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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 October 2017 - 23 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
TOXI-COOP ZRT. 8230 Balatonfüred, Arácsi út 97. Hungary
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
highly viscous, semi-solid mass

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 / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from the Phenobarbital and β-Naphthoflavone-induced rat liver
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 (Informatory Toxicity Test).
The following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate in Experiment I (Plate incorporation method) and Experiment II (pre-incubation method).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ultrapure water, Dimethyl sulfoxide (DMSO)

- Justification for choice of solvent/vehicle: Test item was completely dissolved in ultrapure water. In the study two vehicle control groups were used depending on the solubility of the test item and the solubility of positive control chemicals.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: NPD (4-Nitro-1,2-phenylenediamine)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
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 at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: 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 E.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 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 revertant 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-toxic dose levels is required to evaluate assay data).
A dose level is considered toxic if:
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- 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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
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
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
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was completely dissolved in ultrapure water.
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

RANGE-FINDING/SCREENING STUDIES:
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. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
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 obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

HISTORICAL CONTROL DATA (Please refer to "Any other information on results incl.tables")
In the Initial Mutation Test and Confirmatory Mutation Test most of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges.

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Range Finding Test

Range Finding Test (Informatory Toxicity Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Mean values of revertants per plate and
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

18.3

1.04

24.3

0.80

101.3

1.05

108.0

1.12

DMSO Control

16.0

1.00

27.7

1.00

93.0

1.00

Ultrapure Water Control

17.7

1.00

30.3

1.00

96.3

1.00

96.7

1.00

5000

25.0

1.42

24.7

0.81

82.3

0.85

118.0

1.22

1600

23.3

1.32

24.0

0.79

79.3

0.82

106.0

1.10

500

26.0

1.47

25.7

0.85

91.3

0.95

106.3

1.10

160

20.3

1.15

53.0

1.75

79.7

0.83

117.0

1.21

50

24.3

1.38

24.3

0.80

90.3

0.94

113.0

1.17

16

19.0

1.08

26.0

0.86

88.3

0.92

111.7

1.16

5

19.0

1.08

29.0

0.96

98.0

1.02

123.7

1.28

NPD (4mg)

257.7

16.10

SAZ (2mg)

1353.3

14.05

2AA (2mg)

1341.3

48.48

1212.0

13.03

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azid

2AA:2-aminoanthracene

 Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.

 

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

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-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

25.3

1.07

27.0

0.98

87.3

0.99

115.0

1.13

12.3

1.03

12.0

1.00

8.7

1.04

9.3

1.47

34.0

0.94

43.3

0.86

DMSO Control

20.3

1.00

26.0

1.00

94.7

1.00

12.0

1.00

6.0

1.00

9.3

1.00

40.0

1.00

Ultrapure Water Control

23.7

1.00

27.7

1.00

88.3

1.00

101.3

1.00

12.0

1.00

12.0

1.00

8.3

1.00

6.3

1.00

36.0

1.00

50.7

1.00

5000

31.3

1.32

30.3

1.10

95.3

1.08

101.0

1.00

14.3

1.19

10.3

0.86

10.3

1.24

7.7

1.21

45.7

1.27

59.0

1.16

1600

22.3

0.94

28.3

1.02

100.3

1.14

80.7

0.80

12.7

1.06

10.0

0.83

9.3

1.12

7.7

1.21

46.0

1.28

56.0

1.11

500

29.3

1.24

25.0

0.90

89.3

1.01

80.0

0.79

11.7

0.97

10.7

0.89

6.7

0.80

6.3

1.00

36.7

1.02

39.3

0.78

160

23.0

0.97

29.3

1.06

89.7

1.02

88.7

0.88

15.0

1.25

11.7

0.97

7.0

0.84

7.0

1.11

36.7

1.02

47.0

0.93

50

26.3

1.11

35.3

1.28

93.7

1.06

92.7

0.91

11.0

0.92

11.7

0.97

7.7

0.92

6.3

1.00

39.0

1.08

59.0

1.16

16

23.7

1.00

27.3

0.99

93.0

1.05

93.7

0.92

10.0

0.83

11.7

0.97

8.3

1.00

7.7

1.21

40.3

1.12

44.7

0.88

NPD (4mg)

464.3

22.84

SAZ (2mg)

1658.7

18.78

1717.3

143.11

9AA (50mg)

1052.7

175.44

MMS (2mL)

919.3

25.54

2AA (2mg)

2664.0

102.46

2954.7

31.21

258.0

21.50

239.0

25.61

2AA (50mg)

179.0

4.48

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

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

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

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-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

27.3

1.08

29.0

1.19

92.7

1.06

92.0

0.88

11.0

1.00

11.0

0.92

10.3

1.19

9.0

1.13

17.0

0.46

39.3

0.84

DMSO Control

23.0

1.00

22.7

1.00

94.3

1.00

13.0

1.00

7.3

1.00

6.3

1.00

38.7

1.00

Ultrapure Water Control

25.3

1.00

24.3

1.00

87.3

1.00

104.0

1.00

11.0

1.00

12.0

1.00

8.7

1.00

8.0

1.00

36.7

1.00

46.7

1.00

5000

25.3

1.00

37.3

1.53

97.7

1.12

106.0

1.02

10.7

0.97

11.7

0.97

7.3

0.85

12.0

1.50

52.0

1.42

51.3

1.10

1600

23.7

0.93

31.0

1.27

84.0

0.96

110.0

1.06

9.7

0.88

12.3

1.03

9.0

1.04

11.7

1.46

42.3

1.15

48.3

1.04

500

26.0

1.03

37.3

1.53

82.3

0.94

98.7

0.95

10.3

0.94

12.3

1.03

8.0

0.92

10.0

1.25

35.3

0.96

43.7

0.94

160

23.7

0.93

27.3

1.12

79.3

0.91

100.3

0.96

11.0

1.00

13.7

1.14

7.7

0.88

9.3

1.17

40.7

1.11

43.7

0.94

50

27.7

1.09

33.3

1.37

77.7

0.89

94.3

0.91

11.3

1.03

10.0

0.83

9.0

1.04

10.3

1.29

40.3

1.10

39.7

0.85

16

26.0

1.03

31.0

1.27

88.0

1.01

103.3

0.99

9.7

0.88

12.3

1.03

9.3

1.08

11.0

1.38

35.0

0.95

47.0

1.01

NPD (4mg)

253.3

11.01

SAZ (2mg)

797.3

9.13

509.3

46.30

9AA (50mg)

528.0

72.00

MMS (2mL)

741.3

20.22

2AA (2mg)

1532.0

67.59

2826.7

29.96

164.0

12.62

121.7

19.21

2AA (50mg)

243.7

6.30

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

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

Table 4: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.0

105.0

10.5

8.1

25.4

SD

3.7

25.7

1.4

2.3

5.2

Minimum

9

66

3

2

11

Maximum

39

155

23

19

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.5

117.1

11.8

9.0

33.9

SD

4.3

18.1

1.4

1.9

5.2

Minimum

12

75

4

2

17

Maximum

46

166

23

20

56

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

20.4

100.1

10.3

7.9

24.7

SD

3.6

24.8

1.3

2.4

4.6

Minimum

10

64

3

2

11

Maximum

38

147

23

20

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

26.5

113.8

11.8

8.8

33.7

SD

4.1

18.3

1.5

1.9

5.0

Minimum

15

71

3

3

16

Maximum

47

162

25

20

57

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.9

104.7

10.5

7.6

26.1

SD

3.7

25.9

1.5

2.2

5.5

Minimum

12

68

3

2

12

Maximum

35

154

24

16

48

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.4

117.3

11.4

8.7

34.9

SD

4.0

18.5

1.3

2.2

4.9

Minimum

15

83

4

3

18

Maximum

43

167

22

16

57

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

260.1

977.2

847.3

478.6

724.5

SD

31.8

150.6

126.3

104.5

65.0

Minimum

123

521

359

110

320

Maximum

664

1970

1855

1601

1313

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

1222.7

1436.4

164.1

147.0

257.7

SD

274.9

318.3

33.1

20.1

72.5

Minimum

386

583

85

69

140

Maximum

2676

2988

498

399

477

                           SD: Standard deviation

 

 

Applicant's summary and conclusion

Conclusions:
The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

A bacterial reverse mutation assay according OECD TG 471, EU method B.13/14 and EPA OTS 789.5100 was performed to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate.

Five bacterial strains were used to investigate the mutagenic potential in two independent experiments. Each assay was conducted with and without 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.

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.