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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:
22 January 2009 and 10 February 2009.
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:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Oxooil LS9
- Physical state: Clear colourless liquid
-- Stability under test conditions: Stable
- Storage condition of test material: Room temperature in the dark

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
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: Acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Remarks:
2-Aminoanthracene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
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 rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
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.


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 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
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 micro.g/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 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.
- 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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The vehicle (acetone) 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. No test material precipitate was observed on the plates at 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. 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

95

 

20

 

32

 

23

 

13

 

98

(99)

29

(24)

30

(29)

29

(29)

10

(12)

103

 

24

 

26

 

36

 

12

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

86

 

25

 

18

 

23

 

10

 

118

(104)

32

(25)

30

(26)

24

(24)

13

(12)

109

 

19

 

29

 

25

 

13

 

Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 01 February 2009

To: 04 February 2009

With out 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

101

103

88

(97)

8.1#

22

22

25

(23)

1.7

26

24

29

(26)

2.5

20

16

15

(17)

2.6

12

9

9

(10)

1.7

-

50

86

89

87

(87)

1.5

19

23

22

(21)

2.1

22

22

31

(25)

5.2

18

14

16

(16)

2.0

9

14

8

(10)

3.2

-

150

89

91

102

(94)

7.0

16

25

26

(22)

5.5

25

23

30

(26)

3.6

18

18

15

(17)

1.7

10

8

7

(8)

1.5

-

500

106

95

90

(97)

8.2

25

20

27

(24)

3.6

25

23

26

(25)

1.5

16

15

18

(16)

1.5

10

10

9

(10)

0.6

-

1500

90

101

91

(94)

6.1

25

18

19

(21)

3.8

30

21

32

(28)

5.9

18

19

18

(18)

0.6

7

10

10

(9)

1.7

-

5000

79

81

81

(80)

1.2

21

18

21

(20)

1.7

30

32

26

(29)

3.1

16

16

20

(17)

2.3

13

8

9

(10)

2.6

Positive controls S9-Mix

-

Name Concentration (μg/plate)

No. colonies per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

670

759

826

(752)

78.3

658

467

395

(507)

135.9

384

352

348

(361)

19.7

198

246

192

(212)

29.6

521

531

531

(528)

5.8

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 01 February 2009

To: 04 February 2009

With 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

96

100

(101)

5.0#

13

14

10

(12)

2.1

35

35

31

(34)

2.3

33

22

30

(28)

5.7

9

9

9

(9)

0.0

+

50

99

89

98

(95)

5.5

9

12

15

(12)

3.0

34

30

34

(33)

2.3

33

32

27

(31)

3.2

11

10

7

(9)

2.1

+

150

84

90

99

(91)

7.5

7

9

11

(9)

2.0

34

33

34

(34)

0.6

30

21

32

(28)

5.9

9

8

8

(8)

0.6

+

500

89

88

101

(93)

7.2

14

15

10

(13)

2.6

34

31

32

(32)

1.5

21

36

21

(26)

8.7

10

8

7

(8)

1.5

+

1500

85

95

97

(92)

6.4

11

10

11

(11)

0.6

32

33

34

(33)

1.0

25

33

22

(27)

5.7

8

8

10

(9)

1.2

+

5000

89

98

86

(91)

6.2

10

11

12

(11)

1.0

36

33

34

(34)

1.5

23

33

31

(29)

5.3

8

9

8

(8)

0.6

Positive controls S9-Mix

+

Name Concentration (μg/plate)

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2779

2359

2834

(2657)

259.8

181

206

217

(201)

1.8.4

1720

1652

1646

(1673)

41.1

225

241

267

(244)

21.2

523

511

563

(532)

27.2

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

 

Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 07 February 2009

To: 10 February 2009

With out 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

120

103

104

(109)

9.5#

27

34

31

(31)

3.5

33

33

31

(32)

1.2

18

22

22

(21)

2.3

12

12

14

(13)

1.2

-

50

119

104

103

(109)

9.0

27

31

26

(28)

2.6

31

36

31

(33)

2.9

20

24

20

(21)

2.3

10

11

12

(11)

1.0

-

150

117

118

84

(106)

19.3

21

37

29

(29)

8.0

24

26

25

(25)

1.0

16

12

19

(16)

3.5

10

12

10

(11)

1.2

-

500

101

101

99

(100)

1.2

23

30

31

(28)

4.4

29

27

29

(28)

1.2

23

19

15

(19)

4.0

10

11

11

(11)

0.6

-

1500

128

109

110

(116)

10.7

30

24

27

(27)

3.0

35

29

33

(32)

3.1

19

23

18

(20)

2.6

14

10

9

(11)

2.6

-

5000

115

121

90

(109)

16.4

30

26

25

(27)

2.6

34

32

34

(33)

1.2

19

21

22

(21)

1.5

10

12

14

(12)

2.0

Positive controls S9-Mix

-

Name Concentration (μg/plate)

No. colonies per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

1190

1279

1220

(1230)

45.3

1771

1697

1518

(1662)

130.1

2145

2228

2073

(2149)

77.6

213

233

207

(218)

13.6

1226

1215

1227

(1223)

6.7

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 07 February 2009

To: 10 February 2009

With 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

97

99

(98)

1.2#

10

14

9

(11)

2.6

41

40

34

(38)

3.8

30

30

30

(30)

0.0

14

16

15

(15)

1.0

+

50

92

84

91

(89)

4.4

12

8

15

(12)

3.5

35

41

34

(37)

3.8

24

24

31

(26)

4.0

15

16

14

(15)

1.0

+

150

88

93

95

(92)

3.6

9

11

13

(11)

2.0

40

46

30

(39)

8.1

30

24

31

(28)

3.8

15

12

15

(14)

1.7

+

500

93

92

97

(94)

2.6

11

12

9

(11)

1.5

42

37

43

(41)

3.2

24

31

24

(26)

4.0

10

10

12

(11)

1.2

+

1500

102

93

95

(97)

4.7

9

16

11

(12)

3.6

35

40

36

(37)

2.6

22

26

35

(28)

6.7

11

12

11

(11)

0.6

+

5000

91

102

96

(96)

5.5

14

11

13

(13)

1.5

38

44

35

(39)

4.6

27

25

25

(26)

1.2

12

10

10

(11)

1.2

Positive controls S9-Mix

+

Name Concentration (μg/plate)

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2481

3433

3619

(3178)

610.5

184

273

245

(234)

45.5

468

622

552

(547)

77.1

145

227

216

(196)

44.5

328

588

552

(489)

140.9

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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

Introduction. Thethod conforms 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 typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrA-were treated with 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 (acetone) 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. No test material precipitate was observed on the plates at 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.

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