Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data from three in vitro assays (reverse gene mutation assay in bacteria, in vitro micronucleus test and mammalian cell gene mutation assay) show that the substance does not have a genotoxic potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 October 2009 to 13 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: These mutant strains of Salmonella are incapable of synthesising histidine
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Escherichia coli (WP2uvrA-) requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence. This strain also has a deletion in an excision repair gene.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ ß­naphthoflavone induced rate liver S9
Test concentrations with justification for top dose:
Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was expanded to 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
Additional dose levels (1.5, 5 and 15 µg/plate) and an expanded dose range were selected for Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test material.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Other positive controls were: 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA) and Benzo(a)pyrene
Details on test system and experimental conditions:
See below
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
In Experiment 1, the test material did not cause a visible reduction in the growth of the bacterial background lawn at any dose level. However, in the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on the Experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 17 October 2009

To: 20 October 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

117

128

125

(123)

5.7#

30

28

30

(29)

1.2

19

27

37

(28)

9.0

22

25

16

(21)

4.6

10

10

20

(13)

5.8

-

50

117

120

132

(123)

7.9

35

27

31

(31)

4.0

21

24

24

(23)

1.7

25

21

24

(23)

2.1

10

16

15

(14)

3.2

-

150

106

113

124

(114)

9.1

31

25

29

(28)

3.1

32

29

30

(30)

1.5

25

25

12

(21)

7.5

14

12

21

(16)

4.7

-

500

134

108

117

(120)

13.2

24

20

24

(23)

2.3

23

22

18

(21)

2.6

23

29

16

(23)

6.5

10

15

10

(12)

2.9

-

1500

102

106

142

(117)

22.0

20

33

28

(27)

6.6

20

21

21

(21)

0.6

19

19

20

(19)

0.6

13

8

8

(10)

2.9

-

5000

126

124

117

(122)

4.7

26

28

29

(28)

1.5

15

13

16

(15)

1.5

20

19

21

(20)

1.0

10

9

11

(10)

1.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(µg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

675

662

705

(681)

22.1

574

526

471

(524)

51.5

867

883

907

(886)

20.1

164

168

150

(161)

9.5

866

398

617

(627)

234.2

 

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 17 October 2009

To: 20 October 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

121

139

123

(128)

9.9#

16

22

15

(18)

3.8

35

24

27

(29)

5.7

27

29

32

(29)

2.5

12

18

16

(15)

3.1

+

50

118

121

102

(114)

10.2

21

16

13

(17)

4.0

25

31

21

(26)

5.0

26

23

23

(24)

1.7

15

18

16

(16)

1.5

+

150

92

122

114

(109)

15.5

22

18

13

(18)

4.5

27

29

34

(30)

3.6

26

26

23

(25)

1.7

19

15

13

(16)

3.1

+

500

101

98

107

(102)

4.6

18

24

21

(21)

3.0

29

26

21

(25)

4.0

26

26

23

(25)

1.7

9

12

10

(10)

1.5

+

1500

87

86

106

(93)

11.3

20

15

15

(17)

2.9

16

24

29

(23)

6.6

25

20

27

(24)

3.6

13

15

11

(13)

2.0

+

5000

97

107

87

(97)

10.0

8

15

14

(12)

3.8

15

23

16

(18)

4.4

20

18

20

(19)

1.2

15

10

7

(11)

4.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(µg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1344

2044

1499

(1629)

367.7

139

198

197

(178)

33.8

265

282

249

(265)

16.5

192

197

199

(196)

3.6

301

407

448

(385)

75.9

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Experiment 2 – Without Metabolic Activation

Test Period

From: 10 November 2009

To: 13 November 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

106

121

119

(115)

8.1#

20

23

27

(23)

3.5

23

26

23

(24)

1.7

22

19

19

(20)

1.7

14

13

12

(13)

1.0

-

1.5

101

121

95

(106)

13.6

21

21

22

(21)

0.6

24

23

26

(24)

1.5

20

20

20

(20)

0.0

15

16

12

(14)

2.1

-

5

119

98

136

(118)

19.0

22

27

23

(24)

2.6

25

24

27

(25)

1.5

24

19

20

(21)

2.6

11

13

14

(13)

1.5

-

15

98

102

90

(97)

6.1

24

22

24

(23)

1.2

26

22

26

(25)

2.3

19

24

15

(19)

4.5

11

15

11

(12)

2.3

-

50

99

120

110

(110)

10.5

26

22

18

(22)

4.0

22

20

18

(20)

2.0

23

20

20

(21)

1.7

14

15

13

(14)

1.0

-

150

107

102

106

(105)

2.6

20

26

24

(23)

3.1

27

22

22

(24)

2.9

18

23

20

(20)

2.5

13

15

11

(13)

2.0

-

500

89 S

71 S

85 S

(82)

9.5

20 S

19 S

15 S

(18)

2.6

0 V

0 V

0 V

(0)

0.0

11 S

13 S

3 S

(9)

5.3

10 S

2 S

3 S

(5)

4.4

-

1500

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(µg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

771

739

551

(687)

118.9

267

278

332

(292)

34.8

1027

967

975

(990)

32.6

122

95

122

(113)

15.6

690

338

370

(466)

194.6

 

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 10 November 2009

To: 13 November 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

88

102

91

(94)

7.4#

15

21

18

(18)

3.0

31

36

32

(33)

2.6

24

22

22

(23)

1.2

14

13

15

(14)

1.0

+

1.5

84

82

102

(89)

11.0

15

22

18

(18)

3.5

30

22

30

(27)

4.6

21

23

19

(21)

2.0

15

11

11

(12)

2.3

+

5

101

90

89

(93)

6.7

12

15

12

(13)

1.7

27

34

36

(32)

4.7

20

14

19

(18)

3.2

15

15

10

(13)

2.9

+

15

89

103

101

(98)

7.6

20

14

18

(17)

3.1

33

30

29

(31)

2.1

19

19

21

(20)

1.2

14

13

15

(14)

1.0

+

50

95

87

85

(89)

5.3

14

13

15

(14)

1.0

35

27

30

(31)

4.0

18

19

18

(18)

0.6

16

15

13

(15)

1.5

+

150

99

113

97

(103)

8.7

14

21

19

(18)

3.6

34

25

23

(27)

5.9

21

18

19

(19)

1.5

13

13

16

(14)

1.7

+

500

98

82

90

(90)

8.0

15 S

15 S

14 S

(15)

0.6

21

29

24

(25)

4.0

19

24

22

(22)

2.5

11

15

11

(12)

2.3

+

1500

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(µg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1393

1269

1372

(1345)

66.4

200

205

172

(192)

17.8

503

486

514

(501)

14.1

173

139

123

(145)

25.5

388

374

383

(382)

7.1

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Conclusions:
Interpretation of results :
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The test material was considered to be non-mutagenic under the conditions of this test conducted according to EU Method B13/14.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study start: 25 March 2019; End of experimental phase: 29 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Adopted July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The test item was assayed for the ability to induce micronuclei in human lymphocytes cultured in vitro, after treatment in the absence and presence of S9 metabolism.
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
Two batches of human whole blood for Main Assay I and II and one batch for Main Assay III provided by Biopredic International (France), were used in this study.

Cytokinesis block (if used):
the inhibitor of actin polymerisation: cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue fraction, provided by Trinova Biochem GmbH, from Rats pretrerated with Phenobarbital and 5,6-Benzoflavone
Test concentrations with justification for top dose:


The maximum dose level of 1300 µg/mL corresponding to 10mM and lower dose levels of 867, 578, 385, 257, 171, 114, 76.1 and 50.7 µg/mL were used.
The additional dose level of 33.8 µg/mL was included for the continuous treatment in the absence of S9 metabolism.

Following treatment in the presence of S9 and the continuous treatment in the absence of S9, no adequate toxicity was achieved to select dose levels for scoring micronuclei, therefore treatments were repeated using lower and more closely spaced concentrations.

Dose levels of 550, 500, 455, 413, 376, 342, 311, 282 and 257 µg/mL were used in the presence of S9 metabolism and dose levels of 210, 191, 174, 158, 143, 130, 119, 108, 97.9 and 89.0 µg/mL were used in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.
Vehicle / solvent:
Solutions of the test item were prepared in dimethylsulfoxide (DMSO).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Colchicine 0.080 and 0.040 µg/mL
Details on test system and experimental conditions:
Culture media:
The culture medium for the lymphocytes had the following composition:

RPMI 1640 1x (Dutch modification) 500mL
Foetal Calf Serum 100mL
L-Glutamine (200mM) 6.25mL
Antibiotic solution 1.25mL

The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the following proportion: 10mL of PHA was added to 500mL of medium.

Preparation of the test cultures and treatment:

For each treatment series two replicate cultures were prepared at each test point including negative and positive controls.
Lymphocyte cultures were treated approximately fourty-eight hours after they were initiated.
Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.
For the short treatment series, the composition of the treatment media was as follows:

Presence of S9 metabolism
Test item solution or solvent/vehicle 0.05mL
S9 mix 1.00mL
Culture medium (without PHA) 3.95mL

Absence of S9 metabolism
Test item solution or solvent/vehicle 0.05mL
Culture medium (without PHA) 4.95mL

For the continuous treatment, due to the concurrent addition of Cytochalasin B dissolved in DMSO, it was necessary to reduce the volume of test item solutions (25 µL/tube instead of 50 µL/tube), to maintain the final concentration of organic solvents to 1%.

The composition of the treatment medium was as follows:

Test item solution or solvent/vehicle 0.025mL
Culture medium (without PHA) 4.975mL
The composition of the treatment media for the positive controls was as indicated in the Study Protocol.

For the short treatment series, the treatment mediawere added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.

For the continuous treatment series, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Harvesting and slide preparation

The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed.
The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this waywere dropped onto clean,wet, greasefree glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture.
The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Slide evaluation

The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI =
mononucleated +2×binucleated +3×multinucleated/ total number of cells counted

where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells. CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed. ForMain Assays 1 and 2, since negligible cytotoxcity was observed, the evaluation of CBPI of the lowest dose level was omitted. The highest dose level for genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI.
The percentage cytotoxicity was evaluated according to the following formula:

%Cytotoxicity =100-100 [ CBPIT -1 / CBPIC -1]

where:
T = test item treated culture
C = untreated/solvent control culture
The highest dose level for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50% and 60%. In the absence of cytotoxicity, the highest treatment level is selected as the highest dose level for
scoring.
Two lower dose levels were also selected for the scoring of micronuclei.
For the three selected doses, for the solvent and the positive control Cyclophosphamide, 1000 binucleated cells per cell culture were scored to assess the
frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.

The criteria for identifying micronuclei were as follows:

1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin


Evaluation criteria:
Acceptance criteria:
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

Criterion for outcome:
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the concentrations shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data.
Statistics:
Statistical analysis:
For the statistical analysis, a modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
On the basis of the above results, it is concluded that 2- Octanol does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions
Executive summary:

The test item 2-OCTANOL was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.

In Main Assay 1, cells were exposed to the test item for 3 hours, in the absence and presence of S9 metabolism and sampled at approximately 32 hours, corresponding to about two cell cycle lengths. A long term(continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.

The maximum dose level of 1300 µg/mL corresponding to 10 mM was selected in agreement with the Study Protocol. Lower dose levels of 867, 578, 385, 257, 171, 114, 76.1 and 50.7 µg/mL were used. The additional dose level of 33.8 µg/mL was included for the continuous treatment in the absence of S9 metabolism.

Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

Following treatment in the presence of S9 and the continuous treatment in the absence of S9, no adequate toxicity was achieved to select dose levels for scoring micronuclei, therefore treatments were repeated using lower and more closely spaced concentrations.

Dose levels of 550, 500, 455, 413, 376, 342, 311, 282 and 257 µg/mL were used in the presence of S9 metabolism and dose levels of 210, 191, 174, 158, 143, 130, 119, 108, 97.9 and 89.0 µg/mL were used in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.

The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from moderate to slight or no toxicity. Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

S9    Treatment time (hours)  Harvest time (hours)  Concentration (µg/mL)
 -  3 32  385, 257 and 171
 +  3 32  455, 413 and 376
- 31

31

  

158, 119 and 89.0

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control values and no concentration related increase was seen.

It is concluded that 2-OCTANOL does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2019 - October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes
Type of assay:
other: gene mutation in mammalian cells
Target gene:
The mutation assay method used in this study is based on the identification of L5178Y colonies
which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT).
This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides,
which are used in nucleic acid synthesis resulting in the death of TK-competent cells.
TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot
metabolise trifluorothymidine and thus survive and grow in its presence.
In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on
chromosome 11. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single
step forward mutation to the TK-/- genotype in which little or no TK activity remains.
The cells used, L5178Y TK+/-, are derived from one of the two clones originated from a thymic
tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation
system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive
et al., 1979) and is accepted by most of the regulatory authorities.
The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant
colonies designated as small or large. It has been evaluated that point mutations and deletions
within the active allele (intragenic event) produce large colonies. Small colonies result
in part from lesions that affect not only the active TK allele but also a flanking gene whose
expression modulates the growth rate of cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Numbers: 3512 and 3488
Test concentrations with justification for top dose:
A preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation. The test item was assayed at the maximum dose level of 1300 µg/mL, corresponding to 10mM, and at the lower dose levels of 650, 325, 163, 81.3, 40.6, 20.3, 10.2 and 5.08 µg/mL.

Based on the results obtained in the preliminary trial, a firstMain Assay for mutation at the TK locus was performed using the dose levels described below:
Main Assay 1 (+/-S9, 3 hour treatment): 300, 240, 192, 154, 102, 68.3 and 45.5 µg/mL.

Two experiments were performedand interrupted at the end or during the expression period since the total suspension growth (TSG) of the negative control cultures was not acceptable. Main Assay 4 was performed using the following dose levels: 240, 160, 107, 53.3, 26.7, 13.3 and 6.67 µg/mL.
Main Assay 5 was interrupted because very low number of cells was recovered in the negative control cultures.
Main Assay 6 was performed using the following concentration range 240, 209, 181, 158, 137 and 119 µg/mL.
Vehicle / solvent:
Test item solutions were prepared using dimethylsulfoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
Cytotoxicity assay
In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation.
A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8
cells/mL using complete medium (20%) and, for each dose level, 0.2 mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye and couted.

Mutation assay
Treatment of cell cultures
The mutation assayswere performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, additional experiments in the absence of S9
metabolism were performed, using a longer treatment time (24 hours).
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Expression period: two days after treatment
During the expression period the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10^5 cells/mL.
Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 × 10^3 cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 13 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.
Plating for viability
After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 13 days and wells containing clones were identified and counted.
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

Mutation results:
No statistically significant increase in mutant frequency was observed at any dose level and no concentration related response was seen in any treatment series, in the absence or presence of S9 metabolic activation. Despite the problems encountered in conducting the long treatment series, the overall results indicated that the test item can be concluded to be negative since no significant increase in mutation frequency was observed at concentrations inducing toxicity from
slight to mild (Main 4) and there was a negative data point below 10% RTG in Main Assay 6.

see Final Report

Conclusions:
It is concluded that 2-OCTANOL does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item 2-OCTANOLwas examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

The test item was found to be soluble in dimethylsulfoxide (DMSO).

A preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation. The test item was assayed at the maximum dose level of 1300 µg/mL, corresponding to 10mM, and at the lower dose levels of 650, 325, 163, 81.3, 40.6, 20.3, 10.2 and 5.08 µg/mL. Using the 3 hour treatment time, no cell survived treatment at the three highest dose levels in the absence or presence of S9 metabolic activation. At the next lower concentration of 163 µg/mL, 2-OCTANOL treatment yielded mild toxicity reducing relative survival (RS) to 57% and 51% of the concurrent solvent/vehicle control values in the absence and presence of S9 metabolism, respectively. After 24 hour treatment, no cell survived from 325 µg/mL onwards, while moderate toxicity was noticed at the next lower concentration of 163 µg/mL (RS=27%). Based on the results obtained in the preliminary trial, a firstMain Assay for mutation at the TK locus was performed using the dose levels described below:

Main Assay 1 (3 hours +/- S9): 300, 240, 192, 154, 102, 68.3 and 45.5 µg/mL.

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in both treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. As negative resultswere obtained, a mutation assay using a longer treatment time (24 hours) was performed. Since an interaction between the test item and the polystyrene tubes/flasks (slight opacity) was observed at 200 µL/mL during the preliminary solubility test, treated cell cultures were kept in falcon tubes (polypropylene) for the whole treatment period (24 hours). In this conditions the growth at the end of treatment could be slightly lower, nevertheless the

recovery during the period of phenotype expression is generally very good. Twoexperimentswere performedand interrupted at the end or during the expression period since the total suspension growth (TSG) of the negative control cultures was not acceptable. This unexpected result was attributed both to the different experimental conditions and to the possibility that the test item has spread inside the incubator due to its phisico-chemical properties.

An additional experiment (Main Assay 4) was performed using two different cell culture incubators, one for negative and positive control cultures, the other for test item treated cultures by keeping those at different concentrations adequately separated. The test item was assayed at the following dose levels: 240, 160, 107, 53.3, 26.7, 13.3 and 6.67 µg/mL. No cell survived treatment at the highest dose level, while mild toxicity was noted at the next lower concentration of 160 µg/mL (Relative Total Growth = 48%). No relevant toxicity was seen over the remaining dose levels. No increase in mutation frequency was observed at any analysable concentration. However, the TSG of the negative control was slightly

lower than the acceptable value and the test item did not induce an adequate level of cytotoxicity (RTG=10-20%). Based on these results an additional mutation assay (Main Assay 5) was performed using the modified procedures and a narrowed concentration range to investigate more closely those doses of 2-OCTANOL most likely to exhibit an adequate toxicity. At the end of treatment a very low number of cells was recovered in the negative control cultures, thus the experiment was interrupted.

Based on the overall results obtained using the long treatment, it was decided to carried out an additional experiment (Main Assay 6) using the selected concentration range (240, 209, 181, 158, 137 and 119 µg/mL) and employing standard treatment conditions since the selected top dose was significantly lower than the concentration showing interaction with polystyrene tubes. No cell survived treatment at the three highest concentrations; severe toxicity was noted at the remaining dose levels, reducing RTG to 5% of the concurrent negative control at 119 µg/mL. At this concentration no relevant increase in mutation frequency was noted.

Despite the problems encountered in conducting the long treatment series, the overall results indicated that the test item can be concluded to be negative since no significant increase in mutation frequency was observed at concentrations inducing toxicity from slight to mild (Main 4) and there was a negative data point below 10% RTG in Main Assay 6.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. In Main Assay 1, 4 and 6 the mutant frequencies in the solvent control cultures fell within the normal range and marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

Based on the results obtained, it is concluded that 2-OCTANOL does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reverse gene mutation assay

In a bacterial reverse mutation assay (OECD guideline 471), octan-2-ol was tested in a GLP study in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and E. Coli WP2 uvr A both in the presence and absence of exogenous metabolic activation (phenobarbitone/ ß­naphthoflavone induced rate liver S9) (Bowles, 2009). The experiment was conducted in triplicate.

In a first experiment, five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was expanded to 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate. Additional dose levels (1.5, 5 and 15 µg/plate) and an expanded dose range were selected for Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

In a preliminary Toxicity Test, the test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

In the mutation Test, prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

Results for the negative controls (spontaneous mutation rates) were considered acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

In Experiment 1, the test material did not cause a visible reduction in the growth of the bacterial background lawn at any dose level. However, in the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on the Experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

In vitro micronucleus test

In an vitro micronucleus test performed according to the OECD test guideline NO 487 and in compliance with Good Laboratory Practice, the test substance was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.

In Main Assay 1, cells were exposed to the test item for 3 hours, in the absence and presence of S9 metabolism and sampled at approximately 32 hours, corresponding to about two cell cycle lengths. A long term(continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.

The maximum dose level of 1300 µg/mL corresponding to 10 mM was selected in agreement with the Study Protocol. Lower dose levels of 867, 578, 385, 257, 171, 114, 76.1 and 50.7 µg/mL were used. The additional dose level of 33.8 µg/mL was included for the continuous treatment in the absence of S9 metabolism.

Following treatment in the presence of S9 and the continuous treatment in the absence of S9, no adequate toxicity was achieved to select dose levels for scoring micronuclei, therefore treatments were repeated using lower and more closely spaced concentrations.

Dose levels of 550, 500, 455, 413, 376, 342, 311, 282 and 257 µg/mL were used in the presence of S9 metabolism and dose levels of 210, 191, 174, 158, 143, 130, 119, 108, 97.9 and 89.0 µg/mL were used in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.

The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from moderate to slight or no toxicity. Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

S9

Treatment time

(hours)

Harvest time

(hours)

 

Dose level

(µg/mL)

 

Cytotoxicity

(%)

 

-

3

32

385, 257 and 171

53, 19 and 12

+

455, 413 and 376

48, 19 and 0

-

31

31

158, 119 and 89.0

56, 44 and 16

 

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating

the correct functioning of the test system. The study was accepted as valid.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control values and no concentration related increase was seen.

It is concluded that 2-OCTANOL does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

mammalian cell gene mutation assay

The test item 2-OCTANOL was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The test item was found to be soluble in dimethylsulfoxide (DMSO).

A preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation. The test item was assayed at the maximum dose level of 1300µg/mL, corresponding to 10mM, and at the lower dose levels of 650, 325, 163, 81.3, 40.6, 20.3, 10.2 and 5.08µg/mL.

Using the 3 hour treatment time, no cell survived treatment at the three highest dose levels in the absence or presence of S9 metabolic activation. At the next lower concentration of 163µg/mL, 2-OCTANOL treatment yielded mild toxicity reducing relative survival (RS) to 57% and 51% of the concurrent solvent/vehicle control values in the absence and presence of S9 metabolism, respectively. After 24 hour treatment, no cell survived from 325µg/mL onwards, while moderate toxicity was noticed at the next lower concentration of 163µg/mL (RS=27%).

Based on the results obtained in the preliminary trial, a first Main Assay for mutation at the TK locus was performed using the dose levels described in the following table:

Assay

Assay

No.:

S9

Treatment

time (hours)

Dose level

(µg/mL)

1

-

3

300, 240, 192, 154, 102, 68.3 and 45.5

1

+

3

 

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity,were observed in both treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.

As negative results were obtained, a mutation assay using a longer treatment time (24 hours) was performed. Since an interaction between the test item and the polystyrene tubes/flasks (slight opacity) was observed at 200µL/mL during the preliminary solubility test, treated cell cultures were kept in falcon tubes (polypropylene) for the whole treatment period (24 hours). In this condition, the growth at the end of treatment could be slightly lower nevertheless, the recovery during the period of phenotype expression is generally very good.

Two experiments were performed and interrupted at the end or during the expression period since the total suspension growth (TSG) of the negative control cultures was not acceptable.

This unexpected result was attributed both to the different experimental conditions and to the possibility that the test item has spread inside the incubator due to its physico-chemical properties.

An additional experiment (Main Assay 4) was performed using two different cell culture incubators, one for negative and positive control cultures, the other for test item treated cultures by keeping those at different concentrations adequately separated. The test item was assayed at the following dose levels: 240, 160, 107, 53.3, 26.7, 13.3 and 6.67µg/mL.

No cell survived treatment at the highest dose level, while mild toxicity was noted at the next lower concentration of 160µg/mL (Relative Total Growth=48%). No relevant toxicity was seen over the remaining dose levels. No increase in mutation frequency was observed at any analysable concentration. However, the TSG of the negative control was slightly lower than the acceptable value and the test item did not induce an adequate level of cytotoxicity (RTG=10-20%). Based on these results, an additional mutation assay (Main Assay 5) was performed using the modified procedures and a narrowed concentration range to investigate more closely those doses of 2-OCTANOL most likely to exhibit an adequate toxicity. At the end of treatment a very low number of cells was recovered in the negative control cultures, thus the experiment was interrupted.

Based on the overall results obtained using the long treatment, it was decided to carried out an additional experiment (Main Assay 6) using the selected concentration range (240,209, 181, 158, 137 and 119µg/mL) and employing standard treatment conditions since the selected top dose was significantly lower than the concentration showing interaction with polystyrene tubes. No cell survived treatment at the three highest concentrations; severe toxicity was noted at the remaining dose levels, reducing RTG to 5% of the concurrent negative control at 119µg/mL. At this concentration no relevant increase in mutation frequency was noted.

Despite the problems encountered in conducting the long treatment series, the overall results indicated that the test item can be concluded to be negative since no significant increase in mutation frequency was observed at concentrations inducing toxicity from slight to mild (Main IV) and there was a negative data point below 10% RTG in Main Assay VI.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. InMain Assay I, IV and VI the mutant frequencies in the solvent control cultures fell within the normal range and marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

Based on the results obtained, it is concluded that 2-OCTANOL does not induce mutation at the TK locus of L5178Y mouse lymphoma cellsin vitroin the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Justification for classification or non-classification

According to the available data and CLP criteria, no classification is warranted for the genetic toxicity.