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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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

Constituent 1
Reference substance name:
Manganese Oxalate, dihydrate
IUPAC Name:
Manganese Oxalate, dihydrate

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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.
Controlsopen allclose all
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
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 resultsopen allclose all
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
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 ± 2P

14 ± 5P

33 ± 7P

136 ± 18P

51 ± 7P

 

5000 µg

 

12 ± 1P

15 ± 2P

31 ± 2P

138 ± 6P

55 ± 6P

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 ± 3P

24 ± 2P

43 ± 6P

132 ± 20P

58 ± 8P

 

5000 µg

 

14 ± 3P

15 ± 2P

47 ± 6P

148 ± 13P

66 ± 10P

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 ± 3P

10 ± 4P

29 ± 3P

109 ± 9P

53 ± 3P

 

5000 µg

 

10 ± 1P M

9 ± 3P M

30 ± 3P

113 ± 10P

50 ± 13P

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 ± 2P

15 ± 1P

38 ± 8P

124 ± 4P

67 ± 5P

 

5000 µg

 

16 ± 6P M

14 ± 4P

38 ± 7P

108 ± 16P

55 ± 8P

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.