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Toxicological information

Endpoint summary

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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay; OECD 471; Dreher, D. (2018); Positive (mutagenic)

Mammalian cell cytogeneticity assay; OECD 473; Lyoyd, M. (2018); Positive (clastogenic)

Link to relevant study records

Referenceopen allclose all

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.
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
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
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.
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.
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

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

 

 

 

 

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 March 2017 - 08 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Commission Regulation (EC) N0. 440/2008 of 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)
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: 11-25 ºC (23 Dec 2017 - 11 Jan 2018) and 2-8 ºC (11 Jan 2018 onwards), in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble at 200 mg/mL in acetone which was used to prepare the maximum required treatment concentration.
- 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 (where required), to give the maximum required treatment concentration. Subsequent dilutions were made using acetone. The test article solutions were protected from light and used within approximately 1.5 hours of initial formulation.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Stock solution prepared at 200 mg/mL 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
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood from three healthy, non-smoking male volunteers from a panel of donors at the testing facility. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes on the day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
- Suitability of cells: Screened as described above
- Cell cycle length, doubling time or proliferation index: The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours
- Sex, age and number of blood donors if applicable: Male; aged 24-34
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator set to 37±1 °C) HEPES-buffered RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum and 0.52 % penicillin / streptomycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2 % of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1 °C for approximately 48 hours and rocked continuously.
- Number of passages if applicable: 1
- Methods for maintenance in cell culture if applicable: Described above
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 13±2 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: HEPES-buffered RPMI medium containing 1 0% (v/v) heat inactivated foetal calf serum and 0.5 2% penicillin / streptomycin. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2 % of culture to stimulate the lymphocytes to divide.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No
- Periodically checked for karyotype stability: No
- Periodically 'cleansed' against high spontaneous background: No
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary solubility data indicated that the test article was soluble in acetone at a concentration of at least 600 mg/mL. The solubility limit in culture medium was in the range of 263.6 to
527.1 μg/mL, as indicated by precipitation at the higher concentration which persisted for 20 hours after test article addition, with warming at 37 °C. A maximum concentration of 2000 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the recommended maximum concentration for in vitro chromosome aberration studies according to current regulatory test guidelines (OECD, 2016). Concentrations selected for the Chromosome Aberration Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
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.
Untreated negative controls:
yes
Remarks:
Untreated; culture medium alone
Negative solvent / vehicle controls:
yes
Remarks:
acetone, batch MKBX7488V (Honeywell: expiry date 3 Apr 2018)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:

1. Compared to the concurrent negative control a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) at one or more concentrations is observed (p ≤ 0.05).

2. The incidence of cells with structural aberrations (excluding gaps) at such a concentration exceeds the normal range in both replicate cultures and falls outside the distribution of the historical negative control data.

3. A concentration-related increase in the proportion of cells with structural aberrations (excluding gaps) is observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.
Statistics:
After completion of scoring and decoding of slides the numbers of aberrant cells in each culture were categorised as follows:
1. Cells with structural aberrations including gaps
2. Cells with structural aberrations excluding gaps
3. Polyploid or endoreduplicated cells.
The totals for category 2 in vehicle control cultures were compared with the 95th percentile of the current historical vehicle control (normal) ranges to determine whether the assay was acceptable or not (see Acceptance criteria). The proportion of cells with structural chromosome aberrations excluding gaps were compared with the proportion in vehicle controls by using Fisher’s exact test (Richardson et al., 1989). In addition, a Cochran-Armitage Trend Test was performed to aid determination of concentration response relationships. Probability values of p≤0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to historical vehicle control range.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). Probability values of p≤0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

 Table 1       3 hour treatment in the absence of S9 with 17 hour recovery (3+17),– range finder

 

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH* (%)

Vehicle

A

7.7

 

B

8.6

Total

8.2

-

UTC

A

8.2

-

7.256

A

8.0

2

12.09

A

5.8

29

20.16

A

5.0

39

33.59

A

3.7

55

55.99

A

3.2

61

93.31

A

1.1

87

155.5

A

0.2

98

259.2

A

0.2

98P

432.0

A

0.2

98P

720.0

A

0.1

99P

1200

A

0.2

98P

2000

A

0.0

-PE

UTC: untreated control

P: indicates precipitation observed at the beginning of the test

E: indicates precipitation at the end of the test

* mitotic inhibition (%)= [1-(mean MIT/ mean MIC)] x 100 %

(where T = treatment and C = negative control)

 

Table 2       3 hour treatment in the presence of S9 with 17 hour recovery (3+17),– range finder

 

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH* (%)

Vehicle

A

8.5

 

B

9.5

Total

9.0

-

UTC

A

8.3

-

7.256

A

10.0

0

12.09

A

7.3

19

20.16

A

9.2

0

33.59

A

6.2

31

55.99

A

5.6

38

93.31

A

2.9

68

155.5

A

0.4

96

259.2

A

0.2

98P

432.0

A

0.2

98P

720.0

A

0.4

96P

1200

A

0.3

97P

2000

A

0.4

96PE

UTC: untreated control

P: indicates precipitation observed at the beginning of the test

E: indicates precipitation at the end of the test

* mitotic inhibition (%)= [1-(mean MIT/ mean MIC)] x 100 %

(where T = treatment and C = negative control)UTC: untreated control

 

Table 3       20 hour treatment in the absence of S9 with 0 hour recovery (20+0),– range finder

 

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH* (%)

Vehicle

A

7.6

 

B

8.0

Total

7.8

-

UTC

A

8.4

-

7.256

A

6.6

15

12.09

A

6.2

21

20.16

A

5.0

36

33.59

A

4.1

47

55.99

A

0.0

-

93.31

A

0.0

-

155.5

A

0.0

-

259.2

A

0.0

-P

432.0

A

0.0

-P

720.0

A

0.2

97P

1200

A

0.3

96P

2000

A

0.0

-PH

UTC: untreated control

P: indicates precipitation observed at the beginning of the test

E: indicates precipitation at harvest

* mitotic inhibition (%)= [1-(mean MIT/ mean MIC)] x 100 %

(where T = treatment and C = negative control)UTC: untreated control

 

No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (2000 μg/mL), compared to the concurrent vehicle and negative controls.

 

The results of the cytotoxicity Range-Finder Experiment were used to select suitable concentrations for the Chromosome Aberration Experiment. Marked cytotoxicity (>60 % MIH) was observed at 55.99 μg/mL and above for the 3+17 and 20+0 treatments in the absence of S-9 and at 93.31 μg/mL and above for the 17+3 hour treatment in the presence of S-9. Based on these observations and in order to permit a range of cytotoxicity under each treatment condition, the maximum concentrations selected for the Chromosome Aberration Experiment were 80 μg/mL for the 3+17 hour treatments in the absence of S-9, 120 µg/mL for the 17+3 hour treatment in the presence of S-9 and 80 µg/mL for the 17+3 hour treatment in the absence of S-9.

 

The results of the MI determinations from the Chromosome Aberration Experiment were as follows:

 

Table 4       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment

 

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH* (%)

Vehicle

A

6.6

 

B

7.4

C

8.0

D

6.8

Total

7.2

-

UTC

A

6.6

 

B

7.4

Total

7.0

-

5.000

A

6.4

 

B

7.4

Total

6.9

4

10.00

A

7.7

 

B

6.6

Total

7.2

1

15.00

A

8.1

 

B

6.8

Total

7.5

0#

20.00

A

5.4

 

B

6.0

Total

5.7

21

25.00

A

6.3

 

B

5.5

Total

5.9

18#

30.00

A

5.7

 

B

7.3

Total

6.5

10

35.00

A

6.5

 

B

6.5

Total

6.5

10#

40.00

A

5.7

 

B

3.5

Total

4.6

36

45.00

A

5.5

 

B

4.1

Total

4.8

33#

50.00

A

4.2

 

B

2.8

Total

3.5

51#

60.00

A

2.1

 

B

2.3

Total

2.2

69

80.00

A

2.0

 

B

1.3

Total

1.7

77

MMC, 0.30

A

5.2

 

B

5.5

Total

5.4

26

MMC, 0.40

A

3.8

 

B

3.0

Total

3.4

53#

UTC: untreated control

# concentration selected for chromosome aberration analysis

* mitotic inhibition (%)= [1-(mean MIT/ mean MIC)] x 100 %

(where T = treatment and C = negative control)

 

Table 5       3 hour treatment in the presence of S9 with 17 hour recovery (3+17),– range finder

 

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH* (%)

Vehicle

A

8.0

 

B

6.8

C

7.9

D

7.3

Total

7.5

-

UTC

A

8.4

 

B

8.2

Total

8.3

-

10.00

A

6.8

 

B

9.1

Total

8.0

0

20.00

A

6.0

 

B

7.8

Total

6.9

8#

30.00

A

5.3

 

B

5.5

Total

5.4

28#

40.00

A

6.1

 

B

4.5

Total

5.3

29

50.00

A

6.9

 

B

5.4

Total

6.2

18

60.00

A

5.5

 

B

4.9

Total

5.2

31

70.00

A

3.9

 

B

4.0

Total

4.0

47#

80.00

A

3.4

 

B

4.5

Total

4.0

47

90.00

A

2.7

 

B

4.0

Total

3.4

55#

100.0

A

2.5

 

B

2.2

Total

2.4

69

120.0

A

2.4

 

B

2.6

Total

2.5

67

CPA, 1.00

A

5.5

 

B

5.8

Total

5.7

25#

CPA, 2.00

A

3.0

 

B

3.1

Total

3.1

59

UTC: untreated control

# concentration selected for chromosome aberration analysis

* mitotic inhibition (%)= [1-(mean MIT/ mean MIC)] x 100 %

(where T = treatment and C = negative control)

 

Table 6       20 hour treatment in the absence of S9 with 0 hour recovery (20+0),– range finder

 

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH* (%)

Vehicle

A

3.7

 

B

4.8

C

5.0

D

4.5

Total

4.5

-

UTC

A

6.0

 

B

4.9

Total

5.5

-

5.000

A

3.5

 

B

4.2

Total

3.9

14#

10.00

A

2.7

 

B

4.1

Total

3.4

24

15.00

A

3.6

 

B

2.8

Total

3.2

29#

20.00

A

2.5

 

B

3.4

Total

3.0

34#

25.00

A

3.1

 

B

2.3

Total

2.7

40

27.50

A

2.6

 

B

2.3

Total

2.5

46#

30.00

A

2.7

 

B

3.8

Total

3.3

28

32.50

A

3.1

 

B

2.7

Total

2.9

36

35.00

A

2.2

 

B

2.2

Total

2.2

51#

40.00

A

2.8

 

B

2.1

Total

2.5

46

45.00

A

1.4

 

B

1.3

Total

1.4

70

50.00

A

0.6

 

B

0.9

Total

0.8

83

MMC, 0.05

A

3.3

 

B

4.8

Total

4.1

10#

MMC, 0.10

A

3.5

 

B

3.2

Total

3.4

26

UTC: untreated control

# concentration selected for chromosome aberration analysis

* mitotic inhibition (%)= [1-(mean MIT/ mean MIC)] x 100 %

(where T = treatment and C = negative control)

 

Study validity 

1. The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.

2. The proportions of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal ranges.

3. At least 300 cells were suitable for analysis at each concentration, unless 15 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis.

4. The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated structural aberration cell frequencies (excluding gaps) that clearly exceeded the current historical vehicle control ranges

5. The maximum concentration analysed under each treatment condition was a concentration inducing approximately 50% cytotoxicity.

 

Structural aberrations 

Treatment of cells with the test article for 3+17 hours in the absence of S-9 resulted in frequencies of cells with structural chromosome aberrations that were significantly higher (p≤0.001, using Fisher’s exact test), compared to the concurrent vehicle controls at the highest three concentrations analysed (35, 45 and 50 μg/mL). The aberration frequencies (excluding gaps) exceeded the normal range in both cultures at 35 and 50 μg/mL and in one culture at 45 μg/mL and the Cochran-Armitage linear trend test was statistically significant (p≤0.001).

 

Treatment of cells for 3+17 hours in the presence of S-9 resulted in frequencies of structurally aberrant cells that were significantly higher (p≤0.05), compared to the concurrent vehicle controls at the highest two concentrations analysed (70 and 90 μg/mL). The aberration frequencies (excluding gaps) exceeded the normal range in one culture at 20 μg/mL and in both cultures at 70 and 90 μg/mL, with a statistically significant Cochran-Armitage linear trend test (p≤0.001).

 

Treatment of cells for 20+0 hours in the absence of S-9 resulted in frequencies of structurally aberrant cells that were significantly higher (p≤0.001), compared to the concurrent vehicle controls at the highest two concentrations analysed (27.5 and 35 μg/mL). The aberration frequencies (excluding gaps) exceeded the normal range in one culture at 5 μg/mL and in both cultures at 27.5 and 35 μg/mL, with a statistically significant Cochran-Armitage linear trend test (p≤0.001).

 

Table 7       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)

Replicate

Cells scored

Cells with aberrations inc. gaps

Cells with aberrations exc. gaps

Significance§

MIH* (%)

Vehicle

A

150

0

0

 

 

B

150

0

0

Total

300

0 (0 %)

0 (0 %)

-

-

15

A

150

0

0

 

 

B

150

0

0

Total

300

0 (0 %)

0 (0 %)

NS

0

25

A

150

1

1

 

 

B

150

0

0

Total

300

1 (0.33 %)

1 (0.33 %)

NS

18

35

A

150

6#

6#

 

 

B

150

7#

7#

Total

300

13 (4.33 %)

13 (4.33 %)

p≤0.001

10

45

A

150

10#

10#

 

 

B

150

4

4

Total

300

14 (4.67 %)

14 (4.67 %)

p≤0.001

33

50

A

150

15#

14#

 

 

B

150

17#

16#

Total

300

32 (10.67 %)

30 (10.00 %)

p≤0.001

51

MMC 0.4

A

150

21#

20#

 

 

B

145

22#

20#

Total

295

43 (14.58 %)

40 (13.56 %)

p≤0.001

53

NS: not significant

§statistical significance

# numbers exceed historical vehicle control range

* mitotic inhibition

 

Table 8       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – cells with structural aberrations

 

Treatment (µg/mL)

Replicate

Cells scored

Cells with aberrations inc. gaps

Cells with aberrations exc. gaps

Significance§

MIH* (%)

Vehicle

A

150

1

1

 

 

B

150

0

0

Total

300

1 (0.33 %)

1 (0.33 %)

-

-

20

A

150

4#

4#

 

 

B

150

0

0

Total

300

4 (1.33 %)

4 (1.33 %)

NS

8

30

A

150

3

3

 

 

B

150

2

2

Total

300

5 (1.67 %)

5 (1.67 %)

NS

28

70

A

150

15#

13#

 

 

B

150

22#

20#

Total

300

37 (12.33 %)

33 (11.00 %)

p≤0.001

47

90

A

136

30#

30#

 

 

B

150

23#

22#

Total

286

53 (18.53 %)

52 (18.18 %)

p≤0.001

55

CPA, 1.00

A

150

8#

8#

 

 

B

150

11#

9#

Total

300

19 (6.33 %)

19 (5.67 %)

p≤0.001

25

NS: not significant

§statistical significance

# numbers exceed historical vehicle control range

* mitotic inhibition

 

Table 9       20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – cells with structural aberrations

 

Treatment (µg/mL)

Replicate

Cells scored

Cells with aberrations inc. gaps

Cells with aberrations exc. gaps

Significance§

MIH* (%)

Vehicle

A

150

1

1

 

 

B

150

2

2

Total

300

3 (1.00 %)

3 (1.00 %)

-

-

UTC

A

150

1

1

 

 

B

150

3

3

Total

300

4 (1.33 %)

4 (1.33 %)

NS

-

5

A

150

6#

5#

 

 

B

150

2

2

Total

300

8 (2.67 %)

7 (2.33 %)

NS

14

15

A

150

3

3

 

 

B

150

4

2

Total

300

7 (2.33 %)

5 (1.67 %)

NS

29

27.5

A

150

11#

10#

 

 

B

150

10#

10#

Total

300

21 (7.00 %)

20 (6.67 %)

p≤0.001

46

35

A

150

25#

23#

 

 

B

150

24#

24#

Total

300

49 (16.33 %)

47 (15.67 %)

p≤0.001

51

MMC, 0.05

A

150

17#

17#

 

 

B

150

15#

14#

Total

300

32 (10.67 %)

31 (10.33 %)

p≤0.001

10

NS: not significant

§statistical significance

# numbers exceed historical vehicle control range

* mitotic inhibition

 

Table 10       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)

Rep

Cells*

G

Chr del.

Chr exch.

Ctd del.

Ctd exch.

Other

Abs +g

Abs -g

Vehicle

A

150

0

0

0

0

0

0

0

0

B

150

0

0

0

0

0

0

0

0

Total

300

0

0

0

0

0

0

0

0

15.0

A

150

0

0

0

0

0

0

0

0

B

150

0

0

0

0

0

0

0

0

Total

300

0

0

0

0

0

0

0

0

25.0

A

150

0

0

0

1

0

0

1

1

B

150

0

0

0

0

0

0

0

0

Total

300

0

0

0

1

0

0

1

1

35.0

A

150

0

0

0

6

0

0

6

6

B

150

0

2

0

5

2

0

9

9

Total

300

0

2

0

11

2

0

15

15

45.0

A

150

0

1

0

5

4

0

10

10

B

150

0

0

0

4

0

0

4

4

Total

300

0

1

0

9

4

0

14

14

50.0

A

150

1

3

0

12

3

0

19

18

B

150

1

4

1

18

3

0

17

16

Total

300

2

7

1

120

6

0

36

34

MMC, 0.40

A

150

2

5

0

11

6

0

24

22

B

145

4

3

0

17

3

0

27

23

Total

295

6

8

0

28

9

0

51

45

* total cells examined for structural aberrations

G: gaps

Chr del: chromosome deletion

Chr exch: chromosome exchanges

Ctd del: chromatid deletion

Ctd exch: chromatid exchanges

Abs: aberrations

 

Table 11       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)

Rep

Cells*

G

Chr del.

Chr exch.

Ctd del.

Ctd exch.

Other

Abs +g

Abs -g

Vehicle

A

150

0

0

0

1

0

0

1

1

B

150

0

0

0

0

0

0

0

0

Total

300

0

0

0

1

0

0

1

1

20.0

A

150

0

1

0

3

0

0

4

4

B

150

0

0

0

0

0

0

0

0

Total

300

0

1

0

3

0

0

4

4

30.0

A

150

0

0

0

3

1

0

4

4

B

150

0

1

0

1

0

0

2

2

Total

300

0

1

0

4

1

0

6

6

70.0

A

150

3

3

0

6

4

0

16

13

B

150

3

1

0

18

3

0

25

22

Total

300

6

4

0

24

7

0

41

35

90.0

A

136

2

8

0

28

2

0

40

38

B

150

3

2

0

17

6

0

28

25

Total

286

5

10

0

45

8

0

68

63

CPA, 1.00

A

150

3

3

0

8

0

0

14

11

B

150

2

2

0

9

0

0

13

11

Total

300

5

5

0

17

0

0

27

22

* total cells examined for structural aberrations

G: gaps

Chr del: chromosome deletion

Chr exch: chromosome exchanges

Ctd del: chromatid deletion

Ctd exch: chromatid exchanges

Abs: aberrations

 

Table 12       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)

Rep

Cells*

G

Chr del.

Chr exch.

Ctd del.

Ctd exch.

Other

Abs +g

Abs -g

Vehicle

A

150

0

0

0

1

0

0

1

1

B

150

0

1

0

1

0

0

2

2

Total

300

0

1

0

2

0

0

3

3

UTC

A

150

0

1

0

0

0

0

1

1

B

150

0

0

0

3

0

0

3

3

Total

300

0

1

0

3

0

0

4

4

5

A

150

1

1

0

5

0

0

7

6

B

150

1

1

0

1

0

0

3

2

Total

300

2

2

0

6

0

0

10

8

15

A

150

0

1

0

3

0

0

4

4

B

150

2

2

0

1

0

0

5

3

Total

300

2

3

0

4

0

0

9

7

27.5

A

150

1

0

0

19

0

0

20

19

B

150

0

3

0

10

0

0

13

13

Total

300

1

3

0

29

0

0

33

32

35

A

150

2

6

0

26

3

0

37

35

B

150

2

2

0

32

1

2

39

37

Total

300

4

8

0

58

4

2

76

72

MMC, 0.05

A

150

0

2

0

14

3

0

19

19

B

150

1

1

0

12

2

0

16

15

Total

300

1

3

0

26

5

0

35

34

* total cells examined for structural aberrations

G: gaps

Chr del: chromosome deletion

Chr exch: chromosome exchanges

Ctd del: chromatid deletion

Ctd exch: chromatid exchanges

Abs: aberrations

 

Numerical Aberrations

 

Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and normal ranges were observed under all treatment conditions. There were no concentration-related responses but numerical aberrations were not analysed quantitatively. Increases in numerical aberration frequency in chromosome aberration assays in vitro may be attributable to aneugenicity, but polyploidy alone does not indicate aneugenic potential and may simply indicate cell cycle perturbation or cytotoxicity. As such, although numerical aberrations can be detected, this assay is not specifically designed for quantitative evaluation of aneugens.

 

Table 13       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of numerical aberrations observed

 

Treatment (µg/mL)

Rep

Cells*

E

P

Total abs.

% with numerical abs.

Vehicle

A

150

0

0

0

0

B

150

0

0

0

0

Total

300

0

0

0

0

15

A

150

0

0

0

0

B

151

0

1

1

0.7

Total

301

0

1

1

0.3

25

A

150

0

0

0

0

B

152

0

2#

2

1.3

Total

302

0

2

2

0.7

35

A

154

0

4#

4

2.6

B

159

0

9#

9

5.7

Total

313

0

13

13

4.2

45

A

154

0

4#

4

2.6

B

151

0

1

1

0.7

Total

305

0

5

5

1.6

50

A

150

0

0

0

0

B

151

0

1

1

0.7

Total

301

0

1

1

0.3

MMC 0.4

A

150

0

0

0

0

B

145

0

0

0

0

Total

295

0

0

0

0

* total cells examined for numerical aberrations

# number exceeds historical vehicle control range

E: endoreduplicated

P: polyploid (> 68 chromosomes)

 

Table 14       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of numerical aberrations observed

 

Treatment (µg/mL)

Rep

Cells*

E

P

Total abs.

% with numerical abs.

Vehicle

A

150

0

0

0

0

B

150

0

0

0

0

Total

300

0

0

0

0

20

A

150

0

0

0

0

B

150

0

0

0

0

Total

300

0

0

0

0

30

A

156

0

6#

6

3.8

B

150

0

0

0

0

Total

306

0

6

6

2.0

70

A

155

0

5#

5

3.2

B

154

1#

3#

4

2.6

Total

309

1

8

9

2.9

90

A

136

0

0

0

0

B

151

0

1

1

0.7

Total

287

0

1

1

0.3

CPA, 1.0

A

150

0

0

0

0

B

152

0

2#

2

1.3

Total

302

0

2

2

0.7

* total cells examined for numerical aberrations

# number exceeds historical vehicle control range

E: endoreduplicated

P: polyploid (> 68 chromosomes)

 

Table 15       20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – numbers and types of numerical aberrations observed

 

Treatment (µg/mL)

Rep

Cells*

E

P

Total abs.

% with numerical abs.

Vehicle

A

151

1

0

1

0.7

B

150

0

0

0

0

Total

301

1

0

1

0.3

UTC

A

150

0

0

0

0

B

151

0

1

1

0.7

Total

301

0

1

1

0.3

5

A

150

0

0

0

0

B

150

0

0

0

0

Total

300

0

0

0

0

15

A

151

0

1

1

0.7

B

150

0

0

0

0

Total

301

0

1

1

0.3

27.5

A

155

0

5#

5

3.2

B

157

0

7#

7

4.5

Total

312

0

12

12

3.8

35

A

158

0

8#

8

5.1

B

156

0

6#

6

3.8

Total

314

0

14

14

4.5

MMC 0.05

A

150

0

0

0

0

B

152

0

2#

2

1.3

Total

302

0

2

2

0.7

* total cells examined for numerical aberrations

# number exceeds historical vehicle control range

E: endoreduplicated

P: polyploid (> 68 chromosomes)

 

Table 16       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – statistical analysis

 

Treatment (µg/mL)

Cells scored

Aberrant cells

Proportion

Fisher’s exact test

Significance

Vehicle

300

0

0.000

-

-

15

300

0

0.000

0.500

NS

25

300

1

0.003

0.250

NS

35

300

13

0.043

0.000

p≤0.001

45

300

14

0.047

0.000

p≤0.001

50

300

30

0.100

0.000

p≤0.001

MMC 0.4

295

40

0.136

0.000

p≤0.001

Binomial dispersion testc2: 3.929            DF: 6               p-value: 0.686                Significance: NS

Cochran-Armitage Linear Trend                                     p-value: 0.000                Significance: p≤0.001

NS: not significant

DF: degrees of freedom

 

Table 17       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – statistical analysis

 

Treatment (µg/mL)

Cells scored

Aberrant cells

Proportion

Fisher’s exact test

Significance

Vehicle

300

1

0.003

-

-

20

300

4

0.013

0.109

NS

30

300

5

0.017

0.062

NS

70

300

33

0.110

0.000

p≤0.001

90

286

52

0.182

0.000

p≤0.001

CPA, 1.0

300

17

0.057

0.000

p≤0.001

Binomial dispersion testc2: 9.459           DF: 5               p-value: 0.089              Significance: NS

Cochran-Armitage Linear Trend                                     p-value: 0.000                Significance:p≤0.001

NS: not significant

DF: degrees of freedom

 

Table 18       20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – statistical analysis

 

Treatment (µg/mL)

Cells scored

Aberrant cells

Proportion

Fisher’s exact test

Significance

Vehicle

300

3

0.010

-

-

UTC

300

4

0.013

0.363

NS

5

300

7

0.023

0.112

NS

15

300

5

0.017

0.253

NS

27.5

300

20

0.067

0.000

p≤0.001

35

300

47

0.157

0.000

p≤0.001

MMC 0.05

300

31

0.103

0.000

p≤0.001

Binomial dispersion testc2: 1.882            DF: 5               p-value: 0.865                Significance: NS

Cochran-Armitage Linear Trend                                     p-value: 0.000                Significance:p≤0.001

NS: not significant

DF: degrees of freedom

 

 

Conclusions:
It is concluded that Reaction Mass of N,N,N',N’-tetrabutylmethylenediamine and Dibutylamine induced structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9 and is therefore considered to have clastogenic potential in this chromosome aberration test. Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and the normal ranges, were observed under all treatment conditions, but this assay is not specifically designed for the quantitative evaluation of polyploidy.
Executive summary:

OECD 473 (2018) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine at concentrations of 0, 15, 20, 25, 30, 35, 45, 50, 70 and 90 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 5, 15, 27.5 and 35 µg/mL.

 

Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine was tested up to precipitating and cytotoxic concentrations of 55.99 and 93.31 µg/mLfor the 3 h exposure, in the absence and presence of S9 mix, respectively and 93.31 µg/mL for the 20 h exposure with S9 mix. Positive controls induced the appropriate response.

 

There was evidence that the test item induced structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9 and is therefore considered to have clastogenic potential in this chromosome aberration test. Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and the normal ranges, were observed under all treatment conditions, but this assay is not specifically designed for the quantitative evaluation of polyploidy.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Micronucleus screen OECD 474 incorporated into OECD 442; N Barraclough (2018) - Negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
This assay was incorporated into OECD 422 study design.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 May 2017 - 11 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Similar to OECD 474 guideline - the study was conducted similar to a guideline study, but incorporated into an OECD 422. All validity criteria were met under both guidelines. The study was conducted under GLP.
Justification for type of information:
The assay was incorporated into OECD 422 study design but it meet all the criteria for the OECD 474 guideline.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Assessment incorporated into OECD 442 - ICH S2(R1) (2011).
Deviations:
no
Principles of method if other than guideline:
An integrated standard genotoxicity assessment into a general toxicology study (OECD, 2016) & ICH S2(R1) (2011).
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian erythrocyte micronucleus test (migrated information)
Specific details on test material used for the study:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-19
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: Males: 307.4 and 385.4 g; Females: 174.1 and 218.9 g
- Fasting period before study: Not reported
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of four by sex. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels that may have interfered with achieving the objective of the study
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels that may have interfered with achieving the objective of the study.
- Acclimation period: 16 days prior to initiation of dosing (males) or 9 days prior to initiation of smearing (females).

DETAILS OF FOOD AND WATER QUALITY: As described above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): Minimum of 15 hrs
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 15th Feb 2018 To: 13th Apr 2018
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Formulations (excluding the control group) were stirred continuously for at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
Males were dosed for 42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]). Females were dosed for up to 57 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, throughout gestation and until LD 13, inclusive, or 25 days post-coitum for females which did not litter. Females were sent to necropsy on LD 14 or 26 days post‑coitum).
Animal R0404 (Group 1 female) and Animals R0705 and R0707 (Group 4 females) were not dosed on GD 22/23 due to signs of parturition.
A dose volume of 5 mL/kg was used. Dose volumes were calculated using the most recent recorded body weight for each animal.
Duration of treatment / exposure:
Males were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43.

Females were dosed for up to 57 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD 13).
Frequency of treatment:
Once daily
Post exposure period:
None
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 3 (Intermediate)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 4 (High)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
10 mg/kg/day cyclophosphamide (dissolved in saline).
Tissues and cell types examined:
Bone marrow from isolated femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on dose range finder study 8359421



TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Bone marrow at necropsy, no later than 24 hours after the last administration. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL foetal bovine serum and filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose. Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, room temperature).



DETAILS OF SLIDE PREPARATION: The pellet was mixed into small volume of serum using a Pasteur pipette, one drop of suspension was placed on the end of each of three uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water.

Slides were dried at room temperature. Two male slides per animal were stained on the day of fixation. Female slides (two per animal) were stored at room temperature for subsequent staining. Slides were stained in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, dried and stored protected from light at room temperature prior to analysis.
Male and female positive control slides were stained and coded alongside the study slides

METHOD OF ANALYSIS: Scoring was carried out using fluorescence microscopy at an appropriate magnification (x400 for ratios of PCE (polychromatic erythrocytes):NCE (normochromatic erythrocytes) and either x600 or x1000 (under oil immersion) for MN PCE).
 


OTHER: The following criteria were applied during slide assessment:
1. Cells were to be of normal cell morphology
2. Areas where erythrocytes overlap were to be ignored
3. A MN was to be round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN which were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeds the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE (where more than two dose levels are analysed) is observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.
Statistics:
Statistical analyses (ANCOVA) were performed.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Table 2: Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine: Summary and Statistical Analysis of Micronucleus Data – Bone  Marrow – Males.

Treatment

(mg/kg/day)

PCE Cell Total

% PCE

MN PCE

% MN PCE†

SD

Heterogeneity

Contingency X2C

X2

S

P-value

Significance

0

20000

47.16

35

0.18

0.07

4.87

NS

30

20000

48.48

36

0.18

0.12

11.80

p≤0.05

0.50

NS

50

20000

50.00

37

0.19

0.06

3.41

NS

0.45

NS

100

20000

50.08

23

0.12

0.09

12.01

p≤0.05

0.93

NS

CPA (10)*

12000

50.13

41.8

3.48

0.26

 

0.00

p≤0.001

* CPA slides coded from previously conducted positive control slide bank study (Covance Study Number 8360181)

† %MN PCE calculated from a total of 500 cells (PCE and NCE counted) per animal

Linear trend test P-value: 0.945, NS

NS        Not significant

MN        Micronucleated

SD        Standard deviation

Table 3: Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine: Summary and Statistical Analysis of Micronucleus Data – Bone Marrow - Females.

Treatment

(mg/kg/day)

PCE Cell Total

% PCE

MN PCE

% MN PCE†

SD

Heterogeneity

Contingency X2C

X2

S

P-value

Significance

0

20000

50.16

44

0.22

0.12

9.89

p≤0.05

30

20000

50.88

44

0.22

0.11

8.29

NS

0.46

NS

50

20000

47.72

44

0.22

0.11

8.06

NS

0.54

NS

100

20000

50.52

44

0.22

0.10

7.84

NS

054

NS

CPA (10)*

12000

45.40

427

3.56

0.29

 

0.00

p≤0.001

* CPA slides coded from previously conducted positive control slide bank study (Covance Study Number 8360181)

† %MN PCE calculated from a total of 500 cells (PCE and NCE counted) per animal

Linear trend test P-value: 0.500, NS

NS        Not significant

MN        Micronucleated

SD        Standard deviation

Conclusions:
The test item was considered non-clastogenic and does not meet the criteria for classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP)
Executive summary:

The genotoxicity potential of the Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was investigated in an  Oral (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422, adopted 29 July 2016). Covance study 8359422. Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle]), 30, 50, or 100 mg/kg/day test item once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg. The positive control slides were prepared under Covance Positive control Slide Bank Study (Covance study 8360181) from animals that had received two daily oral gavage doses of 10 mg/kg/day cyclophosphamide (dissolved in saline). Sampling of these animals was on Day 3 (approximately 24 hours post the last dose administration).

Animals treated with the test article at all doses exhibited group mean %PCE values that were either similar to or slightly higher than the value for the concurrent vehicle control group but which fell within the historical vehicle control range. As such, there was no evidence of bone marrow toxicity. Treatment with the test item exhibited group mean MN PCE frequencies that were similar to and not significantly (p≤0.05) higher than those observed in the concurrent vehicle control group for all doses. Individual MN PCE values were consistent with historical vehicle control distribution ranges and similar to values observed in the concurrent vehicle control group.

Based on the conditions of the study, it can be stated that the test item did not induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of male and female Han-Wistar rats treated at doses of 30, 50 or 100 mg/kg/day. The test item was considered non clastogenic and does not meet the criteria for classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Based on the in vivo OECD 474 data, all measured parameters produced negative results and as such the test item is not considered clastogenic. However, data on mutagenicity is currently inconclusive and as such the overall mode of action for genotoxicity and its relevance to human cannot be confirmed.

Additional information

in Vitro genotoxicity data

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.

OECD 473 (2018) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine at concentrations of 0, 15, 20, 25, 30, 35, 45, 50, 70 and 90 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 5, 15, 27.5 and 35 µg/mL.

 

Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine was tested up to precipitating and cytotoxic concentrations of 55.99 and 93.31 µg/mLfor the 3 h exposure, in the absence and presence f S9 mix, respectively and 93.31 µg/mL for the 20 h exposure with S9 mix. Positive controls induced the appropriate response.

 

There was evidence that the test item induced structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9 and is therefore considered to have clastogenic potential in this chromosome aberration test. Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and the normal ranges, were observed under all treatment conditions, but this assay is not specifically designed for the quantitative evaluation of polyploidy.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

In vivo genotoxicity data

OECD 474 (incorporated into OECD 422 study design) - The genotoxicity potential of the Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was investigated in an  Oral (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422, adopted 29 July 2016). Covance study 8359422. Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle: corn oil]), 30, 50, or 100 mg/kg/day test item once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg. The positive control slides were prepared under the Covance Positive control Slide Bank Study (Covance study 8360181) from animals that had received two daily oral gavage doses of 10 mg/kg/day cyclophosphamide (dissolved in saline). Sampling of these animals was on Day 3 (approximately 24 hours post the last dose administration).

Animals treated with the test item at all doses exhibited group mean %PCE values that were either similar to or slightly higher than the value for the concurrent vehicle control group but which fell within the historical vehicle control range. As such, there was no evidence of bone marrow toxicity. Treatment with the test item exhibited group mean MN PCE frequencies that were similar to and not significantly (p≤0.05) higher than those observed in the concurrent vehicle control group for all doses. Individual MN PCE values were consistent with historical vehicle control distribution ranges and similar to values observed in the concurrent vehicle control group.

Based on the conditions of the study, it can be stated that the test item did not induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of male and female Han-Wistar rats treated at doses of 30, 50 or 100 mg/kg/day. The test item was considered non clastogenic and does not meet the criteria for classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).

Justification for classification or non-classification

OECD 422 study design. The reproductive and developmental screening test (OECD 422) incorporated with micronuclus screen OECD 474; the test item was considered non-clastogenic and does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP). However, data on mutagenicity is currently inconclusive based on the positive in vitro gene mutation and as such the overall classification on genotoxicity is also considered inconclusive.