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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009-07-10 to 2009-08-04
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material : 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:
Histidine synthesis in the Salmonella typhimurium strains and tryptophan synthesis in the E. coli strain used.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: GC base pairing at the primary reversion site.
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: AT base pairing at the primary reversion site.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test material was found to be soluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL. Sterile distilled water was chosen as the solvent.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)


DURATION
- Preincubation period: 10 hours at 36 °C
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not reported
Evaluation criteria:
Several criteria for determining a positive result. Dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however statistical significance will not be the only determining factor for a positive response.
Statistics:
The following was used to statistically evaluate the results from the mutagenicity test. Kirkland DJ (ED) (1989) Statistical Evaluation of Mutagenicity Test Data (UKEMS) sub-committee on Guidelines for Mutagenicity Testing. Report Part III - Cambridge University Press.

Results and discussion

Test results
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: Not reported


RANGE-FINDING/SCREENING STUDIES: The test substance was found to be non-toxic in the range-finding study.


COMPARISON WITH HISTORICAL CONTROL DATA: The historical control values were found to concur with the results from the study for both positive and vehicle controls.


ADDITIONAL INFORMATION ON CYTOTOXICITY: Not reported
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Range finding study – toxicity assay

With (+) or Without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

75

81

78

96

89

77

85

74

70

86P

84P

+

TA100

77

96

84

85

74

75

91

88

74

70P

73P

-

WP2uvrA-

25

23

25

23

27

30

23

35

31

22P

27P

+

WP2uvrA-

37

24

27

34

29

25

36

32

38

30P

24P

P - Precipitate

 

Table 3: Spontaneous Mutation Rates (Concurrent Negative Controls), Experiment 1

Number of Revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

93

15

22

14

10

96 (95)

19 (18)

20 (22)

16 (17)

13 (12)

97

21

24

20

13

 

Table 4: Spontaneous Mutation Rates (Concurrent Negative Controls), Experiment 2

Number of Revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

123

22

33

22

12

123 (118)

25 (23)

25 (29)

25 (24)

12 (12)

109

21

29

24

13

 

Table 5: Test Results, Experiment 1 – Without Metabolic Activation

 

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

95

111  (102)

99    8.3#

16

20   (18)

19    2.1

23

25    (23)

20     2.5

26

21   (23)

23    2.5

15

13    (14)

13    1.2

50

112

96    (104)

104   8.0

16

20   (19)

20    2.3

18

25     (22)

23      3.6

21

22    (22)

24     1.5

14

13    (12)

10     2.1

150

115

110   (109)

102    6.6

21

13   (18)

21    4.6

26

15    (20)

19     5.6

16

20    (21)

27     5.6

12

11    (12)

13     1.0

500

103

92    (95)

90     7.0

18

19   (17)

14    2.6

21

23     (25)

30      4.7

21

21     (21)

20      0.6

15

15    (12)

15     0.0

1500

110 P

95 P  (103)

104P   7.5

19P

18P  (18)

16P   1.5

22

24P   (23)

22P    1.2

22P

23P   (25)

26P    3.2

13P

15P   (12)

9P      3.1

5000

88P

117P  (105)

111P   15.3

18P

21P  (19)

18P   1.7

26P

21P    (24)

24P     2.5

25P

23P    (25)

26P     1.5

9P

12P   (12)

16P    3.5

Name

Concentration

No. colonies per plate

ENNG

ENG

ENNG

4NQO

9AA

3

5

2

0.2

80

475

526  (492)

476   29.2

99

150  (115)

95     30.7

145

145   (145)

145    0.0

123

118   (119)

115    4.0

345

462   (400)

394    58.8

ENNG – N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO – 4-Nitroquinoline-1-oxide

9AA – 9-Aminoacridine

P – Precipitate

# - Standard deviation

 

Table 6: Test Results, Experiment 1 – With Metabolic Activation

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

97

96     (98)

100   8.3#

12

12   (11)

9     1.7

23

29    (28)

32     4.6

25

24   (23)

20    2.6

14

9     (12)

14    2.9

50

117

101   (106)

100    9.5

11

9     (10)

10    1.0

31

25     (25)

19      6.0

20

21    (20)

18     1.5

12

12    (12)

12     0.0

150

97

102   (101)

103    3.2

10

9    (9)

9    0.6

25

23    (25)

27     2.0

26

22    (24)

24     2.0

15

10    (12)

11     12.6

500

115

74     (94)

92      20.6

13

8   (10)

9    2.6

29

19     (23)

21      5.3

31

20     (24)

20      6.4

16

15    (15)

13     1.5

1500

90 P

82 P  (88)

91P    4.9

12P

9P    (11)

13P   2.1

26P

24P   (23)

20P    3.1

19P

18P   (24)

20P    6.4

11P

15P   (12)

9P      3.1

5000

92P

104P   (95)

90P      7.6

12P

9P     (11)

11P   1.5

24P

24P    (23)

20P     2.3

20P

24P    (22)

22P     2.0

12P

9P     (11)

13P    2.1

Name

Concentration

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2248

2526 (2506)

2743  248.1

209

151  (179)

177    29.1

172

225   (184)

154    36.9

206

184   (197)

202    11.7

278

217   (247)

247    30.5

2AA – 2-Aminoanthracene

BP – Benzo(a)pyrene

P – Precipitate

# - Standard deviation

 

Table 7: Test Results, Experiment 2 – Without Metabolic Activation

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

101

95      (104)

115    10.3#

20

16   (19)

20    2.3

21

24    (23)

25     2.1

26

26   (25)

23    1.7

11

16    (14)

15    2.6

50

106

97    (104)

106   5.2

21

21   (21)

21    0.0

18

26     (24)

29      5.7

20

26    (25)

2 9    4.6

8

15    (12)

13     3.6

150

113

107   (111)

114    3.8

24

20   (21)

20    2.3

22

29    (25)

23     3.8

20

19    (22)

27     4.4

10

8      (9)

10    1.2

500

96

104    (102)

106     5.3

16

22   (17)

14    4.2

23

24    (24)

25     1.0

23

25     (26)

30      3.6

15

8      (9)

10    1.2

1500

118P  (118)

119P   1.0

117       *

24P

20P  (23)

26P   3.1

21P

24P   (22)

22P    1.5

29P

21P   (25)

26P    4.0

10P

11P   (10)

9P      1.0

5000

101P

104P  (105)

110P   4.6

22P

23P  (22)

21P   1.0

27P

24P    (24)

21P     3.0

26P

25P    (25)

24P     1.0

12P

13P   (11)

9P      2.1

Name

Concentration

No. colonies per plate

ENNG

ENG

ENNG

4NQO

9AA

3

5

2

0.2

80

295

312  (309)

320   12.8

209

222    (212)

206     8.5

416

492   (462)

477    40.3

246

217   (228)

220    15.9

1078

2042 (1426)

1157  535.2

ENNG – N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO – 4-Nitroquinoline-1-oxide

9AA – 9-Aminoacridine

P – Precipitate

# - Standard deviation

* p≤0.05

 

Table 8: Test Results, Experiment 2 – With Metabolic Activation

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

108

106     (108)

109      1.5#

10

9     (11)

13    2.1

25

27    (26)

26     1.0

22

21   (22)

22    0.6

16

16   (15)

12    2.3

50

93

91   (106)

107    8.7

15

9     (12)

13    3.1

22

22     (25)

31      5.2

21

26    (22)

22     0.6

13

15    (15)

16     1.5

150

98

89     (99)

110   10.5

13

8     (10)

13    3.1

22

23    (22)

31     0.6

19

24    (22)

22     2.5

14

14    (12)

7       4.0

500

91

106    (102)

110   10.0

12

14   (12)

9     2.5

30

29     (30)

30      0.6

26

21     (23)

22      2.6

16

14    (14)

12     2.0

1500

81P

100P  (96)

110P  10.0

9P

13P    (10)

9P       2.3

25P

31P   (27)

25P    3.5

25P

25P   (24)

22P    1.7

9P

16P     (14)

16P      4.0

5000

90P

84P     (92)

102P    9.2

11P

10P    (10)

8P     1.5

26P

23P    (25)

27P     2.1

24P

21P    (22)

22P     1.5

12P

15P    (12)

10P     2.5

Name

Concentration

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2474

2427 (2510)

2629  105.7

374

332  (332)

291   41.5

342

264   (313)

333    42.7

261

620   (457)

490    181.8

497

494   (498)

504    5.1

2AA – 2-Aminoanthracene

BP – Benzo(a)pyrene

P – Precipitate

# - Standard deviation

 

The test material was found to cause no visible reduction in growth of the bacterial background lawn at any dose and was therefore tested up to the maximum dose level of 5000 µg/plate A particulate precipitate was at 1500 µg/plate and above. This was considered not to prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for and of the bacterial strains, with any dose of the test material, with or without metabolic activation. In the TA100 revertant colony, a small but statistically significant increase was observed on the 1500 µg/plate in Experiment 2 (increase of less than 1.5 times). However these were within the range specified by the Standard Test Method, this increase proved non-reproducible over two separate experiments. This was concluded to have no biological or toxicological relevance. All of the positive control substances induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test material has been found to be non-mutagenic under the test conditions reported.
Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.13/14.

During the study test strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and a tester strain of Escherichia coli (WP2uvrA) were exposed to the test substance both in the presence and the absence of metabolic activation.Vehicle and positive controls were run concurrently. Two separate experiments were conducted, in the first five concentrations of test substance (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. In the second experiment the test substances and vehicle control were dosed using a pre-incubation method.

The test substance was found to cause no visible reduction in growth of the bacterial background lawn at any dose and was therefore tested up to the maximum dose level of 5000 µg/plate A particulate precipitate was at 1500 µg/plate and above. This was considered not to prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, with or without metabolic activation. In the TA100 revertant colony, a small but statistically significant increase was observed on the 1500 µg/plate in Experiment 2 (increase of less than 1.5 times). However the increase was within the range specified by the Standard Test Method, and proved non-reproducible over two separate experiments. This was concluded to have no biological or toxicological relevance. All of the positive control substances induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains. Under the conditions of the study the test material was found to be non-mutagenic.

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.