Assay Conditions:	Date Counted: 17/08/2010

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			13 ± 3	13 ± 1	26 ± 2	121 ± 7	42 ± 9
	Untreated			14 ± 2	14 ± 2	30 ± 9	108 ± 7	47 ± 8
	Manganese	3 µg		15 ± 1	17 ± 3	26 ± 7	117 ± 15	42 ± 9
	oxalate, dihydrate	10 µg		15 ± 1	18 ± 2	32 ± 7	127 ± 9	43 ± 13
		33 µg		15 ± 2	17 ± 0	32 ± 5	124 ± 6	41 ± 7
		100 µg		13 ± 2	15 ± 5	30 ± 4	130 ± 16	43 ± 1
		333 µg		14 ± 0	22 ± 2	23 ± 6	131 ± 15	40 ± 3
		1000 µg		14 ± 2	17 ± 4	32 ± 4	141 ± 23	54 ± 9
		2500 µg		17 ± 2^P	14 ± 5^P	33 ± 7^P	136 ± 18^P	51 ± 7^P
		5000 µg		12 ± 1^P	15 ± 2^P	31 ± 2^P	138 ± 6^P	55 ± 6^P
	NaN3	10 µg		1610 ± 36			1866 ± 30
	4-NOPD	10 µg				455 ± 6
	4-NOPD	50 µg			69 ± 10
	MMS	3.0 µL						870 ± 16
With Activation	DMSO			25 ± 6	21 ± 6	46 ± 6	132 ± 18	59 ± 3
	Untreated			24 ± 7	26 ± 4	52 ± 4	129 ± 5	65 ± 6
	Manganese	3 µg		23 ± 3	23 ± 4	41 ± 4	139 ± 16	66 ± 8
	oxalate, dihydrate	10 µg		24 ± 2	25 ± 2	52 ± 4	142 ± 20	59 ± 12
		33 µg		22 ± 7	23 ± 4	43 ± 8	147 ± 8	61 ± 8
		100 µg		22 ± 3	26 ± 6	43 ± 8	138 ± 7	64 ± 7
		333 µg		23 ± 1	26 ± 2	46 ± 3	147 ± 10	60 ± 3
		1000 µg		24 ± 3	24 ± 7	49 ± 7	139 ± 24	67 ± 4
		2500 µg		14 ± 3^P	24 ± 2^P	43 ± 6^P	132 ± 20^P	58 ± 8^P
		5000 µg		14 ± 3^P	15 ± 2^P	47 ± 6^P	148 ± 13^P	66 ± 10^P
	2-AA	2.5 µg		358 ± 28	355 ± 18	2244 ± 179	2656 ± 60
	2-AA	10.0 µg						380 ± 26

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P	Precipitate

 

Summary of Results Experiment II

Study Name: 1360602	Study Code: Harlan CCR 1360602
Experiment: 1360602 HV2 Pre	Date Plated: 12/08/2010
Assay Conditions:	Date Counted: 17/08/2010

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			16 ± 4	11 ± 4	28 ± 1	112 ± 16	46 ± 5
	Untreated			16 ± 6	13 ± 1	28 ± 6	111 ± 7	40 ± 4
	Manganese	3 µg		14 ± 3	9 ± 3	27 ± 1	110 ± 1	46 ± 2
	oxalate, dihydrate	10 µg		12 ± 1	11 ± 4	21 ± 2	88 ± 4	47 ± 8
		33 µg		12 ± 1	9 ± 3	23 ± 4	96 ± 3	47 ± 5
		100 µg		15 ± 3	9 ± 0	30 ± 4	99 ± 8	46 ± 3
		333 µg		16 ± 2	9 ± 3	24 ± 5	93 ± 12	46 ± 4
		1000 µg		13 ± 5	9 ± 2	26 ± 4	106 ± 8	56 ± 2
		2500 µg		13 ± 3^P	10 ± 4^P	29 ± 3^P	109 ± 9^P	53 ± 3^P
		5000 µg		10 ± 1^P M	9 ± 3^P M	30 ± 3^P	113 ± 10^P	50 ± 13^P
	NaN3	10 µg		1451 ± 26			1549 ± 82
	4-NOPD	10 µg				397 ± 48
	4-NOPD	50 µg			81 ± 6
	MMS	3.0 µL						278 ± 6
With Activation	DMSO			21 ± 1	18 ± 4	40 ± 3	121 ± 15	55 ± 3
	Untreated			27 ± 5	24 ± 3	40 ± 12	138 ± 19	53 ± 9
	Manganese	3 µg		19 ± 3	18 ± 3	41 ± 4	118 ± 11	61 ± 9
	oxalate, dihydrate	10 µg		25 ± 11	20 ± 4	40 ± 5	117 ± 9	59 ± 7
		33 µg		22 ± 4	15 ± 2	44 ± 6	118 ± 20	54 ± 5
		100 µg		27 ± 1	19 ± 3	38 ± 5	116 ± 3	55 ± 5
		333 µg		19 ± 3	20 ± 3	52 ± 3	112 ± 4	49 ± 6
		1000 µg		21 ± 2	19 ± 2	39 ± 9	118 ± 8	59 ± 6
		2500 µg		16 ± 2^P	15 ± 1^P	38 ± 8^P	124 ± 4^P	67 ± 5^P
		5000 µg		16 ± 6^P M	14 ± 4^P	38 ± 7^P	108 ± 16^P	55 ± 8^P
	2-AA	2.5 µg		287 ± 12	185 ± 11	1702 ± 46	1668 ± 86
	2-AA	10.0 µg						390 ± 30

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item Manganese oxalate, dihydrate was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Manganese oxalate, dihydrate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta­tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.