Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - September 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
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

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:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500, and 5000 μg/plate
No toxic effects were observed at 5000 μg/plate in the pre-experiment.
Vehicle / solvent:
DSMO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 (Without metabolic activation)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 1537, TA98 (Without metabolic activation)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
WP2 uvrA (Without metabolic activation)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA (With metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 h at 37 °C

SELECTION AGENT: L-Histidin for S. typhimurium, Tryptophan for E. coli

NUMBER OF REPLICATIONS: 3

Titer of the incubated culture: 10^8 - 10^9 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn
Evaluation criteria:
Since a reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background lawn (only at 5000 μg/plate) were not included into evaluation procedures.
The test item is to be interpreted mutagenic if there is a concentration effect relationship and the induction rate is >=2.

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:
cytotoxicity
Remarks:
at 5000 μg/plate
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:
cytotoxicity
Remarks:
at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
No toxic effects occurred in the Pre-experiment

HISTORICAL CONTROL DATA
see table at "Any other information"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduced background lawn
- TA 100: with and without metabolic activation at 5000 μg/plate cytotoxic effects
- WP2 uvrA:with metabolic activation at 5000 μg/plate cytotoxic effects

Any other information on results incl. tables

Substance

Concentration

(µg/plate)

S9

Mean revertants per plate

TA1535

TA1537

TA98

TA100

WP2 uvrA

Control

0

-

7.0

10.3

29.7

200.7

48.3

Solvent control

0

-

8.7

10.7

26.7

145.3

50.0

Test item

33

-

9.7

9.0

26.3

148.0

46.0

Test item

100

-

7.3

11.0

25.0

155.0

34.7

Test item

333

-

7.0

8.3

25.3

154.3

34.7

Test item

1000

-

11.0

8.0

25.0

100.7

36.0

Test item

2500

-

9.0

8.0

25.0

105.0

34.0

Test item

5000

-

8.3

10.3

17.7

55.3

17.7

NaN3

10

-

111.3

 

 

1805.3

 

4-NOPD

50

-

 

103.3

 

 

 

4-NOPD

10

-

 

 

301.0

 

 

MMS

2

-

 

 

 

 

637.3

 

 

 

 

 

 

 

 

Control

0

+

10.3

12.0

45.7

203.7

58.7

Solvent control

0

+

12.3

15.7

41.3

138.0

52.3

Test item

33

+

9.7

14.7

38.3

153.0

54.7

Test item

100

+

12.3

11.7

38.3

134.0

53.7

Test item

333

+

10.7

12.0

40.0

148.3

51.0

Test item

1000

+

10.0

13.3

50.7

109.0

50.3

Test item

2500

+

8.0

14.3

37.3

89.0

49.3

Test item

5000

+

9.3

11.0

24.3

27.3

26.0

2-AA

2.5

+

316.7

121.0

4196.7

2183.7

 

2-AA

10

+

 

 

 

 

544.7

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

Historical control data

Strain

 

Without S9 mix

With S9 mix

Mean

SD

Min

Max

Mean

SD

Min

Max

TA 1535

Solvent control

12

2.5

6

25

12

2.5

7

26

Untreated control

12

3.1

6

28

12

2.9

7

26

Positive control

1130

143.1

334

1816

388

58.2

176

668

TA1537

Solvent control

10

2.2

6

19

13

3.5

7

30

Untreated control

11

2.7

5

21

14

4.0

7

31

Positive control

82

12.7

43

157

191

60.8

83

434

TA 98

Solvent control

25

4.4

13

43

34

6.2

15

58

Untreated control

27

4.9

12

43

37

6.5

11

57

Positive control

378

73.7

211

627

3949

771.8

360

6586

TA 100

Solvent control

156

26.0

78

209

148

32.3

73

208

Untreated control

176

23.6

79

217

172

25.4

85

218

Positive control

1966

293.2

498

2767

3798

830.4

536

6076

WP2 uvr A

Solvent control

41

5.6

27

63

50

6.8

28

72

Untreated control

42

5.8

30

63

52

6.8

36

88

Positive control

798

362.7

319

4732

378

112.6

167

1265

Applicant's summary and conclusion

Conclusions:
The mutagenicity of the test item was determined in a study according to OECD guideline 471. The results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and to the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.
Executive summary:

The mutagenicity of the test substance was studied with four mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and one strain of Escherichia coli (WP2 uvrA) according to OECD guideline 471 and EU method B13/14. The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Phenobarbital/β-naphthoflavone induced rat liver. The test substance was dissolved in DMSO and tested in concentrations of 33 to 5000 µg per plate. Cytotoxicity was recorded as reduced background lawn with the strain TA 100 at 5000 μg/plate with and without metabolic activation and with the strain WP2 uvrA at 5000 μg/plate with metabolic activation. Sodium azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that under the experimental conditions described, the test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 or to Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.