Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 November - 01 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 471 without any deviation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
dated 21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
28 November 2017
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
UVCB substance
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and, on the day of each experiment, appropriate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer. No correction for purity was required. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Method

Target gene:
histidine locus for Salmonella strains and tryptophan for E. coli strain
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 5, 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate, with and without S9-mix; repeated in TA1535 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours

CONTROLS:
- Vehicle/solvent control: Dimethyl sulphoxide; performed in triplicate
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate; performed in triplicate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation; performed in triplicate.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

NUMBER OF REPLICATIONS: Triplicate

- OTHER: All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed due to colonies spreading and/or artefacts on the plates, thus distorting the actual plate count.
Rationale for test conditions:
The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5, 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate. Eight test item concentrations per bacterial strain (including an intermediate dose concentration of 3000 µg/plate) were selected in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test item following the change in test methodology. However, after incorporating the pre-incubation modification in the second mutation test, the test item induced significant toxicity as weakened bacterial background lawns and substantial reductions in TA1535 revertant colony frequency to the extent where the bacterial strain required repeat analysis (absence of S9 only) employing an amended test item dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
Evaluation criteria:
Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item 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 making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
refer Tables 7.6.1/1 to 7.6.1/5
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (light and greasy in appearance) was noted at and above 3000 µg/plate, this observation did not prevent the scoring of revertant colonies.

MUTAGENICITY
- 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 first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in both the absence and presence S9-mix.
- In the second mutation test (pre incubation method), the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 1500 µg/plate (TA100 and TA98), 3000 µg/plate (TA1537) and at 500 µg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted from 3000 µg/plate (TA1535) and at 500 µg/plate (TA100). Weakened lawns were not observed to WP2uvrA, TA98 and TA1537 in the presence of S9, however a reduction in WP2uvrA revertant colonies (0.4 compared to the concurrent vehicle control) were noted at 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.
- No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method). Minor statistical values were noted in Experiment 1 (WP2uvrA at 5000 µg/plate in the absence of S9-mix) and in Experiment 2 (TA100 at 500 µg/plate and TA98 at 15 µg/plate in the absence of S9-mix). However, these responses were, in all cases, within the in-house historical vehicle/untreated control values for the relevant tester strains and were, therefore considered of no biological relevance.
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Any other information on results incl. tables

Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

Experiment 1

TA100

TA1535

WP2uvrA

TA98

TA1537

95

 

18

 

20

 

16

 

8

 

82

90)

21

(18)

29

(26)

22

(19)

2

(7)

93

 

16

 

29

 

18

 

10

 

Experiment 2

TA100

TA1535

WP2uvrA

TA98

TA1537

120

 

11

 

14

 

23

 

15

 

100

(113)

13

(14)

29

(25)

19

(20)

16

(14)

119

 

19

 

31

 

17

 

12

 

 

 

12

 

 

 

 

 

 

 

 

 

12

(12)†

 

 

 

 

 

 

 

 

12

 

 

 

 

 

 

 

† Repeated at a later date (absence of S9 only) due to an insufficient number of non-toxic concentrations in the original experiment

 

Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation

 

Test Period

From: 16 November 2017

To: 19 November 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

100

91

91

(94)

5.2#

12

17

25

(18)

6.6

22

21

20

(21)

1.0

24

18

14

(19)

5.0

10

18

13

(14)

4.0

1.5 µg

96

101

69

(89)

17.2

20

17

26

(21)

4.6

26

20

25

(24)

3.2

20

19

14

(18)

3.2

5

8

9

(7)

2.1

5 µg

100

89

84

(91)

8.2

16

15

13

(15)

1.5

17

21

25

(21)

4.0

26

18

23

(22)

4.0

9

9

7

(8)

1.2

15 µg

84

117

97

(99)

16.6

16

19

16

(17)

1.7

26

23

28

(26)

2.5

14

15

18

(16)

2.1

11

8

11

(10)

1.7

50 µg

72

100

105

(92)

17.8

21

16

13

(17)

4.0

29

23

24

(25)

3.2

21

20

13

(18)

4.4

12

8

15

(12)

3.5

150 µg

78

81

73

(77)

4.0

12

14

13

(13)

1.0

28

14

33

(25)

9.8

21

19

13

(18)

4.2

16

3

8

(9)

6.6

500 µg

81

65

82

(76)

9.5

8

8

16

(11)

4.6

19

27

18

(21)

4.9

17

16

19

(17)

1.5

8

6

19

(11)

7.0

1500 µg

85

86

71

(81)

8.4

18

10

14

(14)

4.0

31

23

19

(24)

6.1

24

28

21

(24)

3.5

12

12

11

(12)

0.6

5000 µg

58 SP

53 SP

55 SP

(55)

2.5

8 SP

7 SP

8 SP

(8)

0.6

34 P

37 P

36 P

(36)

1.5

30 SP

10 SP

12 SP

(17)

11.0

8 SP

9 SP

7 SP

(8)

1.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

466

532

571

(523)

53.1

321

330

385

(345)

34.6

562

583

598

(581)

18.1

139

139

150

(143)

6.4

409

459

455

(441)

27.8

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

P:        Test ítem precipitate

S:       Sparse bacterial background lawn

#:       Standard deviation

 

Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation

 

Test Period

From: 16 November 2017

To: 19 November 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

122

119

105

(115)

9.1#

17

17

20

(18)

1.7

33

26

30

(30)

3.5

13

23

21

(19)

5.3

14

17

15

(15)

1.5

1.5 µg

96

102

101

(100)

3.2

20

12

13

(15)

4.4

32

18

29

(26)

7.4

19

22

23

(21)

2.1

13

20

3

(12)

8.5

5 µg

88

113

104

(102)

12.7

15

19

17

(17)

2.0

28

22

17

(22)

5.5

16

19

18

(18)

1.5

17

10

10

(12)

4.0

15 µg

100

97

112

(103)

7.9

16

13

10

(13)

3.0

20

34

28

(27)

7.0

21

26

29

(25)

4.0

16

13

9

(13)

3.5

50 µg

98

90

103

(97)

6.6

12

12

10

(11)

1.2

33

31

33

(32)

1.2

18

30

31

(26)

7.2

7

21

10

(13)

7.4

150 µg

80

65

83

(76)

9.6

8

14

11

(11)

3.0

21

25

17

(21)

4.0

14

22

14

(17)

4.6

7

8

14

(10)

3.8

500 µg

92

101

94

(96)

4.7

8

10

11

(10)

1.5

20

33

24

(26)

6.7

24

22

18

(21)

3.1

15

9

8

(11)

3.8

1500 µg

101

101

103

(102)

1.2

11

10

8

(10)

1.5

31

25

28

(28)

3.0

20

15

26

(20)

5.5

11

8

11

(10)

1.7

5000 µg

85 SP

53 SP

64 SP

(67)

16.3

11 SP

12 SP

9 SP

(11)

1.5

46 P

32 P

30 P

(36)

8.7

18 SP

25 SP

11 SP

(18)

7.0

4 SP

15 SP

7 SP

(9)

5.7

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2321

2466

2205

(2331)

130.8

315

262

312

(296)

29.8

149

173

185

(169)

18.3

191

166

191

(183)

14.4

331

347

345

(341)

8.7

2AA:   2-Aminoanthracene

BP:     Benzo(a)pyrene

P:        Test ítem precipitate

S:       Sparse bacterial background lawn

#:       Standard deviation

 

Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation

 

Test Period

From: 24 November 2017
28 November 2017†

To: 27 November 2017
01 December 2017†

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535 †

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

69

99

67

(78)

17.9#

18

18

21

(19)

1.7

29

27

25

(27)

2.0

13

13

12

(13)

0.6

24

16

13

(18)

5.7

0.05 µg

N/T

14

11

12

(12)

1.5

N/T

N/T

N/T

0.15 µg

N/T

12

16

14

(14)

2.0

N/T

N/T

N/T

0.5 µg

N/T

16

12

9

(12)

3.5

N/T

N/T

N/T

1.5 µg

N/T

11

25

16

(17)

7.1

N/T

N/T

N/T

5 µg

105

98

84

(96)

10.7

14

14

10

(13)

2.3

30

29

20

(26)

5.5

17

27

19

(21)

5.3

11

16

23

(17)

6.0

15 µg

87

82

84

(84)

2.5

9

11

8

(9)

1.5

24

30

27

(27)

3.0

23

25

20

(23)

2.5

16

17

17

(17)

0.6

50 µg

72

87

90

(83)

9.6

8 S

8 S

7 S

(8)

0.6

26

21

20

(22)

3.2

20

20

13

(18)

4.0

19

8

13

(13)

5.5

150 µg

86

68

115

(90)

23.7

7 S

9 S

7 S

(8)

1.2

25

26

24

(25)

1.0

13

13

12

(13)

0.6

28

21

12

(20)

8.0

500 µg

117

97

109

(108)

10.1

N/T

25

22

21

(23)

2.1

20

9

12

(14)

5.7

21

9

5

(12)

8.3

1500 µg

0 T

0 T

0 T

(0)

0.0

N/T

17

12

22

(17)

5.0

13 S

7 S

10 S

(10)

3.0

22

6

13

(14)

8.0

3000 µg

0 TP

0 TP

0 TP

(0)

0.0

N/T

29 P

33 P

24 P

(29)

4.5

11 SP

14 SP

14 SP

(13)

1.7

6 SP

10 SP

9 SP

(8)

2.1

5000 µg

0 TP

0 TP

0 TP

(0)

0.0

N/T

30 SP

19 SP

19 SP

(23)

6.4

11 SP

8 SP

19 SP

(13)

5.7

15 SP

15 SP

14 SP

(15)

0.6

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

574

751

664

(663)

88.5

811

697

813

(774)

66.4

1025

994

939

(986)

43.6

170

206

201

(192)

19.5

304

387

405

(365)

53.9

 †:       Repeated at a later date due to an insufficient number of non-toxic concentrations in the original experiment

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

N/T:    Not tested at this dose level

P:       Precipitate

S:        Sparse bacterial background lawn

T:       Toxic, no bacterial background lawn

V:       Very weak bacterial background lawn

#:       Standard deviation

 

Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation

 

Test Period

From: 24 November 2017

To: 27 November 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

99

84

98

(94)

8.4#

10

12

4

(9)

4.2

39

35

45

(40)

5.0

17

19

26

(21)

4.7

15

15

22

(17)

4.0

5 µg

106

94

92

(97)

7.6

5

9

10

(8)

2.6

22

32

32

(29)

5.8

24

25

15

(21)

5.5

20

12

13

(15)

4.4

15 µg

105

90

111

(102)

10.8

12

8

16

(12)

4.0

34

29

32

(32)

2.5

28

26

23

(26)

2.5

30

19

16

(22)

7.4

50 µg

94

92

106

(97)

7.6

7

7

9

(8)

1.2

38

28

31

(32)

5.1

17

28

25

(23)

5.7

19

17

16

(17)

1.5

150 µg

102

71

70

(81)

18.2

7

9

12

(9)

2.5

29

29

36

(31)

4.0

30

29

23

(27)

3.8

24

15

19

(19)

4.5

500 µg

77

84

72

(78)

6.0

7

13

10

(10)

3.0

29

33

38

(33)

4.5

19

33

23

(25)

7.2

13

23

11

(16)

6.4

1500 µg

85

77

75

(79)

5.3

7

9

8

(8)

1.0

35

33

34

(34)

1.0

21

28

21

(23)

4.0

5

12

17

(11)

6.0

3000 µg

76 P

68 P

71 P

(72)

4.0

15 SP

13 SP

13 SP

(14)

1.2

24 P

29 P

25 P

(26)

2.6

33 P

20 P

19 P

(24)

7.8

17 P

15 P

10 P

(14)

3.6

5000 µg

92 SP

91 SP

87 SP

(90)

2.6

14 SP

16 SP

6 SP

(12)

5.3

17 P

17 P

16 P

(17)

0.6

14 P

27 P

19 P

(20)

6.6

10 P

10 P

9 P

(10)

0.6

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1095

1314

1320

(1243)

128.2

279

284

255

(273)

15.5

174

217

227

(206)

28.2

131

135

116

(127)

10.0

342

353

216

(304)

76.1

2AA:   2-Aminoanthracene

BP:      Benzo(a)pyrene

P:        Precipitate

S:        Sparse bacterial background lawn

#:       Standard deviation

 

Table 7.6.1/6: History Profile of Vehicle and Positive Control Values

 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Values†

274

278

504

285

26

13

461

229

526

299

506

282

42

51

39

49

Min

60

61

7

7

222

278

10

12

11

10

4

6

87

98

89

93

Max

166

175

31

29

376

388

58

43

45

46

27

27

237

254

174

177

Mean

91

95

16

14

286

333

24

27

21

24

12

13

156

164

123

137

SD

19.3

19.1

4.5

4.0

48.7

37.6

5.6

5.9

6.2

6.1

3.8

3.4

42.2

35.6

23.1

21.2

POSITIVE CONTROL VALUES 2015

 

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

Values

276

280

252

264

13

13

231

227

262

276

253

261

20

35

20

35

 

Min

222

250

79

118

953

673

116

103

100

78

164

97

430

494

745

325

 

Max

2266

2402

2779

457

3140

1655

2769

550

502

705

2318

823

1696

2264

3662

1174

 

Mean

614

927

472

246

2303

1093

792

266

222

218

911

336

761

1461

2257

569

 

SD

260.6

452.5

434.8

55.7

815.2

376.5

342.1

97.7

70.2

107.6

412.4

135.7

350.0

382.0

790.7

220.3

 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Values

399

401

758

393

60

30

690

345

788

415

762

398

32

32

16

24

Min

63

66

8

8

216

221

10

13

8

12

3

4

97

104

78

52

Max

154

156

34

39

340

375

53

53

49

51

24

23

268

243

148

166

Mean

90

93

15

15

268

310

22

27

21

25

12

13

161

159

118

110

SD

14.5

14.3

4.5

5.2

26.4

31.1

5.8

6.3

4.8

5.7

3.5

3.5

39.2

32.3

17.0

29.3

POSITIVE CONTROL VALUES 2016

 

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

Values

409

406

381

386

30

28

341

335

388

385

379

381

14

24

8

16

 

Min

221

284

84

92

897

629

107

102

100

96

95

101

445

574

1674

372

 

Max

2222

2863

2994

879

2326

2140

1611

637

449

4357

1413

639

1117

1855

2823

945

 

Mean

724

1264

854

240

1633

950

718

240

186

188

406

290

743

1271

2379

535

 

SD

320.4

562.9

664.9

62.1

564.5

382.7

338.6

98.2

49.8

230.8

227.0

92.7

214.6

326.5

426.2

143.3

 

SD:     Standard deviation

Min:     Minimum value

Max:    Maximum value

†:        Number of mean values used to create dataset

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:

- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

- Experiment 2 - Pre-Incubation Method: 5, 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate, with and without S9-mix

After incorporating the pre-incubation modification in the second mutation test, the test item induced significant toxicity as weakened bacterial background lawns and substantial reductions in TA1535 revertant colony frequency to the extent where the bacterial strain required repeat analysis (absence of S9 only) employing an amended test item dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.

Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.

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 first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in both the absence and presence S9-mix.

In the second mutation test (pre incubation method), the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 1500 µg/plate (TA100 and TA98), 3000 µg/plate (TA1537) and at 500 µg/plate (WP2uvrA).  In the presence of S9-mix, weakened bacterial background lawns were noted from 3000 µg/plate (TA1535) and at 500 µg/plate (TA100). Weakened lawns were not observed to WP2uvrA, TA98 and TA1537 in the presence of S9, however a reduction in WP2uvrA revertant colonies (0.4 compared to the concurrent vehicle control) were noted at 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.  

A test item precipitate (light and greasy in appearance) was noted at and above 3000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and in Experiment 2 (pre incubation method). Minor statistical values were noted in Experiment 1 (WP2uvrA at 5000 µg/plate in the absence of S9-mix) and in Experiment 2 (TA100 at 500 µg/plate and TA98 at 15 µg/plate in the absence of S9-mix). However, these responses were, in all cases, within the in-house historical vehicle/untreated control values for the relevant tester strains and were, therefore considered of no biological relevance.

Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.