Registration Dossier

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 study was conducted between 23 February 2007 and 27 March 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

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
Test material form:
other: Liquid
Details on test material:
Sponsor's identification: Herboxane
Description: Clear colourless liquid
Batch number: 812596723001
Storage conditions: Room temperature in the dark

Method

Target gene:
S. typhimurium: Histidine
E. coli: Tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
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.
Range-finding test: 15, 50, 150, 500, 1500 and 5000 µg/plate
Main test: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
Dimethyl sulphoxide
Controls
Untreated negative controls:
no
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
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196 °C in a Statebourne Liquid Nitrogen Freezer, Model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
In this assay, overnight subcultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement for the test guidelines and was therefore not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumina-silicate) i.e. 2 mm pellets with a normal pore diameter of 4E-04 microns.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials used in the series of plates without S9-mix, were as follows:
N-ethyl-N’-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate for WP2uvrA-, 3 µg/plate for TA100 and 5 µg/plate for TA1535
9-Aminoacridine (9AA): 80 µg/plate for TA1537
4-nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA98
In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, were used in the series of plates with S9-mix at the following concentrations:
2AA at 1 µg/plate for TA100
2AA at 2 µg/plate for TA1535 and TA1537
2AA at 10 µg/plate for WP2uvrA-
BP at 5 µg/plate for TA98

Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The assay was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and 2 L of molten trace histidine or tryptophan supplemented, top again and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 h incubation at 37 °C the 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.

Mutation test – Experiment 1 (range-finding test)
Six concentration of the test material (15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
An additional dose level was included to allow for test material induced toxicity, ensuring that a minimum of four non-toxic doses were achieved.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix of phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated in triplicate for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation test – Experiment 2 (Main test)
The second experiment was performed using methodology as described for the range-finding test, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly, based on the results of the range-finding test and was 50 to 5000 µg/plate.
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, e.g. rfa cell wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9E+09 bacterial 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 concentration 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 UK EMS 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.

Results and discussion

Test results
Species / strain:
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The test material was toxic to TA100 at 5000 µg/plate and non-toxic to WP2uvrA-. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data were 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 acceptable. These data were for current untreated control plates performed on the same day as the mutation test.
The test material caused a visible reduction in the growth of the bacterial background lawn of all of the Salmonella strains at 5000 µg/plate in both the absence and presence of S9. No weakening of the bacterial background lawns were noted for Escherichia coli strain WP2urvA-. The test material was therefore tested up to the maximum recommended dose level of 5000 µ/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, 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary toxicity test

The number of revertant colonies for the toxicity assay

With (+) or

without (-)

S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5.0

15

50

150

500

1500

5000

-

TA100

100

66

79

89

77

88

74

84

88

81

44*P

+

TA100

99

103

92

71

96

85

85

94

78

95

46*P

-

WP2uvrA-

27

17

21

24

24

15

30

18

18

24

15P

+

WP2uvrA-

29

23

27

20

30

26

22

18

25

25

30P

P: Precipitate

*: partial or complete absence of bacterial background lawn

Spontaneous mutation rates (concurrent negative controls)

Range-finding test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

87

(84)

22

(22)

31

(26)

33

(38)

9

(8)

79

24

19

43

7

85

21

29

38

7

 

Main test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

122

(113)

19

(28)

26

(24)

35

(33)

11

(12)

101

32

27

31

18

115

34

18

32

8

 

19

(24)*

 

34

18

* An additional experimental procedure performed at late date due to contamination in initial main test of TA98 (with S9).

 

Test results: Range-finding test without metabolic activation

Test period

From: 04 March 2007

To: 07 March 2007

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

101

(90)

11.0#

30

(29)

3.2

13

(19)

6.0

12

(18)

5.5

10

(9)

3.6

79

31

25

22

12

90

25

20

21

5

-

15

100

(90)

11.2

36

(29)

7.5

21

(20)

1.2

12

(18)

5.5

3

(4)

1.0

78

21

19

22

4

93

30

19

21

5

-

50

99

(96)

14.3

30

(30)

1.5

11

(17)

5.6

16

(18)

1.5

9

(7)

2.5

108

32

22

18

4

80

29

18

19

7

-

150

85

(87)

11.1

31

(25)

5.9

22

(17)

4.7

24

(19)

4.2

10

(9)

1.7

99

23

15

18

7

77

32

13

16

10

-

500

98

(97)

10.1

22

(24)

1.5

22

(21)

4.2

16

(17)

2.6

5

(6)

2.6

106

24

24

15

4

86

25

16

20

9

-

1500

86

(95)

8.1

26

(25)

1.2

21

(19)

2.5

20

(16)

3.5

8

(8)

1.5

101

24

16

16

7

99

26

19

13

10

-

5000

87*P

(91)

8.1

34*P

(26)

6.9

16 P

(18)

2.0

23*P

(21)

2.0

5*P

(4)

1.2

85*P

22*P

18 P

21*P

3*P

100*P

22*P

20 P

19*P

5*P

Positive control

S9-mix

 -

Name

EENG

EENG

EENG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

No. colonies per plate

422

(446)

36.5

267

(288)

17.9

507

(530)

27.9

287

(267)

18.6

2479

(2497)

18.6

428

297

561

250

2516

488

299

299

265

2495

EENG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NGO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

P = Precipitate

* = Partial absence of bacterial background lawn

# = standard deviation

Test results: Range-finding test with metabolic activation

Test period

From: 04 March 2007

To: 07 March 2007

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

(89)

4.7#

11

(14)

3.8

27

(24)

4.9

31

(26)

4.4

11

(15)

4.0

91

12

26

23

14

84

18

18

24

19

+

15

88

(87)

1.7

14

(15)

3.1

23

(23)

0.6

29

(25)

3.2

12

(15)

3.8

85

18

24

24

19

88

12

23

23

13

+

50

89

(91)

5.3

16

(13)

2.3

18

(24)

6.0

29

(30)

3.6

15

(13)

2.0

97

12

25

27

11

87

12

30

34

13

+

150

87

(79)

7.2

10

(12)

3.2

22

(24)

2.9

30

(20)

9.1

13

(12)

3.1

77

11

27

16

15

73

16

22

13

9

+

500

74

(81)

8.1

18

(15)

3.5

32

(26)

5.6

33

(27)

6.0

18

(12)

3.1

90

15

25

21

9

80

11

21

26

9

+

1500

75

(79)

3.5

16

(12)

4.0

19

(21)

2.9

23

(24)

0.6

10

(12)

2.1

82

12

19

24

11

79

8

24

24

14

+

5000

77*P

(79)

2.1

15*P

(9)

5.7

22 P

(25)

4.4

21*P

(19)

1.5

15*P

(15)

1.0

78*P

7*P

23 P

18*P

16*P

81*P

4*P

30 P

19*P

14*P

Positive control

S9-mix

 +

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. colonies per plate

1214

(1292)

67.9

272

(271)

2.6

1189

(1134)

52.7

326

(331)

8.7

261

(263)

9.7

1333

268

1129

341

255

1330

273

1084

326

274

  BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

P = Precipitate

* = Partial absence of bacterial background lawn

# = Standard deviation

Test results: Main test without metabolic activation

Test period

From: 18March 2007

To: 21 March 2007

With or without S9-mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per place)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

140

(147)

15.7#

33

(30)

6.7

37

(29)

9.2

38

(32)

7.2

8

(7)

3.6

136

34

31

34

10

165

22

19

24

3

-

50

164

(163)

1.2

30

(27)

2.5

15

(18)

4.9

29

(29)

2.0

3

(6)

2.6

164

25

16

27

7

162

27

24

31

8

-

150

157

(158)

19.0

23

(21)

1.7

20

(18)

2.6

36

(28)

6.9

10

(8)

2.9

139

20

19

24

10

177

20

15

24

5

-

200

141

(145)

3.2

20

(23)

2.6

20

(16)

4.0

24

(21)

7.6

2

(4)

2.6

147

24

12

26

7

146

25

15

12

3

-

1500

151

(151)

8.5

37

(34)

13.2

16

(20)

3.8

13

(18)

4.2

5

(5)

1.5

142

46

22

19

4

159

20

23

21

7

-

5000

157*P

(161)

4.0

27*P

(28)

2.6

26P

(19)

6.1

18*P

(23)

4.7

5*P

(7)

4.7

162*P

26*P

14P

25*P

12*P

165*P

31*P

18P

27*P

3*P

Positive control

S(-mix

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

No. colonies per plate

831

(588)

212.1

150

(181)

27.6

313

(276)

33.8

330

(209)

104.7

1677

(1444)

280.4

438

190

267

156

1133

496

203

247

142

1523

ENNG = N-ethyl-N’-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

P = Precipitate

*= Partial absence of bacterial background lawn

# = 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

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 also meets the requirements of the OECD Guidelines for testing chemicals No.471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC 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 the test material using the Ames plate incorporation method at up to six 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 15 to 5000µg/plate. The experiment was repeated on a separate day using an amended dose range, fresh cultures of the bacterial strains and fresh test material.

An additional dose level was included in the range-finding test to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals 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 a visible reduction in the growth of the bacterial background lawn of all of the Salmonella strains at 5000µg/plate in both the absence and presence of S9. No weakening of the bacterial background lawns were noted for Escherichia coli strain WP2uvrA-. The test material was therefore tested up to the maximum recommended dose level of 5000 µg/plate. A light oily precipitate was observed 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 the test.