Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2016 - 08 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
Council Regulation (EC) No 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
cf. Sofuni, 1993
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 Aroclor 1254 in livers of rats
Test concentrations with justification for top dose:
5, 15.8, 50, 158, 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 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 30%
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:
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 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 1:  Revertants per plate (Mean ± SD) - 1st series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

 

 

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

41± 3

113 ± 13

29 ± 6

23 ± 2

34 ± 8

 

Art A2504

5.00

35 ± 5

115 ± 5

26 ± 2

28 ± 4

31 ± 5

 

 

15.8

33 ± 3

107 ± 15

37 ± 2

27 ± 4

35 ± 4

 

 

50.0

44 ± 1

121 ± 17

36 ± 7

27 ± 9

39 ± 7

 

 

158

39 ± 7

114 ± 10

32 ± 5

28 ± 8

28 ± 1

 

 

500

35 ± 7

108 ± 5

29 ± 2

28 ± 6

34 ± 8

 

 

1580

36 ± 7

115 ± 7

31 ± 7

23 ± 2

29 ± 4

 

 

5000

41 ± 7

109 ± 19

26 ± 2

27 ± 2

35 ± 7

 

 

 

 

 

 

 

 

 

DAUN

1.00

464 ± 94

 

 

 

 

 

NaN3

2.00

 

1609 ± 103

949 ± 96

 

 

 

9-AA

50.0

 

 

 

1926 ± 339

 

 

NQO

2.00

 

 

 

 

2221 ± 218

 

 

 

 

 

 

 

 

With Activation

H2O

 

50 ± 9

125 ± 12

29 ± 5

27 ± 4

33 ± 5

 

Art. A2504

5.00

46 ± 4

134 ± 23

29 ± 6

25 ± 6

37 ± 7

 

 

15.8

43 ± 8

127 ± 7

28 ± 1

32 ± 9

32 ± 5

 

 

50.0

30 ± 1

143 ± 5

30 ± 4

28 ± 3

37 ± 11

 

 

158

48 ± 7

118 ± 14

30 ± 2

30 ± 2

37 ± 1

 

 

500

47 ± 3

125 ± 6

28 ± 4

23 ± 2

38 ± 1

 

 

1580

48 ± 8

135 ± 14

32 ± 2

23 ± 1

37 ± 1

 

 

5000

48 ± 8

135 ± 6

33 ± 1

28 ± 4

36 ± 1

 

 

 

 

 

 

 

 

 

2-AA

2.00

301 ± 35

517 ± 62

 

 

 

 

2-AA

5.00

 

 

170 ± 16

156 ± 5

 

 

2-AA

10.0

 

 

 

 

298 ± 29

NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9 -AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-NitroquinoIine-N-oxide

Table 2:  Revertants per plate (Mean ± SD) - 2nd series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

 

 

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

31± 7

115 ± 5

30 ± 4

25 ± 3

35 ± 6

 

Art A2504

50.0

27 ± 8

98 ± 9

37 ± 4

25 ± 6

32 ± 4

 

 

158

28 ± 4

103 ± 11

31 ± 7

26 ± 6

36 ± 4

 

 

500

21 ± 7

113 ± 10

37 ± 5

25 ± 3

41 ± 9

 

 

1580

27 ± 6

111 ± 14

27 ± 8

27 ± 8

35 ± 6

 

 

5000

22 ± 8

115 ± 4

35 ± 3

25 ± 4

24 ± 4

 

 

 

 

 

 

 

 

 

DAUN

1.00

301 ± 10

 

 

 

 

 

NaN3

2.00

 

1591 ± 73

914 ± 92

 

 

 

9-AA

50.0

 

 

 

1061 ± 196

 

 

NQO

2.00

 

 

 

 

1800 ± 399

 

 

 

 

 

 

 

 

With Activation

H2O

 

30 ± 8

107 ± 7

24 ± 5

30 ± 2

46 ± 7

 

Art. A2504

50.0

28 ± 4

129 ± 10

23 ± 7

26 ± 9

42 ± 12

 

 

158

34 ± 9

118 ± 9

21 ± 7

29 ± 5

44 ± 7

 

 

500

29 ± 10

100 ± 5

25 ± 1

31 ± 5

32 ± 5

 

 

1580

28 ± 5

106 ± 7

26 ± 9

28 ± 7

41 ± 3

 

 

5000

33 ± 3

113 ± 3

23 ± 4

26 ± 7

37 ± 7

 

 

 

 

 

 

 

 

 

2-AA

2.00

164 ± 42

 

 

 

 

 

2-AA

5.00

 

949 ± 108

 

 

 

 

2-AA

10.0

 

 

136 ± 11

226 ± 28

217 ± 36

NaN3: Sodium azide,2-AA: 2-Aminoanthracene,9 -AA:9-Aminoacridine,DAUN:Daunomycin,NQO:4-NitroquinoIine-N-oxide

Table 3: Historical data - negative controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

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

180

179

176

176

304

304

388

400

Number of Values

89

89

39

39

38

38

70

70

91

94

Minimum

28

31

23

19

15

13

23

26

89

95

Maximum

49

54

54

39

29

33

49

51

147

166

Mean

37

42

31

28

24

25

33

38

111

120

Standard Deviation

4.3

5.6

3.9

4.4

3.4

4.7

5.3

5.3

12.2

11.3

Table 4: Historical data - positive controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

DAUN

2-AA

NaN3

2-AA

9-AA

2-AA

NQO

2-AA

NaN3

2-AA

Total Plates

190

190

90

90

88

88

152

152

190

200

Number of Values

89

89

39

39

38

38

70

70

89

94

Minimum

89

201

546

138

120

106

222

126

195

450

Maximum

1197

1544

1562

352

2313

588

2613

737

2289

2229

Mean

362

769

867

262

977

378

1606

394

1360

1365

Standard Deviation

201.9

257.4

172.3

51.8

429.9

142.3

488.8

142.2

305.0

314.9

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 test item was examined for its mutagenic activity in two series of a bacterial reverse mutation assay (OECD 471) employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pre-treated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10 % and 30% S9 were used in the first and second series, respectively.

Treatments of all tester strains were performed using the test item solved in ultrapure water in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.

The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquin-olin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the study was considered valid.

No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. During the experiments there was no evidence of toxicity to the bacteria caused by the test material exposure.

Under the conditions described, there were no relevant increases in revertant numbers after treatment with the test item observed in both, the absence and presence of S9 mix.

In conclusion, the test material was considered to be non-mutagenic under the described experimental conditions.