Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-14 to 2018-08-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (rat liver)
Test concentrations with justification for top dose:
preliminary study: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160, 5000 µg/plate (plate incorporation method, TA-100)
main tests: 31.6, 100, 316, 1000, 3160, 5000 µg/plate (preincubation and plate incorporation test, all strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was complete soluble in DMSO during the preliminary test
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:Two independent tests, one in agar (plate incorporation) and the other as preincubation
- Cell density at seeding (if applicable): 1E+08 - 1E+09 cells/mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h


NUMBER OF REPLICATIONS: 3 per concentration and/or control in 2 independent experiments


DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants by at least 50%, a clearing or dimunition of the background lawn or by the degree of survival of the treated cultures
Rationale for test conditions:
As recommended by the test guideline
Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- Biological relevance of the results should be considered first.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
U-test according to MANN and WHITNEY and the Spearman's rank correlation coefficient

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA-98, TA-100, TA-102, TA-1535, TA-1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: 1 g/L
- Precipitation: no precipitation described

RANGE-FINDING/SCREENING STUDIES: preliminary (plate incorporation test, without and with metabolic activation) with the tester strain TA100 revealed not cytotoxicity up to the top dose of 5000 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see ' Any other information on results incl. tables'
- Negative (solvent/vehicle) historical control data: see ' Any other information on results incl. tables'


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction in the number of spontaneous revertants by at least 50%, a clearing or dimunition of the background lawn or by the degree of survival of the treated cultures

Any other information on results incl. tables

History profile of Negative and Positive Control Values of three years (n=88), Data obtained from plate incorporation and preincubation tests

Negative Reference item

Strain

 

TA98

TA100

TA102

TA1535

TA1537

S9-Mix

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean

30.2

31.9

145.9

145.1

278.1

279.0

19.7

19.9

6.7

6.7

SD

5.6

5.9

19.6

19.9

16.9

17.5

4.4

4.6

1.8

1.8

Min

20

20

95

100

245

203

10

10

2

2

Max

49

49

200

209

323

324

34

36

10

10

Positive Reference item

Strain

 

TA98

TA100

TA102

TA1535

TA1537

S9-Mix

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

2-nitro-fluorene

Benzo[a] pyrene

sodium azide

2-amino-anthracene

Mitomycin C

Benzo[a] pyrene

sodium azide

2-amino-anthracene

9-amino-acridine

Benzo[a] pyrene

Mean

158.8

157.6

954.0

949.5

1020.7

1015.0

144.5

144.5

75.0

75.9

SD

26.2

26.9

102.1

104.3

90.8

95.1

44.7

47.4

26.9

26.4

Min

102

104

701

703

756

759

62

67

28

31

Max

293

291

1365

1238

1263

1311

406

404

185

184

Table 1: Summarised data: Plate incorporation test without metabolic activation

Test item (µg/plate)

 

Plate incorporation test without metabolic activation

 

 

Number of revertant colonies

 

 

TA-98

TA-100

TA-102

TA-1535

TA-1537

 

 

Mean values±SD

TA-3

 

 

 

 

 

 

31.6

mean

24.7

137.3

299.7

28.7

7.3

 

SD

1.2

12.9

6.5

0.6

1.2

100

mean

27.7

136.3

293.7

24.7

6.7

 

SD

1.5

8.4

9.1

2.1

3.5

316

mean

25.0

122.7

292.7

21.7

5.3

 

SD

5.0

7.6

4.7

1.2

2.1

1000

mean

29.0

133.7

292.7

25.0

5.0

 

SD

4.6

12.6

4.9

3.5

1.7

3160

mean

27.7

137.7

300.0

23.3

7.0

 

SD

4.5

14.2

6.1

4.0

0.0

5000

mean

28.0

135.3

274.7

22.3

7.3 

 

SD

6.6

 9.6

4.5

1.2

2.1

Negative reference item 100µL/plate)

mean

27.3

167.7

302.0

21.7

 8.0

 

SD

1.5

7.2

12.5

1.5

1.0

Positive reference item

 

2-Nitrofluorene

Sodium azide

Mitomycin C

Sodium azide

9-Amino-acridine

Concentration [µg/plate]

 

10

10

10

10

100

 

mean

172.0  

1047.0  

1034.0  

169.3  

58.3

 

SD

10.1

21.9

10.8

2.9

14.7

Table 2: Summarised data: Plate incorporation test with metabolic activation

Test item (µg/plate)

 

Plate incorporation test with metabolic activation

 

 

Number of revertant colonies

 

 

TA-98

TA-100

TA-102

TA-1535

TA-1537

 

 

Mean values±SD

TA-3

 

 

 

 

 

 

31.6

mean

29.0

176.3

279.0

29.0

5.3

 

SD

7.8

7.0

4.0

0.0

2.1

100

mean

28.0

165.0

294.7

20.7

8.0

 

SD

7.0

7.0

6.7

0.6

1.7

316

mean

29.3

152.0

272.7

13.7

5.0

 

SD

6.5

6.9

14.2

2.1

1.7

1000

mean

32.7

188.7

280.0

19.0

7.7

 

SD

2.5

3.2

30.3

6.2

2.3

3160

mean

29.7

138.3

285.7

23.7

5.7

 

SD

6.4

14.2

1.5

3.8

3.8

5000

mean

27.7

162.7

260.7

24.0

5.0 

 

SD

5.7

16.0

1.5

1.7

2.6

Negative reference item 100µL/plate)

mean

32.7

164.0

262.7

27.7

8.7

 

SD

5.0

4.4

6.1

1.2

1.2

Positive reference item

 

Benzo[a] pyrene

2-Amino-anthracene

Benzo[a] pyrene

2-Amino-anthracene

Benzo[a] pyrene

Concentration [µg/plate]

 

10

2

10

2

10

 

mean

179.7  

1076.3  

1163.0  

164.7  

59.3

 

SD

5.7

47.9

19.5

14.7

15.5

Table 3: Summarised data: Preincubation test without metabolic activation

Test item (µg/plate)

 

Preincubation test without metabolic activation

 

 

Number of revertant colonies

 

 

TA-98

TA-100

TA-102

TA-1535

TA-1537

 

 

Mean values±SD

TA-3

 

 

 

 

 

 

31.6

mean

25.0

103.7

276.0

27.0

5.0

 

SD

1.0

2.3

3.5

0.0

1.7

100

mean

34.0

124.0

263.7

20.3

6.0

 

SD

5.6

30.3

13.5

4.0

1.7

316

mean

25.3

126.0

262.7

24.3

5.7

 

SD

4.0

13.0

22.0

1.2

0.6

1000

mean

25.7

130.7

266.0

26.3

7.7

 

SD

0.6

23.0

3.5

3.1

2.1

3160

mean

28.3

121.3

278.7

24.7

5.7

 

SD

1.5

14.4

8.4

2.1

1.5

5000

mean

24.3

107.7  

297.3  

26.7  

7.0

 

SD

3.2

4.0

3.5

7.4

3.0

Negative reference item 100µL/plate)

mean

34.7

120.3

261.0

24.7

4.7

 

SD

5.9

3.1

1.7

0.6

0.6

Positive reference item

 

2-Nitro-fluorene

Sodium azide

Mitomycin C

Sodium azide

9-Amino-acridine

Concentration [mg/plate]

 

10

10

10

10

100

 

mean

161.7  

1115.3  

1151.0  

175.0  

68.7

 

SD

1.5

14.6

18.7

7.8

13.8

Table 4: Summarised data: Preincubation test with metabolic activation

Test item (µg/plate)

 

Preincubation test with metabolic activation

 

 

Number of revertant colonies

 

 

TA-98

TA-100

TA-102

TA-1535

TA-1537

 

 

Mean values±SD

TA-3

 

 

 

 

 

 

31.6

mean

29.0

176.3

279.0

29.0

5.3

 

SD

7.8

7.0

4.0

0.0

2.1

100

mean

28.0

165.0

294.7

20.7

8.0

 

SD

7.0

7.0

6.7

0.6

1.7

316

mean

29.3

152.0

272.7

13.7

5.0

 

SD

6.5

6.9

14.2

2.1

1.7

1000

mean

32.7

188.7

280.0

19.0

7.7

 

SD

2.5 3

3.2

30.3

6.2

2.3

3160

mean

29.7

138.3

285.7

23.7

5.7

 

SD

6.4

14.2

1.5

3.8

3.8

5000

mean

27.7

162.7  

260.7  

24.0  

5.0 

 

SD

5.7

16.0

1.5

1.7

2.6

Negative reference item 100µL/plate)

mean

32.7

164.0

262.7

27.7

8.7

 

SD

5.0

4.4

6.1

 1.2

1.2

Positive reference item

 

Benzo[a] pyrene

2-Amino-anthracene

Benzo[a] pyrene

2-Amino-anthracene

Benzo[a] pyrene

Concentration [mg/plate]

 

10

2

10

2

10

 

mean

179.7  

1076.3  

1163.0  

164.7  

59.3

 

SD

5.7

47.9

19.5

14.7

15.5

Applicant's summary and conclusion

Conclusions:
In conclusion, under the present test conditions, the test item tested up to the top concentration of 5000 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.