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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 22 December 2009 and 07 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
Histidine for Salmonella.
Tryptophan 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 mix
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:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 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 fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation Rate for TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA100
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
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1535
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
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of TA1537
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl suphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of TA98
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
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of TA98
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
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of WP2uvrA-
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 10 µg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous Mutation rate of WP2uvrA-
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- 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 hrs


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 and examined for effects on the growth of the bacterial background lawn.
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 not be an excessive loss of plates due to 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:
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
-Dimethyl suphoxide solubility: The test material was fully soluble in sdimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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).

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: None

Any other information on results incl. tables

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.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without

(-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

122

115

116

128

115

106

119

104

107

100

110

+

TA100

94

91

99

104

71

90

97

91

98

88

84

-

WP2uvrA-

17

32

25

16

28

32

25

18

19

27

29

+

WP2uvrA-

30

33

24

28

31

27

22

32

29

24

32

In the range-finding test (Experiment 1 – plate incorporation method) the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level in either the absence or presence of S9-mix. However, in the second experiment (pre-incubation methodology) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester stains dosed in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to any of the bacterial strains dosed in the presence of S9-mix. The toxicity observed was of insufficient severity to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, 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.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation for the Main test are presented in the tables below:

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

104

 

21

 

19

 

19

 

9

 

104

(101)

20

(24)

21

(18)

9

(16)

13

(13)

95

 

32

 

13

 

21

 

16

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

104

 

20

 

19

 

22

 

12

 

102

(101)

28

(22)

25

(25)

16

(19)

11

(11)

98

 

19

 

30

 

19

 

10

 

Table 2 Test Results: Range-Finding Test– Without Metabolic Activation

Test period

From: 30 January 2010

To: 02 February 2010

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

88

92

109

(96)

11.2#

20

23

23

(22)

1.7

16

16

22

(18)

3.5

21

18

18

(19)

1.7

11

14

11

(12)

1.7

-

50

82

70

85

(79)

7.9

20

16

19

(18)

2.1

13

24

19

(19)

5.5

13

12

14

(13)

1.0

13

9

12

(11)

2.1

-

150

85

84

89

(86)

2.6

16

21

15

(17)

3.2

16

22

14

(17)

4.2

14

14

19

(16)

2.9

13

8

13

(11)

2.9

-

500

85

77

95

(86)

9.0

19

16

16

(17)

1.7

11

22

19

(17)

5.7

12

16

14

(14)

2.0

11

13

12

(12)

1.0

-

1500

88

90

86

(88)

2.0

14

24

24

(21)

5.8

12

12

20

(15)

4.6

12

12

12

(12)

0.0

13

11

15

(13)

2.0

-

5000

87

93

104

(95)

8.6

20

15

16

(17)

2.6

16

16

23

(18)

4.0

15

20

15

(17)

2.9

8

9

12

(10)

2.1

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

607

517

531

(552)

48.4

1683

1649

1740

(1691)

46.0

397

428

428

(418)

17.9

113

118

121

(117)

4.0

544

534

551

(543)

8.5

ENNG4NQO9AA#

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table 3 Test Results: Range-Finding Test– With Metabolic Activation

Test period

From: 30 January 2010

To: 02 February 2010

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

99

102

97

(99)

2.5#

12

12

10

(11)

1.2

20

19

18

(19)

1.0

16

24

23

(21)

4.4

11

15

13

(13)

2.0

+

50

88

91

109

(96)

11.4

9

9

11

(10)

1.2

13

18

23

(18)

5.0

17

24

15

(19)

4.7

8

15

14

(12)

3.8

+

150

84

74

81

(80)

5.1

12

12

11

(12)

0.6

19

15

18

(17)

2.1

17

15

19

(17)

2.0

13

15

14

(14)

1.0

+

500

75

76

81

(77)

3.2

9

9

7

(8)

1.2

24

16

16

(19)

4.6

20

19

18

(19)

1.0

13

13

12

(13)

0.6

+

1500

75

85

71

(77)

7.2

9

9

12

(10)

1.7

21

16

16

(18)

2.9

18

22

15

(18)

3.5

15

16

12

(14)

2.1

+

5000

80

79

95

(85)

9.0

8

14

13

(12)

3.2

11

25

24

(20)

7.8

16

18

17

(17)

1.0

11

14

16

(14)

2.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1052

1258

1057

(1122)

117.5

242

213

217

(224)

15.7

166

186

223

(192)

28.9

244

260

289

(264)

22.8

143

231

96

(157)

68.5


BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

#       Standard deviation

Table 4 Test Results: Main Test– Without Metabolic Activation

Test Period

From: 04 February 2010

To: 07 February 2010

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

109

113

80

(101)

18.0#

17

19

19

(18)

1.2

28

17

17

(21)

6.4

22

24

21

(22)

1.5

8

13

13

(11)

2.9

-

50

92

87

97

(92)

5.0

22

21

21

(21)

0.6

21

21

21

(21)

0.0

22

22

20

(21)

1.2

14

12

12

(13)

1.2

-

150

100

105

81

(95)

12.7

17

20

18

(18)

1.5

16

16

21

(18)

2.9

19

19

15

(18)

2.3

11

12

12

(12)

0.6

-

500

111

90

94

(98)

11.2

16

21

21

(19)

2.9

21

20

20

(20)

0.6

21

25

19

(22)

3.1

14

11

11

(12)

1.7

-

1500

110

96

113

(106)

9.1

16

21

17

(18)

2.6

24

15

24

(21)

5.2

18

17

17

(17)

0.6

13

13

12

(13)

0.6

-

5000

104 *

109 *

104 *

(106)

2.9

21 *

21 *

17 *

(20)

2.3

22 *

22 *

18 *

(21)

2.3

20 *

17 *

18 *

(18)

1.5

13 *

12 *

13 *

(13)

0.6

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

515

613

520

(549)

55.2

359

359

365

(361)

3.5

671

690

686

(682)

10.0

153

116

146

(138)

19.7

1012

888

1019

(973)

73.7

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

*         Partial absence of bacterial background lawn

#        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. This study has been selected as the key study because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP).
Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods 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 for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, 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.

In the range-finding test (Experiment 1 – plate incorporation method) the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level in either the absence or presence of S9-mix. However, in the second experiment (pre-incubation methodology) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester stains dosed in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to any of the bacterial strains dosed in the presence of S9-mix. The toxicity observed was of insufficient severity to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

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 or exposure method.

Conclusion.

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