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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2018 - 20 July 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate
EC Number:
259-130-3
EC Name:
2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate
Cas Number:
54380-33-5
Molecular formula:
C17H19ClO7
IUPAC Name:
2-(3-chloro-2-hydroxypropyl)-6-({2-[(2-methylprop-2-enoyl)oxy]ethoxy}carbonyl)benzoic acid
Test material form:
liquid

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9
S9 fraction of Phenobarbital and β-naphthoflavone induced rat liver, Supplier: Trinova Biochem GmbH (Rathenau Str. 2); D-35394 Giessen, Germany
- method of preparation of S9 mix
The preparation of the S9 Mix was performed according to Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium
The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M Sodium phosphate-buffer, pH 7.4 (500.0 mL)
Rat liver homogenate (S9) (100.0 mL)
Salt solution for S9 Mix (400.0 mL)
Test concentrations with justification for top dose:
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test.
±S9 Mix: 5000, 1600, 500, 160; 50 and 16 μg/plate
In experiment I (initial mutation test) of the main study the plate incorporation method was used. In experiment II (confirmatory mutation test) the pre-incubation method was applied.
+S9 Mix: 5000, 4500, 4000, 3000, 2500, 1600, 500 and 160 μg/plate (additional confirmatory mutation test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, ultrapure water
Dimethyl sulfoxide (DMSO) for Test Item, NPD (4-Nitro-1,2-phenylenediamine), 9AA (9-Aminoacridine), 2AA (2-Aminoanthracene)
Ultrapure water for SAZ (Sodium azide) and MMS (Methyl methanesulfonate)

Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours in the dark
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each.
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at
least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle
control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least
three times higher than the reversion rate of the vehicle control.
Conditions for the Validity of the Test
The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and
the deletion in the uvrB gene.
- The TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The E. coli WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the
vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revert
ant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-tox
ic dose levels is required to evaluate assay data).
A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value
and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.
Statistics:
none

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was noticed on the plates at the concentration of 5000 μg/plate (±S9 Mix). The obtained precipitate did not disturb the scoring in any case.

RANGE-FINDING/SCREENING STUDIES
Based on the solubility test, a 50 mg/mL test item solution was prepared in dimethyl sulfoxide (DMSO) and diluted in six steps. The number of revertant colonies and the background lawn of auxotrophic cells of two test strains (S. typhimurium TA98 and S. typhimurium TA100) were determined using seven different test item concentrations (5000; 1600; 500; 160, 50, 16 and 5 μg/plate), in presence and absence of a metabolic activation system (S9). In the informatory toxicity test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the noticed slightly lower or higher revertant colony numbers (when compared to that of the vehicle control) remained within the corresponding historical control data ranges, within the biological variability range of the applied test system.

HISTORICAL CONTROL DATA
see table 4

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations
(µg/plate)

Salmonella typhimurium tester strains

Escherichia coli
WP2 uvrA

 

 

TA 98

TA100

TA 1535

TA 1537

 

 

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of
revertants per plate
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

19.0

1.30

19.3

0.88

70.7

1.05

102.7

1.03

9.3

0.97

13.0

0.85

7.7

0.85

8.3

1.25

33.0

1.13

35.7

1.03

DMSO Control

14.7

1.00

22.0

1.00

67.0

1.00

100.0

1.00

9.7

1.00

15.3

1.00

9.0

1.00

6.7

1.00

29.3

1.00

34.7

1.00

Ultrapure Water
Control

-

-

-

-

74.3

1.00

-

-

13.0

1.00

-

-

-

-

-

-

27.7

1.00

-

-

5000

12.0

0.82

25.7

1.17

104.0

1.55

139.3

1.39

20.7

2.14

44.0

2.87

7.7

0.85

9.0

1.35

32.3

1.10

43.0

1.24

1600

15.7

1.07

25.0

1.14

86.7

1.29

116.7

1.17

17.3

1.79

25.0

1.63

9.3

1.04

7.7

1.15

37.3

1.27

38.0

1.10

500

17.0

1.16

23.3

1.06

83.0

1.24

91.3

0.91

18.7

1.93

20.0

1.30

8.0

0.89

9.7

1.45

33.7

1.15

45.7

1.32

160

17.3

1.18

25.0

1.14

85.3

1.27

112.0

1.12

12.7

1.31

16.0

1.04

7.0

0.78

7.0

1.05

29.7

1.01

45.7

1.32

50

15.7

1.07

22.7

1-03

71.7

1.07

110.0

1.10

9.0

0.93

13.7

0.89

8.0

0.89

8.0

1.20

36.0

1.23

42.0

1.21

16

17.3

1.18

17.7

0.80

79.7

1.19

97.3

0.97

10.7

1.10

15.3

1.00

8.0

0.89

8.3

1.25

31.3

1.07

36.7

1.06

NPD (4 µg/plate)

436.0

29.73

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

SAZ (2 µg/plate)

-

-

-

-

701.3

9.43

-

-

743.3

57.18

-

-

-

-

-

-

-

-

-

-

9AA (50 µg/plate)

-

-

-

-

-

-

-

-

-

-

-

-

599.3

66.59

-

-

-

-

-

-

MMS (2 µL/plate)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

858.7

31.04

-

-

2AA (2 µg/plate)

-

-

997.3

45.33

-

-

1245.3

12.45

-

-

186.7

12.17

-

-

131.3

19.70

-

-

-

-

2AA (50 µg/plate)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

184.7

5.33

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoactidine; MMS: Methyl methanesulfonate; 2.4A: 2-aminoanthracene
Remarks:
Dimethyl sulfoxide (DMSO) was applied as vehicle for the test item, for NPD, 9AA and 2AA and ultrapure water was applied as vehicle of the positive control substances SAZ and MMS. The mutation rate of the test item, the untreated control, NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of SAZ and MMS is given referring to ultrapure water.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-incubation Test)

Concentrations
(µg/plate)

Salmonella typhimurium tester strains

Escherichia coli
WP2 uvrA

 

 

TA 98

TA 100

TA 1535

TA 1537

 

 

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of
revertauts per plate
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

12.7

0.86

17.7

0.70

69.3

1.07

91.0

1.07

11.0

1.03

11.3

0.94

6.7

1.11

8.7

0.93

26.7

1.11

37.3

1.06

DMSO control

14.7

1.00

25.3

1.00

65.0

1.00

85.3

1.00

10.7

1.00

12.0

1.00

6.0

1.00

9.3

1.00

24.0

1.00

35.3

1.00

Ultrapure Water
Control

-

-

-

-

63.0

1.00

-

-

14.0

1.00

-

-

-

-

-

-

26.3

1.00

-

-

5000

12.7

0.86

21.7

0.86

60.3

0.93

110.3

1.29

29.3

2.75

45.3

3.78

2.0

0.33

4.7

0.50

42.0

1.75

44.0

1.25

1600

21.0

1.43

25.3

1.00

63.7

0.98

115.0

1.35

22.0

2.06

26.3

2.19

2.3

0.39

7.0

0.75

28.7

1.19

36.7

1.04

500

17.7

1.20

25.7

1.01

80.0

1.23

107.7

1.26

15.7

1.47

21.0

1.75

5.3

0.89

7.7

0.82

26.0

1.08

34.3

0.97

160

18.7

1.27

22.0

0.87

73.0

1.12

103.0

1.21

13.7

1.28

14.7

1.22

6.0

1.00

8.0

0.86

28.3

1.18

35.7

1.01

50

10.3

0.70

18.7

0.74

59.7

0.92

103.3

1.21

13.0

1.22

12.7

1.06

6.7

1.11

8.7

0.93

27.7

1.15

28.3

0.80

16

20.3

1.39

24.7

0.97

68.7

1.06

108.3

1.27

11.3

1.06

14.0

1.17

10.3

1.72

12.7

1.36

22.0

0.92

30.3

0.86

NPD (4 µg/plate)

460.7

31.41

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

SAZ (2 µg/plate)

-

-

-

-

625.3

9.93

-

-

1157.3

82.67

-

-

-

-

-

-

-

-

-

-

9AA (50 µg/plate)

-

-

-

-

-

-

-

-

-

-

-

-

364.7

60.78

-

-

-

-

-

-

MMS (2 µL/plate)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1258.7

47.80

-

-

2AA (2 µg/plate)

-

-

677.3

26.74

-

-

726.7

8.52

-

-

107.0

8.92

-

-

145.7

15.61

-

-

-

-

2AA (50 µg/plate)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

156.3

4.42

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine, SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Dimethyl sulfoxide (DMSO) was applied as vehicle for the test item, for NPD, 9AA and 2AA and ultrapure water was applied as vehicle of the positive control substances SAZ and MMS. The mutation rate of the test item, the untreated control, NPD, 9AA and 2AA is given referring to the DMSO, and the mutation rate of SAZ and MMS is given referring to ultrapure water.

Table 3: Summary Table of the Results of the Additional Confirmatory Mutation Test

 

Additional Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations
(µg/plate)

Salmonella typhimurium tester strain

 

 

TA 1535

 

 

+S9

Mean values of
revertants per plate
Mutation rate (MR)

Mean

MR

Untreated Control

 

0.92

DMSO Control

8.3

1.00

5000

57.0

6.84

4500

42.3

5.08

4000

44.7

5.36

3000

40.3

4.84

2500

30.3

3.64

1600

22.3

2.68

500

10.0

1.20

160

11.3

1.36

2AA (2 µg/plate)

176.0

21.12

MR: Mutation Rate

2AA: 2-aminoanthracene

Remarks: Dimethyl sulfoxide (DMSO) was applied as vehicle for the test item and for the positive control item: 2AA. The mutation rate of the test item, the untreated control and 2AA is given referring to the DMSO.

 

Table 4: Historical Control Values for Revertants Plate (for the Period of 2015-2017)

 

Bacterial strains

Historical

control data

of untreated

control

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

19.7

94.6

10.9

8.9

25.2

-S9

SD

1.7

2.4

0.4

1.3

5.2

 

Minimum

8

67

4

3

11

 

Maximum

40

133

21

20

52

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

24.5

112.9

11.0

9.2

31.7

+S9

SD

1.4

6.1

0.5

1.2

6.4

 

Minimum

11

74

3

3

13

 

Maximum

43

159

20

20

60

 

Bacterial strains

Historical

control data

of DMSO

control

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

18.1

87.0

10.8

8.5

24.6

-S9

SD

1.1

3.6

0.6

1.3

3.3

 

Minimum

9

58

4

3

10

 

Maximum

36

131

23

20

54

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

23.0

102.4

11.0

9.2

31.4

+S9

SD

0.8

7.7

0.4

1.3

5.8

 

Minimum

11

69

3

3

12

 

Maximum

42

14S

23

21

59

 

Bacterial strains

Historical

control data of Water

control

 

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

19.5

92.8

11.3

9.1

26.6

-S9

SD

1.4

5.0

0.5

1.8

6.6

 

Minimum

12

62

4

3

10

 

Maximum

30

139

22

IS

52

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

24.4

109.9

11.2

9.5

34.0

+S9

SD

1.2

6.7

0.8

1.9

6.1

 

Minimum

13

83

5

4

16

 

Maximum

37

149

18

18

63

 

Bacterial strains

 

 

 

 

Historical

control data

of positive

controls

 

 

 

 

 

 

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

285.9

1214.0

1024.3

594.4

858.4

-S9

SD

25.5

80.0

120.3

36.4

164.7

 

Minimum

152

609

407

136

330

 

Maximum

598

2272

2597

2048

1760

 

 

TA98

TA100

TA1535

TA1537

E. coli

 

Average

1395.7

1727.4

160.7

145.8

204.9

+S9

SD

261.4

244.1

30.0

18.9

3.9

 

Minimum

286

712

91

70

133

Maximum

3211

3435

328

315

367

Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100. TA1535. TA1537;

E. coli: Escherichia coli WP2 uvrA
SD: Standard deviation

 

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, the test item (acting as promutagen, in presence of exogenous metabolic activation) induced gene mutations by base-pair substitution in the genome of the Salmonella typhimurium TA1535 tester strain investigated. Therefore, the test item is considered mutagenic in this bacterial reverse mutation assay.
Executive summary:

A study according OECD TG 471 (Ames test) was performed. The test item was dissolved in dimethyl sulfoxide (DMSO). In the initial and confirmatory mutation tests the following concentrations were examined:

±S9 Mix: 5000, 1600, 500, 160; 50 and 16 μg/plate.

Because of the positive results observed in the confirmatory mutation test an additional confirmatory mutation test was performed (in Salmonella typhimurium TA1535, +S9 Mix) with following concentration levels:

+S9 Mix: 5000, 4500, 4000, 3000, 2500, 1600, 500 and 160 μg/plate.

In the initial and confirmatory mutation tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. In the additional confirmatory mutation test the Salmonella typhimurium TA1535 strain was investigated.

Five bacterial strains were used to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). For confirmation and repetition of the positive results obtained in the confirmatory mutation test an additional confirmatory mutation test was carried out. Each assay was conducted with and without metabolic activation (±S9 Mix); however in the additional confirmatory mutation test the Salmonella typhimurium TA1535 strain was investigated in the presence of exogenous metabolic activation (+S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

In the performed experiments all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled.

In the confirmatory mutation test following treatment with the test item significant, biological relevant revertant colony number increases, positive result was noticed at the concentration of 5000 μg/plate in S. typhimurium TA1535 in the presence of exogenous metabolic activation (+S9 Mix). The revertant colony number increases showed clear dose-relationship at the further, lower concentration levels. The dose related tendencies, the unequivocal positive results were repeated in a subsequent experiment: in the additional pre-incubation test (additional confirmatory mutation test).

In the confirmatory mutation test slight, rather equivocal inhibitory effect of the test item was observed in Salmonella typhimurium strains at 5000 μg/plate (in TA1537 also at 1600 μg/plate) in absence of exogenous metabolic activation (-S9 Mix). The slight inhibitory effect of the test item was indicated mainly by affected (slightly reduced) background lawn development. Microdrops (colloid-chemical phenomenon) were noticed in the examined strains at the highest examined concentration of 5000 μg/plate, in the absence (-S9 Mix) of exogenous metabolic activation following the pre-incubation procedure (confirmatory mutation test).

The reported data of this mutagenicity assay shows that under the experimental conditions reported, the test item (acting as promutagen, in presence of exogenous metabolic activation) induced gene mutations by base-pair substitution in the genome of the Salmonella typhimurium TA1535 tester strain investigated.