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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the requirements of REACh Annex VII the genotoxicity assessment is restricted to an in vitro gene mutation study in bacteria. When tested in vitro, Oct-7-en-1-ol was negative in an Ames test. It was concluded that Oct-7-en-1-ol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 1250 µg/mL (a concentration limited by toxicity), in the absence and presence of a rat liver metabolic activation system (S9) using pre-incubation methodology.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-28 to 2015-10-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Chemical name: Oct-7-en-1-ol (7-OEA)
- CAS no.: 13175-44-5
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / OEA-162736NC
- Expiration date of the lot/batch: not stated
- Molecular weight: 128.213 g/mol
- Purity: 94.61%
Target gene:
see below
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital / 5,6-benzoflavone
Test concentrations with justification for top dose:
Dose range finder: -/+S9: 0, 19.5, 78.1, 313, 1250, 5000 ug/plate
Main study: -/+S9: 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 ug/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
+S9: TA1535, WP2uvrA
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
+S9: TA100, TA98, TA1537
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
-S9: TA100, TA98, WP2uvrA
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
-S9: TA1535
Positive controls:
yes
Positive control substance:
other: ICR-191 (6-Chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride)
Remarks:
-S9: TA1537
Details on test system and experimental conditions:
The pre-incubation methodology was used for the dose range finder and main tests 1 and 2. Duplicate plates were used for the dose range finder and triplicate plates were used for the main tests.

Test tubes containing 0.1 mL of vehicle, positive control or test article formulation were mixed with 0.5 mL S9 mix (or PBS in the absence of metabolic activation).. 0.1 mL fresh bacterial culture was added and the mixture was incubated while shaking at 37°C for 20 minutes.

After pre-incubation, 2 mL of top agar (kept at 45°C) was added to each tube and this mixture was shaken and overlaid onto Vogel-Bonner agar plates (minimal glucose agar plates). E.coli containing tubes were poured onto minimal agar plates.

Agar plates were incubated at 37°C for 48 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.

The background lawns of the plates were examined for signs of toxicity. Other toxicity indicators that may have been noted included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.
Evaluation criteria:
The test chemical was considered positive in this assay if the following criteria were met:
- dose-related and reproducible increase in the number of revertant colonies (i.e. doubling of the spontaneous mutation rate in at least one tester strain either –S9 or +S9)

A test substance wass generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
Statistics not warranted
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

A. Dose range finder:

No precipitation was observed at any dose level, - or + S9. Growth inhibition (evaluation by bacterial background lawn) was observed at 313 ug/plate and above for strains TA1535 and TA1537 in both the absence and presence of S9 and at 1250 ug/plate and above for strains TA98, TA100 and WP2uvrA in both the absence and presence of S9. Oct-7-en-1-ol was deemed not mutagenic in the dose range finder experiment following a pre-incubation methodology. For main mutation tests 1 and 2, as growth inhibition was the limiting factor the maximum dose level was set at 313 ug/plate for TA1535 and TA1537 (-/+ S9) and at 1250 ug/plate for TA98, TA100 and WP2uvrA (-/+S9).

 

Table 7.6.1/01-1:
Bacterial (reverse) gene mutation pre-incubation data – Dose range finder

Conc. (ug/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

97

117

12

7

14

25

13

30

10

7

19.5

112

113

8

12

17

25

14

25

6

10

78.1

107

111

8

5

19

17

13

24

5

9

313

87

115

6B

7

19

24

10

16

5B

11

1250

0B

0B

0B

0B

0B

0B

0B

0B

0B

0B

5000

0B

0B

0B

0B

0B

0B

0B

0B

0B

0B

+ve

591

777

254

204

55

643

298

321

990

82

B: reduced background lawn

+ve controls:

-S9 (absence of metabolic activation):

TA100, WP2uvrA, TA98: furylfuramide

TA1535: sodium azide

TA1537: ICR-191

+S9 (presence of metabolic activation):

TA100, TA98, TA1537: Benzo[a]pyrene

TA1537, WP2urvA: 2-aminoanthracene

B. Main mutation tests

No precipitation was observed at any dose level, - or + S9. Growth inhibition (evaluation by bacterial background lawn) was observed at 313 ug/plate and above for strains TA1535 and TA1537 in both the absence and presence of S9 and at 625 ug/plate and above for strains TA98, TA100 and WP2uvrA in both the absence and presence of S9. Oct-7-en-1-ol was deemed not mutagenic in the dose range finder experiment following a pre-incubation methodology. For main mutation tests 1 and 2, as growth inhibition was the limiting factor the maximum dose level was set at 313 ug/plate for TA1535 and TA1537 (-/+ S9) and at 1250 ug/plate for TA98, TA100 and WP2uvrA (-/+S9).

 

The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system.

 

Following Oct-7-en-1ol treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control in two independent experiments. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system (refer to Table 7.6.1/01-2, -3).

 

Table 7.6.1/01-2:
Bacterial (reverse) gene mutation pre-incubation data – Main mutation experiment 1

Conc. (ug/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

100

114

11

8

18

19

19

33

8

10

9.77

-

-

11

8

-

-

-

-

9

10

19.5

-

-

11

7

-

-

-

-

7

9

39.1

84

120

9

9

16

19

14

29

7

8

78.1

107

113

9

8

14

19

17

28

5

7

156

103

111

9

5

16

19

14

30

5

6

313

95

114

6B

5B

15

12

15

26

5B

7B

625

0B

66B

-

-

0B

11B

0B

0B

-

-

1250

0B

0B

-

-

0B

0B

0B

0B

-

-

+ve

565

825

209

230

64

504

314

330

948

77

B: reduced background lawn

+ve controls:

-S9 (absence of metabolic activation):

TA100, WP2uvrA, TA98: furylfuramide

TA1535: sodium azide

TA1537: ICR-191

+S9 (presence of metabolic activation):

TA100, TA98, TA1537: Benzo[a]pyrene

TA1537, WP2urvA: 2-aminoanthracene

 Table 7.6.1/01-3:
Bacterial (reverse) gene mutation pre-incubation data – Main mutation experiment 2

Conc. (ug/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

99

127

9

10

28

30

15

31

9

12

9.77

-

-

9

9

-

-

-

-

9

10

19.5

-

-

9

7

-

-

-

-

6

10

39.1

101

114

9

7

28

28

20

27

7

13

78.1

100

129

9

11

32

27

14

26

7

15

156

81

125

9

11

31

27

14

29

4

8

313

82

109

6B

12B

31

33

14

29

6B

10B

625

0B

65B

-

-

0B

14B

0B

17B

-

-

1250

0B

0B

-

-

0B

0

0B

0

-

-

+ve

476

803

222

255

70

713

286

351

792

102

B: reduced background lawn

+ve controls:

-S9 (absence of metabolic activation):

TA100, WP2uvrA, TA98: furylfuramide

TA1535: sodium azide

TA1537: ICR-191

+S9 (presence of metabolic activation):

TA100, TA98, TA1537: Benzo[a]pyrene

TA1537, WP2urvA: 2-aminoanthracene

Table 7.6.1/01-4: Bacterial (reverse) gene mutation data – Historical vehicle and positive control data

Conc. (ug/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Vehicle control

Mean ±SD

99

±16.3

126

±16.2

10

±3.02

10

±3.12

18

±4.29

21

±5.13

16

±3.65

32

±7.28

6

±2.64

10

±3.30

Lower limit

54

387

1

61

7

8

6

13

1

1

Upper limit

143

720

18

424

29

33

25

50

14

19

Positive control

Mean ±SD

554

±59.5

923

±108

243

±62.9

277

±70.2

62

±8.8

841

±121

374

±50

376

±43.6

1026

±143

95

±13.7

Lower limit

81

646

61

69

40

538

240

263

657

61

Upper limit

646

1200

424

485

84

1144

508

489

1396

130

-S9 (absence of metabolic activation):

TA100, WP2uvrA, TA98: furylfuramide

TA1535: sodium azide

TA1537: ICR-191

+S9 (presence of metabolic activation):

TA100, TA98, TA1537: Benzo[a]pyrene

TA1537, WP2urvA: 2-aminoanthracene

C. Deficiencies:

None.

Conclusions:
It was concluded that Oct-7-en-1-ol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 1250 µg/mL (a concentration limited by toxicity), in the absence and presence of a rat liver metabolic activation system (S9) using pre-incubation methodology.
Executive summary:

In a reverse gene mutation assay in bacteria, S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA were exposed to Oct-7-en-1-ol formulated in dimethyl sulphoxide. The pre-incubation methodology was used. Following a dose range-finder experiment, two main mutation experiments were conducted using the same dose levels. For main mutation tests 1 and 2, as growth inhibition was the limiting factor the maximum dose level was set at 313 ug/plate for TA1535 and TA1537 (-/+ S9) and at 1250 ug/plate for TA98, TA100 and WP2uvrA (-/+S9). In the main mutation tests treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of Oct-7-en-1-ol at 39.1, 78.1, 156, 313, 625, 1250 µg/plate for strains TA100, WP2uvrA and TA98 and at 9.77, 19.5, 39.5, 78.1, 156, 313 ug/plate for strains TA1535 and TA1537. No precipitation was observed at any dose level, - or + S9. Growth inhibition (evaluation by bacterial background lawn) was observed at 313 ug/plate and above for strains TA1535 and TA1537 in both the absence and presence of S9 and at 625 ug/plate and above for strains TA98, TA100 and WP2uvrA in both the absence and presence of S9. The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system. Following Oct-7-en-1-ol treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system. It was concluded that Oct-7-en-1-ol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 1250 µg/mL (a concentration limited by toxicity), in the absence and presence of a rat liver metabolic activation system (S9) using pre-incubation methodology.

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

Genetic toxicity in vivo

Description of key information

Under the requirements of REACh Annex VII the genotoxicity assessment is restricted to an in vitro gene mutation study in bacteria.

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Under the requirements of REACh Annex VII the genotoxicity assessment is restricted to an in vitro gene mutation study in bacteria. When tested in vitro, Oct-7-en-1-ol was negative in an Ames test.

Additional information

Justification for classification or non-classification

Comparison with the CLP criteria

There was no indication that Oct-7-en-1-ol has a mutagenic effect in the in vitro bacterial (reverse) gene mutation assay. However, no data were available with respect to the germ cell mutation potential of Oct-7 -en-1 -ol in human cells. Therefore, criteria for classification for germ cell mutagenicity could not be met based on data lacking.

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