Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2018 to 13 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No. 471, “Bacterial Reverse Mutation Test”, adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No 440/2008, part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14 “Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA Health Effects Test Guidelines, OPPTS 870.5100, “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998
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: particulate/powder
Details on test material:
Name: BAL0001026
CAS number: 376653-41-7
Batch/Lot number: 012
Appearance: Off-white powder
Purity: Content by mass balance: 84.9% w/w
Expiry date: 30 June 2020
Storage conditions: Under inert gas, protected from light and humidity (tight closed container), frozen (≤-15 °C)
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report.

Method

Target gene:
histidine (his) and tryptophan (trp)
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Examined concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test same concentrations were used.
Vehicle:
All dilutions in the main tests of test item were made in the testing laboratory using Dimethyl sulfoxide (DMSO). Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 2 hours after preparation.
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine (NPD); 2-aminoanthracene (2AA)
Details on test system and conditions:
DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test item was insoluble in Distilled water at 100 mg/mL concentration. The test item was soluble at this concentration using DMSO and DMF (clear pale yellow colour solution was detected in each case). Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock solution (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilution in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Procedure for Exposure in the Initial Mutation Test
A standard plate incorporation procedure was performed as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No .2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per test item concentration and for each control) were appropriately labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar: 2000 µL
vehicle or test item formulation (or reference controls): 50 µL
overnight culture of test strain: 100 µL
phosphate buffer (pH 7.4) or S9 mix: 500 µL
This solution was mixed and subsequently poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
For the pre-incubation method, bacteria (cultured in Nutrient Broth No. 2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates were appropriately labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 µL of the test item formulation or its vehicle (or 50 µL of positive reference controls or their solvents), 100 µL of the overnight culture of bacterial cells and 0.5 mL of the S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into appropriate tubes to provide direct contact between bacteria and the test item. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated whilst being shaken for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar was added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.
Rationale for test conditions:
The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of Citoxlab Hungary Ltd.
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
-the number of revertant colonies of the negative (solvent) and positive controls were in the relevant historical control range in all tester strains of the main tests;
-at least five analysable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
-a dose–related increase in the number of revertants occurred and/or;
-a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
-the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
-the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response in any of the dose groups, with or without metabolic activation.
Statistics:
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
PRELIMINARY CONCENTRATION RANGE FINDING TEST
In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 strains in the presence and absence of metabolic activation system (± S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Preliminary Concentration Range Finding Test.
In the preliminary experiment, the numbers of revertant colonies were generally within the normal range (any minor differences were without biological significance and considered to be within the normal biological variability of the test system).
Precipitate/slight precipitate was observed on the plates in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation at the concentrations of 5000, 2500 and 1000 μg/plate*.
*Note: Due to the observed precipitate, on the plates at 5000 μg/plate concentration with and without metabolic activation, it was difficult to clearly evaluate the background lawn at that concentration. The precipitation did not adversely affect the colony counting.
No inhibitory or toxic effects of the test item were detected in the preliminary experiment.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
In the Initial Mutation Test (using plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA100 bacterial strain at 1581 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.20). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In the Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 500 and 158.1 μg/plate concentrations without metabolic activation (the observed mutation factor value was: MF: 1.17). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
Precipitate/slight precipitate was observed on the plates in the main tests in all examined bacterial strains with and without metabolic activation at the concentrations of 5000 and 1581 μg/plate*.
*Note: Due to the observed precipitate, on the plates at 5000 μg/plate concentration with and without metabolic activation, it was difficult to clearly evaluate the background lawn at that concentration. The precipitation did not adversely affect the colony counting.
No inhibitory or toxic effects of the test item were detected in the main tests.

Any other information on results incl. tables

Summary Tables of the Results

 

Summary Table of the Preliminary Concentration Range Finding Test

Concentrations

(μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

TA98

TA100

-S9

+S9

-S9

+S9

Untreated control

Mean

19.7

25.0

107.7

110.3

MF

0.95

1.07

1.06

1.06

DMSO control

Mean

20.7

23.3

102.0

104.3

MF

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

103.7

--

MF

--

--

1.02

--

5000

Mean

20.3

22.3

109.7

120.3

MF

0.98

0.96

1.08

1.15

2500

Mean

21.3

22.3

106.7

117.0

MF

1.03

0.96

1.05

1.12

1000

Mean

20.0

21.7

114.0

111.0

MF

0.97

0.93

1.12

1.06

316

Mean

20.0

23.0

110.0

115.7

MF

0.97

0.99

1.08

1.11

100

Mean

20.7

21.3

114.0

112.7

MF

1.00

0.91

1.12

1.08

31.6

Mean

21.3

23.7

112.7

115.0

MF

1.03

1.01

1.10

1.10

10

Mean

22.3

20.3

109.3

114.0

MF

1.08

0.87

1.07

1.09

NPD (4 μg)

Mean

417.3

--

--

--

MF

20.19

--

--

--

2AA (2 μg)

Mean

--

2477.3

--

2386.7

MF

--

106.17

--

22.88

SAZ (2 μg)

Mean

--

--

2118.7

--

MF

--

--

11.76

--

 

Summary Table of the Initial Mutation Test

Concentrations

(μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

20.0

22.3

96.3

98.7

12.3

10.0

9.3

8.0

49.3

54.0

MF

1.03

1.05

0.99

1.04

1.00

0.94

1.12

0.86

0.98

1.00

DMSO control

Mean

19.3

21.3

97.3

94.7

12.3

10.7

8.3

9.3

50.3

54.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

95.3

--

12.7

--

--

--

50.7

--

MF

--

--

0.98

--

1.03

--

--

--

1.01

--

5000

Mean

18.7

22.0

94.7

87.0

9.3

8.3

8.3

8.7

40.0

48.3

MF

0.97

1.03

0.97

1.02

0.76

0.78

1.00

0.93

0.79

0.90

1581

Mean

19.3

22.7

111.0

114.0

9.7

9.3

8.0

8.3

39.7

53.7

MF

1.00

1.06

1.14

1.20

0.78

0.88

0.96

0.89

0.79

0.99

500

Mean

18.3

21.3

100.0

98.7

10.0

11.7

8.7

8.7

38.3

51.7

MF

0.95

1.00

1.03

1.04

0.81

1.09

1.04

0.93

0.76

0.96

158.1

Mean

18.7

21.0

105.3

96.7

11.0

11.7

9.0

8.3

41.0

53.0

MF

0.97

0.98

1.08

1.02

0.89

1.09

1.08

0.89

0.81

0.98

50

Mean

19.3

19.0

102.0

99.3

10.3

8.7

8.0

8.7

41.3

51.7

MF

1.00

0.89

1.05

1.05

0.84

0.81

0.96

0.93

0.82

0.96

15.81

Mean

17.3

21.0

96.0

107.7

12.3

10.3

9.0

8.7

39.0

53.3

MF

0.90

0.98

0.99

1.14

1.00

0.97

1.08

0.93

0.77

0.99

NPD (4 μg)

Mean

397.3

--

--

--

--

--

--

--

--

--

MF

20.55

--

--

--

--

--

--

--

--

--

2AA (2 μg)

Mean

--

2421.3

--

2441.3

--

203.3

--

218.0

--

--

MF

--

113.50

--

25.79

--

19.06

--

23.36

--

--

2AA (50 μg)

Mean

--

--

--

--

--

--

--

--

--

260.0

MF

--

--

--

--

--

--

--

--

--

4.81

SAZ (2 μg)

Mean

--

--

1181.3

--

1069.3

--

--

--

--

--

MF

--

--

12.39

--

84.42

--

--

--

--

--

9AA (50 μg)

Mean

--

--

--

--

--

--

377.3

--

--

--

MF

--

--

--

--

--

--

45.23

--

--

--

MMS (2 μL)

Mean

--

--

--

--

--

--

--

--

1093.3

--

MF

--

--

--

--

--

--

--

--

21.58

--

 

Summary Table of the Confirmatory Mutation Test

Concentrations

(μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

22.0

24.0

100.3

103.3

13.3

13.3

12.3

12.3

44.7

44.3

MF

0.96

0.95

0.96

1.01

1.14

0.93

1.06

1.12

1.09

0.99

DMSO control

Mean

23.0

25.3

104.3

102.0

11.7

14.3

11.7

11.0

41.0

45.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

103.7

--

11.3

--

--

--

43.7

--

MF

--

--

0.99

--

0.97

--

--

--

1.07

--

5000

Mean

23.0

26.0

114.3

100.0

10.7

12.3

11.0

10.3

43.0

45.7

MF

1.00

1.03

1.10

0.98

0.91

0.86

0.94

0.94

1.05

1.01

1581

Mean

24.0

26.7

98.7

99.7

13.0

12.7

9.0

11.3

42.3

45.3

MF

1.04

1.05

0.95

0.98

1.11

0.88

0.77

1.03

1.03

1.01

500

Mean

23.7

26.3

95.3

107.3

13.7

12.3

11.0

10.7

42.0

46.7

MF

1.03

1.04

0.91

1.05

1.17

0.86

0.94

0.97

1.02

1.04

158.1

Mean

23.7

23.3

96.0

101.7

13.7

12.0

10.3

10.0

42.7

46.7

MF

1.03

0.92

0.92

1.00

1.17

0.84

0.89

0.91

1.04

1.04

50

Mean

24.3

36.0

97.0

105.0

13.3

13.0

11.3

10.3

42.7

46.0

MF

1.06

10.3

0.93

1.03

1.14

0.91

0.97

0.94

1.04

1.02

15.81

Mean

22.3

25.7

96.7

110.0

12.3

10.3

10.3

11.0

44.0

47.0

MF

0.97

1.01

0.93

1.08

1.06

0.72

0.89

1.00

1.07

1.04

NPD (4 μg)

Mean

436.0

--

--

--

--

--

--

--

--

--

MF

18.96

--

--

--

--

--

--

--

--

--

2AA (2 μg)

Mean

--

2457.3

--

2500.0

--

207.7

--

210.3

--

--

MF

--

97.00

--

24.51

--

14.49

--

19.12

--

--

2AA (50 μg)

Mean

--

--

--

--

--

--

--

--

--

261.3

MF

--

--

--

--

--

--

--

--

--

5.81

SAZ (2 μg)

Mean

--

--

1041.3

--

1097.3

--

--

--

--

--

MF

--

--

10.05

--

96.82

--

--

--

--

--

9AA (50 μg)

Mean

--

--

--

--

--

--

496.0

--

--

--

MF

--

--

--

--

--

--

42.51

--

--

--

MMS (2 μL)

Mean

--

--

--

--

--

--

--

--

1057.3

--

MF

--

--

--

--

--

--

--

--

24.21

--

 

Historical Control Data

(Period of 2011-2017)

Untreated control data

 

Without metabolic activation (-S9 Mix)

With metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

22.5

102.3

12.0

7.7

35.2

29.0

109.7

11.5

9.4

40.4

St. dev.

5.6

20.3

4.8

3.5

10.8

6.8

19.2

3.7

3.9

10.4

Range

9-50

54-210

1-46

1-26

11-82

10-56

65-204

1-39

1-29

16-89

n

1650

1636

1647

1653

1662

1668

1656

1667

1671

1662

DMSO control data

 

Without metabolic activation (-S9 Mix)

With metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

21.5

98.0

12.1

7.6

34.0

28.1

107.3

11.3

9.1

39.4

St. dev.

5.5

19.7

4.7

3.4

10.5

6.9

20.2

3.6

3.8

10.36

Range

6-55

40-217

1-43

1-27

7-81

11-67

53-229

2-33

1-29

9-85

n

1770

1761

1770

1776

1779

1787

1776

1790

1791

1782

Distilled water control data

 

Without metabolic activation (-S9 Mix)

With metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

23.3

101.7

12.1

8.5

36.2

29.7

109.7

11.3

9.9

41.5

St. dev.

5.7

21.3

4.6

3.5

10.7

6.9

21.1

3.5

3.8

10.3

Range

11-45

45-215

2-47

2-24

12-84

10-53

64-222

3-39

1-24

13-91

n

351

1644

1650

357

1683

354

1668

1677

354

1677

DMF control data

 

Without metabolic activation (-S9 Mix)

With metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

20.4

90.0

11.4

7.5

36.6

27.4

98.8

11.1

8.7

39.4

St. dev.

5.3

17.0

4.4

3.4

12.8

6.9

18.4

3.4

3.5

10.9

Range

8-38

54-152

1-34

1-19

16-99

11-49

60-156

3-21

1-23

17-76

n

258

258

258

258

249

258

258

258

255

249

Acetone control data

 

Without metabolic activation (-S9 Mix)

With metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

22.5

98.0

12.1

7.5

35.6

28.9

107.5

11.1

8.8

40.9

St. dev.

5.1

15.1

5.8

3.0

9.7

6.7

14.5

3.4

3.4

9.2

Range

11-39

62-160

4-49

1-17

17-63

15-52

66-177

4-22

1-19

17-70

n

290

291

291

291

288

291

291

294

291

291

Positive reference control data

 

Without metabolic activation (-S9 Mix)

With metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

363.9

1216.4

1167.3

447.3

1028.0

2409.2

2423.8

230.9

219.7

255.1

St. dev.

105.6

193.5

188.7

155.7

133.5

290.5

267.0

123.9

51.7

104.1

Range

152-2336

536-2120

208-2440

149-2104

488-1708

312-4918

1192-5240

101-2216

117-838

125-2512

n

1650

1638

1647

1653

1665

1668

1656

1671

1671

1662

TA98:Salmonella typhimuriumTA98, TA100:Salmonella typhimuriumTA100, TA1535:Salmonella typhimuriumTA1535, TA1537:Salmonella typhimuriumTA1537, E. coli:Escherichia coliWP2 uvrA, n: number of cases

Applicant's summary and conclusion

Conclusions:
The data from this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial strains used.

In conclusion, the test item BAL0001026 (Batch Number: 017) had no mutagenic activity on the applied bacterial strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test item was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were tested in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains with and without metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

 

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. There were no dose-related trends and no indication of any treatment-related effect.

 

Precipitate/slight precipitate was observed on the plates in the main tests in all examined bacterial strains with and without metabolic activation at the concentrations of 5000 and 1581 μg/plate. Due to the observed precipitate, on the plates at 5000 μg/plate concentration with and without metabolic activation, it was difficult to clearly evaluate the background lawn at that concentration. The precipitation did not adversely affect the colony counting.

No inhibitory or toxic effects of the test item were detected in the study.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The data from this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial strains used.

 

In conclusion, the test item BAL0001026 (Batch Number: 017) had no mutagenic activity on the applied bacterial strains under the test conditions used in this study.