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:
2014-04-24 to 2014-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented, according to OECD guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his trp
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle:
- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Remarks:
With metabolic activation: 2-aminoanthracene (all strains), Without metabolic activation: sodium azide (TA 1535), 2-aminoanthracene , 4-nitro-o-phenylene-diamine(TA 1537, TA 98), methyl methane sulfonate (E. coli)
Details on test system and conditions:
DETAILS ON TEST SYSTEM
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 37°C for 48 - 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer


Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in both experiments with and without S9 mix. The undissolved particles had no influence on the data recording.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in both experiments

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

strain

TA 1535

strain

TA 1537

Strain

TA 98

strain

TA 100

E. coli WP2 uvrA

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

deionized water

20

16

8

21

29

46

184

193

46

50

0

12

16

7

17

27

49

178

195

36

47

3

19

17

11

21

27

48

200

189

39

44

10

17

17

10

21

32

45

193

199

42

47

33

16

18

10

19

32

50

204

210

40

52

100

18

16

7

17

26

42

190

205

39

48

333

16

20

9

21

27

41

194

203

41

46

1000

15

14*

9*

24*

30*

46*

216*

197*

48*

38*

2500

15

18*

6*

22*

25*

39*

199*

196*

39*

46*

5000

11

12*

7*

11*

24*

37*

190*

209*

47*

56*

Sodium azide

10 µg

3339

 

 

 

 

 

1871

 

 

 

4-nitro-o-phenylene-diamine

10 µg

 

 

 

 

294

 

 

 

 

 

4-nitro-o-phenylene-diamine

50 µg

 

 

62

 

 

 

 

 

 

 

methyl methane sulfonate

2 µL

 

 

 

 

 

 

 

 

822

 

2-aminoanthracene

2.5 µg

 

619

 

371

 

3510

 

3403

 

 

2-aminoanthracene

10 µg

 

 

 

 

 

 

 

 

 

470

*Precipitate

 

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

strain

TA 1535

strain

TA 1537

Strain

TA 98

strain

TA 100

E. coli WP2 uvrA

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

deionized water

17

24

10

18

29

44

176

199

40

53

0

12

18

11

17

25

33

193

206

44

41

10

14

22

10

22

27

41

205

217

39

47

33

19

21

9

19

27

41

209

205

35

52

100

19

24

9

9

29

46

180

232

40

55

333

15

19

9

21

27

42

199

198

43

43

1000

21*

15*

10*

15*

29*

40*

204*

215*

42*

53*

2500

8*

13*

8*

17*

29*

50*

189*

200*

44*

49*

5000

11*

13*

8*

14*

22*

34*

178*

210*

35*

53*

Sodium azide

10 µg

3206

 

 

 

 

 

1667

 

 

 

4-nitro-o-phenylene-diamine

10 µg

 

 

 

 

274

 

 

 

 

 

4-nitro-o-phenylene-diamine

50 µg

 

 

63

 

 

 

 

 

 

 

methyl methane sulfonate

2 µL

 

 

 

 

 

 

 

 

461

 

2-aminoanthracene

2.5 µg

 

531

 

316

 

3348

 

2735

 

 

2-aminoanthracene

10 µg

 

 

 

 

 

 

 

 

 

264

*Precipitate

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative