Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October 2016 - 25 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
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:
1997
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix induced by ß-Naphthoflavone/Phenobarbital in livers of rats
Test concentrations with justification for top dose:
1st series: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
2nd series: 50, 281, 500, 1580 and 5000 µg/plate
top dose: in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD 471, 1997)
Vehicle / solvent:
- Vehicle/solvent used: ultrapure water
- Justification for choice of solvent/vehicle: based on the available information from the preliminary solubility test
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: daunomycin, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 to 3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%
Evaluation criteria:
A test material was to be defined as negative or non-mutagenic in this assay if
- The assay was to be considered valid, and
- "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)

For valid data, the test material was considered to be positive or mutagenic if:
- a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
- "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Results and discussion

Test resultsopen allclose all
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:
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:
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:
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:
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Any other information on results incl. tables

Table 2: Summary 1st series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

Without Activation

H2O

 

43 ± 8

133 ± 10

25 ± 6

29 ± 4

36 ± 10

Test item

5.00

41 ± 9

154 ± 10

33 ± 10

31 ± 2

36 ± 2

15.8

44 ± 6

135 ± 17

27 ± 4

31 ± 3

39 ± 3

50.0

36 ± 6

135 ± 18

27 ± 6

32 ± 5

34 ± 10

158

41 ± 6

145 ± 7

25 ± 10

28 ± 7

37 ± 2

500

40 ± 5

127 ± 5

28 ± 7

33 ± 7

34 ± 4

1580

41 ± 4

125 ± 11

30 ± 2

37 ± 6

32 ± 2

5000

44 ± 9

115 ± 8

28 ± 5

31 ± 6

41 ± 6

DAUN

1.00

213 ± 29

 

 

 

 

NaN3

2.00

 

1621 ± 31

983 ± 48

 

 

9-AA

50.0

 

 

 

623 ± 97

 

NQO

2.00

 

 

 

 

1548 ± 38

With Activation

H2O

 

56 ± 18

143 ± 13

26 ± 2

32 ± 6

41 ± 9

Test item

5.00

43 ± 5

137 ± 13

30 ± 3

35 ± 2

50 ± 10

15.8

57 ± 4

151 ± 17

30 ± 1

29 ± 5

39 ± 4

50.0

46 ± 3

152 ± 21

30 ± 2

35 ± 14

43 ± 8

158

58 ± 2

148 ± 12

30 ± 5

33 ± 2

39 ± 2

500

63 ± 3

146 ± 12

33 ± 8

38 ± 10

42 ± 3

1580

51 ± 12

142 ± 8

25 ± 3

38 ± 13

40 ± 6

5000

54 ± 5

116 ± 8

30 ± 4

31 ± 4

36 ± 6

2-AA

2.00

986 ± 30

1575 ± 61

 

 

 

2-AA

5.00

 

 

229 ± 26

559 ± 9

 

2-AA

10.0

 

 

 

 

277 ± 8

Key to Positive Control: NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-Nitroquinoline-N-oxide

 

Table 3: Summary 2nd series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

Without Activation

H2O

 

45 ± 4

138 ± 18

27 ± 4

31 ± 4

38 ± 8

Test item

50.0

37 ± 6

137 ± 13

32 ± 3

29 ± 8

39 ± 10

281

34 ± 6

122 ± 12

27 ± 10

30 ± 7

34 ± 9

500

39 ± 8

123 ± 7

31 ± 3

39 ± 6

29 ± 9

1580

42 ± 11

135 ± 3

31 ± 3

34 ± 6

33 ± 5

5000

39 ± 11

118 ± 10

27 ± 2

32 ± 5

33 ± 5

DAUN

1.00

424 ± 117

 

 

 

 

NaN3

2.00

 

1329 ± 45

790 ± 36

 

 

9-AA

50.0

 

 

 

1156 ± 534

 

NQO

2.00

 

 

 

 

1618 ± 51

With Activation

H2O

 

48 ± 7

135 ± 14

21 ± 3

34 ± 4

36 ± 9

Test item

50.0

50 ± 7

131 ± 7

24 ± 4

41 ± 5

45 ± 3

281

52 ± 9

137 ± 5

24 ± 3

36 ± 10

38 ± 3

500

50 ± 1

140 ± 5

27 ± 4

38 ± 8

35 ± 11

1580

56 ± 11

130 ± 8

26 ± 2

43 ± 8

42 ± 8

5000

52 ± 7

106 ± 17

17 ± 6

40 ± 9

38 ± 9

2-AA

2.00

479 ± 39

708 ± 65

 

 

 

2-AA

5.00

 

 

96 ± 2

207 ± 24

 

2-AA

10.0

 

 

 

 

149 ± 32

Key to Positive Control: NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-Nitroquinoline-N-oxide

 

Table 4: Historical data - Negative Controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Total Plates

380

379

388

400

180

179

176

176

304

304

Number of Values

89

89

91

94

39

39

38

38

70

70

Minimum

28

31

89

95

23

19

15

13

23

26

Maximum

49

54

147

166

54

39

29

33

49

51

Mean

37

42

111

120

31

28

24

25

33

38

Standard Deviation

4.3

5.6

12.2

11.3

6.9

4.4

3.4

4.7

5.3

5.3

The historical data have been obtained in experiments between 01/2015 and 12/2015

 

Table 5: Historical data - Positive Controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

DAUN

2-AA

NaN3

2-AA

NaN3

2-AA

9-AA

2-AA

NQO

2-AA

Total Plates

190

190

190

200

90

90

88

88

152

152

Number of Values

89

89

89

94

39

39

38

38

70

70

Minimum

89

201

195

450

546

138

120

106

222

126

Maximum

1197

1544

2289

2229

1562

352

2313

588

2613

737

Mean

362

769

1360

1365

867

262

977

378

1606

394

Standard Deviation

201.9

257.4

305.0

314.9

172.3

51.8

429.9

142.3

488.8

142.2

The historical data have been obtained in experiments between 01/2015 and 12/2015

Applicant's summary and conclusion

Conclusions:
The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.
Executive summary:

The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolising system (S9 mix).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537,and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from ß-Naphthoflavone/Phenobarbital-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10% S9 mix were used in the 1st and 20% S9 in the 2nd series, respectively.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

No relevant increases in revertant numbers were observed after treatment in any of the tester strains in the absence and presence of S9 mix.