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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January 2017 - 31 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-18
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: No
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by formulating the test article under subdued lighting in acetone with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using acetone.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: 100 mg/mL, dilutions of which were prepared in acetone.
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as a liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A

Method

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
Test concentrations with justification for top dose:
A maximum concentration of 5000 µg/plate was selected for Experiment 1, to ensure treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD, 1997). For Experiment 2 the maximum concentration tested was selected on the basis of toxicity limitations seen in Experiment 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Maron, D., Katzenellenbogen, J., and Ames, B.N. (1981). Compatibility of Organic Solvents
with the Salmonella/Microsome Test. Mutation Res., 88, 343-350.
Controls
Untreated negative controls:
no
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: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 2-3 days
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3 per concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: N/A

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: thinning/ absence of bacterial lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A
Rationale for test conditions:
The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.

2. Any observed response was reproducible under the same treatment conditions.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria were met.
Statistics:
The statistical significance of results was analysed using Dunnett’s test to aid in the evaluation of potential positive responses.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1       Raw plate counts and calculated mutagenicity data, experiment 1 (- S9 mix)

 

Strain

Compound

Conc. level

(µg/plate)

Mean revertants per plate

SD

Fold increase

Dunnett’s T-value

Statistic. sig.

Revertant numbers/plate

TA98

Acetone

-

27.3

10.2

-

-

-

23, 39, 20

Test item

5

31.0

2.6

1.1

0.75

NS

28, 32, 33

16

29.7

2.5

1.1

0.52

NS

27, 30, 32

50

30.7

6.0

1.1

0.67

NS

37, 30, 25

160

42.3

4.5

1.5

2.54

NS

38, 47, 42

500

45.0

14.7

1.6

2.83

*

61, 42, 32

1600

33.0

6.1

1.2

1.07

NS

40S, 30S, 29S

5000

-

-

-

-

-

-T, -T, -T

2NF

5

334.3

96.1

12.2

-

-

314, 439, 250

TA100

Acetone

-

118.7

1.5

-

-

-

119, 120, 117

Test item

5

104.0

7.5

0.9

-1.73

NS

96, 111, 105

16

108.3

2.1

0.9

-1.20

NS

110, 109, 134

50

112.0

19.7

0.9

-0.83

NS

96, 106, 134

160

145.7

17.6

1.2

2.88

*

151, 126, 160

500

122.7

4.9

1.0

0.45

NS

126, 117, 125

1600

171.7

9.1

1.4

5.46

**

182S, 168S, 165S

5000

-

-

-

-

-

-T, -T, -T

NaN3

2

489.7

19.3

4.1

-

-

468, 505, 496

TA1535

Acetone

-

12.3

4.0

-

-

-

8, 16, 13

Test item

5

17.0

2.6

1.4

1.03

NS

16, 20, 15

16

26.3

1.2

2.1

2.68

*

27, 27, 25

50

18.7

4.6

1.5

1.33

NS

24, 16, 16

160

13.0

3.0

1.1

0.18

NS

10, 13, 16

500

20.0

6.6

1.6

1.55

NS

14, 27, 19

1600

14.3

12.7

1.2

0.12

NS

8S, 29S, 6S

5000

-

-

-

-

-

-T, -T, -T

NaN3

2

458.0

62.4

37.1

-

-

466, 516, 392

TA1537

Acetone

-

5.0

1.7

-

-

-

6, 3, 6

Test item

5

5.3

2.5

1.1

0.14

NS

8, 5, 3

16

9.0

1.0

1.8

1.98

NS

9, 8, 10

50

8.3

5.8

1.7

1.44

NS

5, 15, 5

160

7.7

1.5

1.5

1.8

NS

9, 6, 8

500

6.0

2.0

1.2

0.54

NS

6, 8, 4

1600

4.7

0.6

0.9

-0.13

NS

5S, 5S, 4S

5000

-

-

-

-

-

-T, -T, -T

AAC

50

87.0

15.5

17.4

-

-

102, 71, 88

TA102

Acetone

-

293.3

16.2

-

-

-

312, 284, 284

Test item

5

282.0

17.6

1.0

-1.00

NS

269, 302, 275

16

287.7

15.2

1.0

-0.49

NS

274, 285, 304

50

285.3

7.8

1.0

-0.69

NS

279, 294, 283

160

308.0

4.6

1.1

1.27

NS

312, 309, 303

500

399.3

8.5

1.4

8.50

**

399, 391, 408

1600

401.0

25.1

1.4

8.60

**

430S, 387S, 386S

5000

-

-

-

-

-

-T, -T, -T

MMC

0.2

542.3

22.5

1.8

-

-

568, 526, 533

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

* <0.05

**p<0.01

 

Table 2       Raw plate counts and calculated mutagenicity data, experiment 1 (+ S9 mix)

 

Strain

Compound

Conc. level

(µg/plate)

Mean revertants per plate

SD

Fold increase

Dunnett’s T-value

Statistic. sig.

Revertant numbers/plate

TA98

Acetone

-

27.7

2.5

-

-

-

30, 25, 28

Test item

5

43.0

5.6

1.6

2.84

*

44, 37, 48

16

30.7

10.0

1.1

0.52

NS

42, 23, 27

50

40.0

6.2

1.4

2.32

NS

35, 38, 47

160

35.3

4.7

1.3

1.49

NS

37, 30, 39

500

55.3

8.0

2.0

4.77

**

56, 47, 63

1600

29.0

7.0

1.0

0.22

NS

32S, 34S, 21S

5000

-

-

-

-

-

-T, -T, -T

B[a]P

10

344.3

41.0

12.4

-

-

391, 314, 328

TA100

Acetone

-

120.3

9.0

-

-

-

125, 110, 126

Test item

5

130.3

15.5

1.1

0.85

NS

115, 146, 130

16

136.0

12.1

1.1

1.34

NS

143, 143, 122

50

133.7

4.5

1.1

1.16

NS

129, 138, 134

160

136.3

12.7

1.1

1.37

NS

134, 125, 150

500

178.0

16.1

1.5

4.60

**

173, 165, 196

1600

118.3

22.9

1.0

-0.24

NS

129V, 134V, 92V

5000

-

-

-

-

-

-T, -T, -T

AAN

5

746.7

139.6

6.2

-

-

875, 767, 598

TA1535

Acetone

-

21.0

3.6

-

-

-

18, 25, 20

Test item

5

14.0

1.0

0.7

-1.82

NS

13, 15, 14

16

20.3

4.0

1.0

-0.17

NS

25, 18, 18

50

17.3

7.1

0.8

-1.02

NS

16, 11, 25

160

16.0

7.0

0.8

-1.42

NS

8, 19, 21

500

23.0

4.0

1.1

0.46

NS

27, 19, 23

1600

9.7

1.2

0.5

-3.20

NS

9S, 9S, 11S

5000

-

-

-

-

NS

-T, -T, -T

AAN

5

270.0

19.0

12.9

-

-

251, 289, 270

TA1537

Acetone

-

19.0

5.0

-

-

-

24, 14, 19

Test item

5

16.0

7.0

0.8

-0.87

-

24, 13, 11

16

22.3

3.1

1.2

0.85

NS

25, 19, 23

50

16.7

6.0

0.9

-0.65

NS

16, 11, 23

160

18.0

2.6

0.9

-0.22

NS

19, 20, 15

500

14.7

1.5

0.8

-1.12

NS

16, 15, 13

1600

-

-

-

-

NS

-T, -T, -T

5000

-

-

-

-

-

-T, -T, -T

AAN

 

405.7

44.0

21.4

-

-

421, 356, 440

TA102

Acetone

-

356.7

21.8

-

-

-

376, 361, 333

Test item

5

381.0

12.5

1.1

1.74

-

371, 377, 395

16

373.0

3.5

1.0

1.19

NS

377, 371, 371

50

344.7

9.2

1.0

-0.86

NS

350, 334, 350

160

375.3

23.5

1.1

1.33

NS

401, 370, 355

500

448.0

30.3

1.3

6.23

**

467, 464, 413

1600

395.3

3.1

1.1

2.74

*

398S, 396S, 392S

5000

-

-

-

-

-

-T, -T, -T

AAN

20

1250.3

333.5

3.5

-

-

1464, 866, 1421

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

V: very thin background bacterial lawn

*p<0.05

** p<0.01

 

Table 3       Raw plate counts and calculated mutagenicity data, experiment 2 (- S9 mix)

 

Strain

Compound

Conc. level

(µg/plate)

Mean revertants per plate

SD

Fold increase

Dunnett’s T-value

Statistic. sig.

Revertant numbers/plate

TA98

Acetone

-

22.7

6.5

-

-

-

16, 23, 29

Test item

31.25

20.3

6.1

0.9

-0.54

NS

19, 27, 15

62.5

16.3

2.5

0.7

-1.50

NS

16, 14, 19

125

24.3

7.1

1.1

0.37

NS

23, 32, 18

250

18.7

3.2

0.8

-0.90

NS

20, 21, 15

400

18.3

2.5

0.8

-0.98

NS

16, 21, 18

500

21.7

4.7

1.0

-0.19

NS

18, 27, 20

750

20.7

7.6

0.9

-0.50

NS

19, 29, 14

1000

33.0

5.2

1.5

2.17

NS

39S, 30S, 30S

2000

-

-

-

-

-

-T, -T, -T

2NF

5

423.7

199.1

18.7

-

-

645, 367, 259

TA100

Acetone

-

82.7

10.2

-

-

-

71, 87, 90

Test item

31.25

97.0

13.1

1.2

1.22

NS

111, 85, 95

62.5

93.3

3.2

1.1

0.94

NS

92, 97, 91

125

105.3

15.3

1.3

1.89

NS

88, 111, 117

250

117.7

12.2

1.4

2.85

*

115, 107, 131

500

147.3

30.6

1.8

4.89

**

124, 182, 136

1000

187.7

24.5

2.3

7.46

**

174S, 173S, 216S

2000

-

-

-

-

-

-T, -T, -T

NaN3

2

448.0

61.6

5.4

-

-

500, 380, 464

TA1535

Acetone

-

6.7

1.2

-

-

-

6, 6, 8

Test item

31.25

10.0

1.0

1.5

1.88

NS

10, 9, 11

62.5

11.0

1.7

1.7

2.37

NS

13, 10, 10

125

8.0

2.6

1.2

0.72

NS

5, 10, 9

250

8.7

3.2

1.3

1.06

NS

10, 11, 5

500

7.0

2.6

1.1

0.11

NS

9, 8, 4

1000

11.0

0.0

1.7

2.39

NS

11, 11, 11

2000

-

-

-

-

-

-T, -T, -T

NaN3

2

352.7

52.7

52.9

-

-

379, 292, 387

TA1537

Acetone

-

4.0

1.0

-

-

-

4MB, 5, 3

Test item

31.25

4.7

1.5

1.2

0.44

NS

6, 5, 3

62.5

8.0

2.6

2.0

2.38

NS

10, 5, 9

125

4.3

0.6

1.1

0.26

NS

4, 4, 5

250

8.0

3.0

2.0

2.37

NS

8, 5, 11

500

10.0

1.0

2.5

3.44

*

10, 11, 9

1000

10.3

4.0

2.6

3.47

**

14S, 6S, 11S

 

2000

-

-

-

-

-

-T, -T, -T

AAC

50

152.7

71.1

38.2

-

-

201, 71, 186

TA102

Acetone

-

299.0

78.6

-

-

-

386, 278, 233

Test item

31.25

315.0

39.1

1.1

0.30

NS

334, 341, 270

62.5

297.3

20.6

1.0

0.02

NS

317, 276, 299

125

344.3

39.1

1.2

0.77

NS

307, 341, 385

250

355.7

27.2

1.2

0.94

NS

339, 387, 341

500

342.3

34.6

1.1

0.74

NS

382, 327, 318

1000

310.3

7.8

1.0

0.24

NS

304, 308, 319

 

2000

188.0

147.8

0.6

-2.38

NS

74V, 355V, 135V

MMC

0.2

954.3

109.0

3.2

-

-

986, 833, 1044

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

V: very thin background bacterial lawn

* p<0.05

** p<0.01

 

Table 4       Raw plate counts and calculated mutagenicity data, experiment 2 (+ S9 mix)

 

Strain

Compound

Conc. level

(µg/plate)

Mean revertants per plate

SD

Fold increase

Dunnett’s T-value

Statistic. sig.

Revertant numbers/plate

TA98

Acetone

-

30.5

3.5

-

-

-

28, -T, 33

Test item

31.25

22.0

1.7

0.7

-1.30

NS

23, 20, 23

62.5

23.7

3.1

0.8

-1.04

NS

27, 23, 21

125

33.3

4.7

1.1

0.39

NS

37, 35, 28

250

43.0

11.5

1.4

1.57

NS

39, 56, 34

400

47.0

7.8

1.5

2.08

NS

43, 42, 56

500

24.3

10.0

0.8

-1.04

NS

14, 34, 25

750

15.3

2.3

0.5

-2.53

NS

18, 14, 14

1000

16.0

10.4

0.5

-2.59

NS

11S, 9S, 28S

2000

-

-

-

-

-

-T, -T, -T

B[a]P

10

373.3

48.3

12.2

-

-

380, 322, 418

TA100

Acetone

-

101.0

18.3

-

-

-

85, 121, 97

Test item

31.25

107.0

4.0

1.1

0.17

NS

111, 107, 103

62.5

125.3

7.5

1.2

0.63

NS

121, 121, 134

125

130.0

39.6

1.3

0.69

NS

165, 138, 87

250

181.3

18.1

1.8

1.85

NS

162, 184, 198

500

269.7

173.2

2.7

3.14

*

462, 221, 126

1000

52.5

27.6

0.5

-1.40

NS

-T, 72V, 33V

2000

-

-

-

-

-

-T, -T, -T

AAN

5

1143.3

78.4

11.3

-

-

1088, 1109, 1233

TA1535

Acetone

-

14.7

3.5

-

-

-

18, 15, 11

Test item

31.25

13.7

2.5

0.9

-0.34

NS

16, 11, 14

62.5

12.0

1.7

0.8

-0.96

NS

10, 13, 13

125

16.0

1.7

1.1

0.50

NS

15, 15, 18

250

18.3

7.6

1.3

1.12

NS

13, 15, 27

500

12.7

1.5

0.9

-0.70

NS

14, 11, 13

1000

-

-

-

-

-

-T, -T, -T

2000

-

-

-

-

-

-T, -T, -T

AAN

5

179.7

35.7

12.3

-

-

139, 194, 206

TA1537

Acetone

-

3.7

1.2

-

-

-

3, 3, 5

Test item

31.25

6.3

4.2

1.7

1.07

NS

3, 11, 5

62.5

6.0

2.0

1.6

1.06

NS

4, 8, 6

125

5.7

2.5

1.5

0.88

NS

3, 6, 8

250

10.0

5.6

2.7

2.36

NS

11, 4, 15

500

7.3

1.2

2.0

1.62

NS

8V, 8V, 6V

1000

-

-

-

-

-

-T, -T, -T

 

2000

-

-

-

-

-

-T, -T, -T

AAN

5

249.3

180.7

68.0

-

-

74, 239, 435

TA102

Acetone

-

233.7

19.1

-

-

-

254, 231, 216

Test item

31.25

218.0

40.8

0.9

-0.43

NS

197, 192, 265

62.5

281.7

51.0

1.2

1.14

NS

281, 231, 333

125

328.0

13.5

1.4

2.21

NS

339, 332, 313

250

264.3

14.3

1.1

0.76

NS

280, 261, 252

500

302.0

67.6

1.3

1.58

NS

237, 372, 297

1000

104.7

53.5

0.4

-4.16

NS

133V, 43V, 138V

 

2000

-

-

-

-

-

-T, -T, -T

AAN

20

745.0

85.8

3.2

-

-

791, 798, 646

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

V: very thin background bacterial lawn

 

 

 

 

Applicant's summary and conclusion

Conclusions:
It was concluded that the test article induced mutation in histidine-requiring strains TA98 and TA100, of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines)
Executive summary:

OECD 471 (2018) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimuriumwere exposed to Reaction mass of N,N,N’,N-tetrabutylmethylenediamine and dibutylamine in acetone at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate a screening experiment followed by concentrations of 31.25, 62.5, 125, 250, 500, 100 and 200 µg/plate (TA98 also tested at 400 and 750 µg/plate) in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix.

 

The test item was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

 

It was concluded that the test article induced mutation in histidine-requiring strains TA98 and TA100, of Salmonella typhimurium when tested under the conditions of this study.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.