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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
Study period:
The experimental phase of this study was performed between 13 May 2008 and 04 June 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide
EC Number:
700-042-6
Cas Number:
177997-13-6
Molecular formula:
LiNiCoAlO2 where stoichiometry of Ni+Co+Al equals 1 and ranges are Li = 0.9 – 1.3, Co = 0.01 – 0.2, Ni = 0.75 – 0.99 and Al = 0.01 – 0.2
IUPAC Name:
aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide

Method

Target gene:
Histidine for Salmonella
Trytptophan for E Coli
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:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main test (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water according to information provided by the sponsor. In solubility checks performed in-house, the test material was found to be insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml. The best doseable suspension was achieved in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: (ENNG)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: (4NQO)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: (9AA)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix Migrated to IUCLID6: (BP)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)


DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 - 72 hours


SELECTION AGENT (mutation assays): Not applicable.



NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None


Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria:

There are several criteria for determining a positive result, such as a 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.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation.

Results and discussion

Test resultsopen allclose all
Key result
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 the maximum recommended dose level of 5000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
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 the maximum recommended dose level of 5000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: The test material was insoluble in sterile distilled water.
- Precipitation: A dark, particulate precipitate was observed under an inverted microscope at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: None.


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.



COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.


ADDITIONAL INFORMATION ON CYTOTOXICITY: none.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results for the negative controls (spontaneous mutation rates) are presented in Table 1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A dark, particulate precipitate was observed under an inverted microscope at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

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.

Table 1               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

128

 

25

 

40

 

19

 

15

 

129

(132)

25

(23)

35

(41)

19

(20)

14

(15)

140

 

19

 

47

 

22

 

15

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

123

 

13

 

23

 

17

 

20

 

113

(120)

17

(15)

35

(31)

23

(23)

17

(19)

124

 

16

 

34

 

30

 

21

 

 

Table 2               Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 24 May 2008

To: 27 May 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

-

0

108

115

129

(117)

10.7#

27

26

26

(26)

0.6

24

25

30

(26)

3.2

15

18

22

(18)

3.5

8

10

10

(9)

1.2

-

50

134

126

130

(130)

4.0

27

30

34

(30)

3.5

29

29

29

(29)

0.0

14

20

22

(19)

4.2

13

13

8

(11)

2.9

-

150

118

118

132

(123)

8.1

40

23

22

(28)

10.1

26

27

27

(27)

0.6

12

10

12

(11)

1.2

10

10

10

(10)

0.0

-

500

117

103

117

(112)

8.1

29

21

21

(24)

4.6

29

27

31

(29)

2.0

16

12

12

(13)

2.3

16

16

8

(13)

4.6

-

1500

128

139

125

(131)

7.4

40

25

20

(28)

10.4

26

26

32

(28)

3.5

23

9

16

(16)

7.0

11

10

10

(10)

0.6

-

5000

130

140

120

(130)

10.0

16

22

31

(23)

7.5

22

35

33

(30)

7.0

18

12

19

(16)

3.8

10

12

11

(11)

1.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

1513

1127

1049

(1230)

248.5

114

119

120

(118)

3.2

651

660

647

(653)

6.7

115

115

114

(115)

0.6

2572

2606

2770

(2649)

105.9

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO   4-Nitroquinoline-1-oxide

9AA     9-Aminoacridine

#            Standard deviation

Table 3               Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 24 May 2008

To: 27 May 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

+

0

114

85

93

(97)

15.0#

15

18

19

(17)

2.1

40

21

35

(32)

9.8

21

27

10

(19)

8.6

22

14

20

(19)

4.2

+

50

91

93

99

(94)

4.2

16

16

21

(18)

2.9

41

25

26

(31)

9.0

36

22

15

(24)

10.7

23

15

8

(15)

7.5

+

150

86

101

112

(100)

13.1

18

15

15

(16)

1.7

33

34

34

(34)

0.6

19

19

21

(20)

1.2

15

23

16

(18)

4.4

+

500

102

100

80

(94)

12.2

14

12

10

(12)

2.0

35

34

33

(34)

1.0

10

24

20

(18)

7.2

27

16

14

(19)

7.0

+

1500

81

85

91

(86)

5.0

11

12

13

(12)

1.0

34

24

29

(29)

5.0

14

24

23

(20)

5.5

12

19

20

(17)

4.4

+

5000

91

93

92

(92)

1.0

19

9

15

(14)

5.0

29

34

26

(30)

4.0

23

19

21

(21)

2.0

22

13

10

(15)

6.2

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2370

2221

2043

(2211)

163.7

319

354

307

(327)

24.4

443

514

510

(489)

39.9

126

151

114

(130)

18.9

207

158

206

(190)

28.0

2AA     2-Aminoanthracene

BP         Benzo(a)pyrene

#            Standard deviation

Table 4               Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 01 June 2008

To: 04 June 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

-

0

106

80

93

(93)

13.0#

17

10

17

(15)

4.0

43

34

35

(37)

4.9

18

21

34

(24)

8.5

18

17

22

(19)

2.6

-

50

91

101

86

(93)

7.6

23

16

14

(18)

4.7

41

31

36

(36)

5.0

25

27

27

(26)

1.2

21

22

21

(21)

0.6

-

150

93

111

104

(103)

9.1

15

13

20

(16)

3.6

41

39

39

(40)

1.2

16

28

23

(22)

6.0

16

19

21

(19)

2.5

-

500

110

107

108

(108)

1.5

15

10

18

(14)

4.0

43

43

42

(43)

0.6

17

23

23

(21)

3.5

17

19

20

(19)

1.5

-

1500

94

117

108

(106)

11.6

15

10

10

(12)

2.9

33

29

37

(33)

4.0

34

22

21

(26)

7.2

18

23

14

(18)

4.5

-

5000

100

118

112

(110)

9.2

11

9

15

(12)

3.1

44

28

35

(36)

8.0

28

30

21

(26)

4.7

22

21

15

(19)

3.8

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

416

586

568

(523)

93.4

85

147

138

(123)

33.5

758

762

745

(755)

8.9

167

146

141

(151)

13.8

2028

2103

1603

(1911)

269.6

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO   4-Nitroquinoline-1-oxide

9AA     9-Aminoacridine

#            Standard deviation

Table 5               Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 01 June 2008

To: 04 June 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

+

0

93

102

175

(123)

45.0#

14

10

14

(13)

2.3

29

26

31

(29)

2.5

14

19

26

(20)

6.0

15

13

21

(16)

4.2

+

50

109

98

108

(105)

6.1

9

9

9

(9)

0.0

21

23

23

(22)

1.2

18

27

21

(22)

4.6

19

25

18

(21)

3.8

+

150

78

72

79

(76)

3.8

11

9

9

(10)

1.2

23

23

29

(25)

3.5

20

19

25

(21)

3.2

23

13

17

(18)

5.0

+

500

99

68

91

(86)

16.1

9

12

12

(11)

1.7

23

22

29

(25)

3.8

20

28

23

(24)

4.0

21

17

22

(20)

2.6

+

1500

102

90

98

(97)

6.1

10

9

9

(9)

0.6

18

33

30

(27)

7.9

17

26

21

(21)

4.5

14

14

12

(13)

1.2

+

5000

92

109

122

(108)

15.0

12

10

9

(10)

1.5

20

22

23

(22)

1.5

25

34

9

(23)

12.7

13

20

16

(16)

3.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2195

2014

2025

(2078)

101.5

445

142

275

(287)

151.9

412

487

486

(462)

43.0

113

188

184

(162)

42.2

271

570

510

(450)

158.2

2AA     2-Aminoanthracene

BP         Benzo(a)pyrene

#            Standard deviation


Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction. 

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-were treated with suspensions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A dark, particulate precipitate was observed under an inverted microscope at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.