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Genetic toxicity in vitro

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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:
22 January 2009 and 10 February 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
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:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Remarks:
2-Aminoanthracene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None
Evaluation criteria:
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 109 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 not be an excessive loss of plates due to contamination.

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:
Standard deviation
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9- mix.



RANGE-FINDING/SCREENING STUDIES:
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.


COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

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.


ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 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. Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

95

 

20

 

32

 

23

 

13

 

98

(99)

29

(24)

30

(29)

29

(29)

10

(12)

103

 

24

 

26

 

36

 

12

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

86

 

25

 

18

 

23

 

10

 

118

(104)

32

(25)

30

(26)

24

(24)

13

(12)

109

 

19

 

29

 

25

 

13

 

Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 01 February 2009

To: 04 February 2009

With out 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

101

103

88

(97)

8.1#

22

22

25

(23)

1.7

26

24

29

(26)

2.5

20

16

15

(17)

2.6

12

9

9

(10)

1.7

-

50

86

89

87

(87)

1.5

19

23

22

(21)

2.1

22

22

31

(25)

5.2

18

14

16

(16)

2.0

9

14

8

(10)

3.2

-

150

89

91

102

(94)

7.0

16

25

26

(22)

5.5

25

23

30

(26)

3.6

18

18

15

(17)

1.7

10

8

7

(8)

1.5

-

500

106

95

90

(97)

8.2

25

20

27

(24)

3.6

25

23

26

(25)

1.5

16

15

18

(16)

1.5

10

10

9

(10)

0.6

-

1500

90

101

91

(94)

6.1

25

18

19

(21)

3.8

30

21

32

(28)

5.9

18

19

18

(18)

0.6

7

10

10

(9)

1.7

-

5000

79

81

81

(80)

1.2

21

18

21

(20)

1.7

30

32

26

(29)

3.1

16

16

20

(17)

2.3

13

8

9

(10)

2.6

Positive controls S9-Mix

-

Name Concentration (μg/plate)

No. colonies per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

670

759

826

(752)

78.3

658

467

395

(507)

135.9

384

352

348

(361)

19.7

198

246

192

(212)

29.6

521

531

531

(528)

5.8

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: 01 February 2009

To: 04 February 2009

With 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

96

100

(101)

5.0#

13

14

10

(12)

2.1

35

35

31

(34)

2.3

33

22

30

(28)

5.7

9

9

9

(9)

0.0

+

50

99

89

98

(95)

5.5

9

12

15

(12)

3.0

34

30

34

(33)

2.3

33

32

27

(31)

3.2

11

10

7

(9)

2.1

+

150

84

90

99

(91)

7.5

7

9

11

(9)

2.0

34

33

34

(34)

0.6

30

21

32

(28)

5.9

9

8

8

(8)

0.6

+

500

89

88

101

(93)

7.2

14

15

10

(13)

2.6

34

31

32

(32)

1.5

21

36

21

(26)

8.7

10

8

7

(8)

1.5

+

1500

85

95

97

(92)

6.4

11

10

11

(11)

0.6

32

33

34

(33)

1.0

25

33

22

(27)

5.7

8

8

10

(9)

1.2

+

5000

89

98

86

(91)

6.2

10

11

12

(11)

1.0

36

33

34

(34)

1.5

23

33

31

(29)

5.3

8

9

8

(8)

0.6

Positive controls S9-Mix

+

Name Concentration (μg/plate)

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2779

2359

2834

(2657)

259.8

181

206

217

(201)

1.8.4

1720

1652

1646

(1673)

41.1

225

241

267

(244)

21.2

523

511

563

(532)

27.2

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

 

Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 07 February 2009

To: 10 February 2009

With out 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

120

103

104

(109)

9.5#

27

34

31

(31)

3.5

33

33

31

(32)

1.2

18

22

22

(21)

2.3

12

12

14

(13)

1.2

-

50

119

104

103

(109)

9.0

27

31

26

(28)

2.6

31

36

31

(33)

2.9

20

24

20

(21)

2.3

10

11

12

(11)

1.0

-

150

117

118

84

(106)

19.3

21

37

29

(29)

8.0

24

26

25

(25)

1.0

16

12

19

(16)

3.5

10

12

10

(11)

1.2

-

500

101

101

99

(100)

1.2

23

30

31

(28)

4.4

29

27

29

(28)

1.2

23

19

15

(19)

4.0

10

11

11

(11)

0.6

-

1500

128

109

110

(116)

10.7

30

24

27

(27)

3.0

35

29

33

(32)

3.1

19

23

18

(20)

2.6

14

10

9

(11)

2.6

-

5000

115

121

90

(109)

16.4

30

26

25

(27)

2.6

34

32

34

(33)

1.2

19

21

22

(21)

1.5

10

12

14

(12)

2.0

Positive controls S9-Mix

-

Name Concentration (μg/plate)

No. colonies per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

1190

1279

1220

(1230)

45.3

1771

1697

1518

(1662)

130.1

2145

2228

2073

(2149)

77.6

213

233

207

(218)

13.6

1226

1215

1227

(1223)

6.7

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 07 February 2009

To: 10 February 2009

With 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

99

97

99

(98)

1.2#

10

14

9

(11)

2.6

41

40

34

(38)

3.8

30

30

30

(30)

0.0

14

16

15

(15)

1.0

+

50

92

84

91

(89)

4.4

12

8

15

(12)

3.5

35

41

34

(37)

3.8

24

24

31

(26)

4.0

15

16

14

(15)

1.0

+

150

88

93

95

(92)

3.6

9

11

13

(11)

2.0

40

46

30

(39)

8.1

30

24

31

(28)

3.8

15

12

15

(14)

1.7

+

500

93

92

97

(94)

2.6

11

12

9

(11)

1.5

42

37

43

(41)

3.2

24

31

24

(26)

4.0

10

10

12

(11)

1.2

+

1500

102

93

95

(97)

4.7

9

16

11

(12)

3.6

35

40

36

(37)

2.6

22

26

35

(28)

6.7

11

12

11

(11)

0.6

+

5000

91

102

96

(96)

5.5

14

11

13

(13)

1.5

38

44

35

(39)

4.6

27

25

25

(26)

1.2

12

10

10

(11)

1.2

Positive controls S9-Mix

+

Name Concentration (μg/plate)

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2481

3433

3619

(3178)

610.5

184

273

245

(234)

45.5

468

622

552

(547)

77.1

145

227

216

(196)

44.5

328

588

552

(489)

140.9

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Conclusions:
Interpretation of results (migrated information):
negative

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

Introduction. Thethod conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrA-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 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.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.
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:
22 January 2009 and 10 February 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
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:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Remarks:
2-Aminoanthracene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None
Evaluation criteria:
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 109 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 not be an excessive loss of plates due to contamination.

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:
Standard deviation
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9- mix.



RANGE-FINDING/SCREENING STUDIES:
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.


COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

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.


ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 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. Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

95

 

20

 

32

 

23

 

13

 

98

(99)

29

(24)

30

(29)

29

(29)

10

(12)

103

 

24

 

26

 

36

 

12

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

86

 

25

 

18

 

23

 

10

 

118

(104)

32

(25)

30

(26)

24

(24)

13

(12)

109

 

19

 

29

 

25

 

13

 

Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 01 February 2009

To: 04 February 2009

With out 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

101

103

88

(97)

8.1#

22

22

25

(23)

1.7

26

24

29

(26)

2.5

20

16

15

(17)

2.6

12

9

9

(10)

1.7

-

50

86

89

87

(87)

1.5

19

23

22

(21)

2.1

22

22

31

(25)

5.2

18

14

16

(16)

2.0

9

14

8

(10)

3.2

-

150

89

91

102

(94)

7.0

16

25

26

(22)

5.5

25

23

30

(26)

3.6

18

18

15

(17)

1.7

10

8

7

(8)

1.5

-

500

106

95

90

(97)

8.2

25

20

27

(24)

3.6

25

23

26

(25)

1.5

16

15

18

(16)

1.5

10

10

9

(10)

0.6

-

1500

90

101

91

(94)

6.1

25

18

19

(21)

3.8

30

21

32

(28)

5.9

18

19

18

(18)

0.6

7

10

10

(9)

1.7

-

5000

79

81

81

(80)

1.2

21

18

21

(20)

1.7

30

32

26

(29)

3.1

16

16

20

(17)

2.3

13

8

9

(10)

2.6

Positive controls S9-Mix

-

Name Concentration (μg/plate)

No. colonies per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

670

759

826

(752)

78.3

658

467

395

(507)

135.9

384

352

348

(361)

19.7

198

246

192

(212)

29.6

521

531

531

(528)

5.8

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: 01 February 2009

To: 04 February 2009

With 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

96

100

(101)

5.0#

13

14

10

(12)

2.1

35

35

31

(34)

2.3

33

22

30

(28)

5.7

9

9

9

(9)

0.0

+

50

99

89

98

(95)

5.5

9

12

15

(12)

3.0

34

30

34

(33)

2.3

33

32

27

(31)

3.2

11

10

7

(9)

2.1

+

150

84

90

99

(91)

7.5

7

9

11

(9)

2.0

34

33

34

(34)

0.6

30

21

32

(28)

5.9

9

8

8

(8)

0.6

+

500

89

88

101

(93)

7.2

14

15

10

(13)

2.6

34

31

32

(32)

1.5

21

36

21

(26)

8.7

10

8

7

(8)

1.5

+

1500

85

95

97

(92)

6.4

11

10

11

(11)

0.6

32

33

34

(33)

1.0

25

33

22

(27)

5.7

8

8

10

(9)

1.2

+

5000

89

98

86

(91)

6.2

10

11

12

(11)

1.0

36

33

34

(34)

1.5

23

33

31

(29)

5.3

8

9

8

(8)

0.6

Positive controls S9-Mix

+

Name Concentration (μg/plate)

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2779

2359

2834

(2657)

259.8

181

206

217

(201)

1.8.4

1720

1652

1646

(1673)

41.1

225

241

267

(244)

21.2

523

511

563

(532)

27.2

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

 

Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 07 February 2009

To: 10 February 2009

With out 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

120

103

104

(109)

9.5#

27

34

31

(31)

3.5

33

33

31

(32)

1.2

18

22

22

(21)

2.3

12

12

14

(13)

1.2

-

50

119

104

103

(109)

9.0

27

31

26

(28)

2.6

31

36

31

(33)

2.9

20

24

20

(21)

2.3

10

11

12

(11)

1.0

-

150

117

118

84

(106)

19.3

21

37

29

(29)

8.0

24

26

25

(25)

1.0

16

12

19

(16)

3.5

10

12

10

(11)

1.2

-

500

101

101

99

(100)

1.2

23

30

31

(28)

4.4

29

27

29

(28)

1.2

23

19

15

(19)

4.0

10

11

11

(11)

0.6

-

1500

128

109

110

(116)

10.7

30

24

27

(27)

3.0

35

29

33

(32)

3.1

19

23

18

(20)

2.6

14

10

9

(11)

2.6

-

5000

115

121

90

(109)

16.4

30

26

25

(27)

2.6

34

32

34

(33)

1.2

19

21

22

(21)

1.5

10

12

14

(12)

2.0

Positive controls S9-Mix

-

Name Concentration (μg/plate)

No. colonies per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

1190

1279

1220

(1230)

45.3

1771

1697

1518

(1662)

130.1

2145

2228

2073

(2149)

77.6

213

233

207

(218)

13.6

1226

1215

1227

(1223)

6.7

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 07 February 2009

To: 10 February 2009

With 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

99

97

99

(98)

1.2#

10

14

9

(11)

2.6

41

40

34

(38)

3.8

30

30

30

(30)

0.0

14

16

15

(15)

1.0

+

50

92

84

91

(89)

4.4

12

8

15

(12)

3.5

35

41

34

(37)

3.8

24

24

31

(26)

4.0

15

16

14

(15)

1.0

+

150

88

93

95

(92)

3.6

9

11

13

(11)

2.0

40

46

30

(39)

8.1

30

24

31

(28)

3.8

15

12

15

(14)

1.7

+

500

93

92

97

(94)

2.6

11

12

9

(11)

1.5

42

37

43

(41)

3.2

24

31

24

(26)

4.0

10

10

12

(11)

1.2

+

1500

102

93

95

(97)

4.7

9

16

11

(12)

3.6

35

40

36

(37)

2.6

22

26

35

(28)

6.7

11

12

11

(11)

0.6

+

5000

91

102

96

(96)

5.5

14

11

13

(13)

1.5

38

44

35

(39)

4.6

27

25

25

(26)

1.2

12

10

10

(11)

1.2

Positive controls S9-Mix

+

Name Concentration (μg/plate)

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2481

3433

3619

(3178)

610.5

184

273

245

(234)

45.5

468

622

552

(547)

77.1

145

227

216

(196)

44.5

328

588

552

(489)

140.9

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Conclusions:
Interpretation of results (migrated information):
negative

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

Introduction. Method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrA-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 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.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August to 28 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate OECD and EC Test Guidelines
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver fraction (S9) from male CD rats
Test concentrations with justification for top dose:
First test
In the absence of S9 mix: 3.78, 6.3, 10.5, 17.5, 29.1, 48.5, 80.9, 134.8, 224.6, 374.4, 264 and 1040 μg/mL
In the presence of S9 mix: 10, 20, 30, 35, 40, 45, 50, 55, 60 and 70 μg/mL

Second test
In the absence of S9 mix: 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50.μg/mL
In the presence of S9 mix: 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Prior to commencing testing, Oxooil LS9 was found to be miscible in ethanol at 104 mg/mL (1M). On dosing a 104 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 1040 μg/mL (10 mM), no precipitate was observed.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system.
The osmolality and pH of the test substance in medium was tested at 1040 μg/mL; no fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0 unit were observed when compared with the solvent control.
In this case, the highest final concentration used for subsequent testing was 1040 μg/mL (10 mM). In this study Oxooil LS9 was added to cultures at 1% v/v (50 μL per 5 mL culture).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
mitomycin C (without S9), Cyclophosphamide (with S9) Migrated to IUCLID6: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 48 hours
- Exposure duration:
The study comprised two independent tests. In the first test, a three hour treatment was used
in both the absence and presence of S9 mix. In the second test, a 21 hour continuous
treatment was used in the absence of S9 mix, and a three hour treatment using an increased
S9 concentration (5% v/v) was used in the presence of S9 mix.

- Fixation time (start of exposure up to fixation or harvest of cells):
First test: three hours after dosing, the cultures were centrifuged at 500g for 5 minutes. The
supernatant removed and the cell pellets resuspended in fresh medium. They were then
incubated for a further 18 hours.
Second test: three hours after dosing, the cultures containing S9 mix were centrifuged. The cell pellets
were rinsed and resuspended in fresh medium. They were then incubated for a further
18 hours. Cultures treated in the absence of S9 mix were incubated continuously for
21 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®

STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: duplicate


NUMBER OF CELLS EVALUATED: 100 each replicate


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index:
The prepared slides were examined by light microscopy using a low power objective. The
proportion of mitotic cells per 1000 cells in each culture was recorded except for positive
control treated cultures, or cultures where there were no signs of cytotoxicity.

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploid and endoreduplicated cells were noted when seen.



Evaluation criteria:
An assay is considered to be acceptable if the negative (solvent) and positive control values lie within
the current historical control range.

The test substance is considered to cause a positive response if the following conditions are
met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant
chromosomes (excluding gaps) are observed at one or more test concentration.

The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.

The increases are reproducible between replicate cultures.

The increases are not associated with large changes in pH, osmolality of the treatment
medium or extreme toxicity.

Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of
aberrant cells above concurrent control frequencies are observed, at any concentration.

A further evaluation may be carried out if the above criteria for a positive or a negative
response are not met.
Statistics:
The number of aberrant metaphase cells in each treatment group was compared with the
solvent control value using the one-tailed Fisher exact test (Fisher 1973).

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test
substance groups. If this is significant at the 1% level, the test is reiterated excluding the
highest dose group - this process continues until the trend test was no longer significant.

Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Human lymphocytes
Remarks:
Migrated from field 'Test system'.

First test

Toxicity data

       

Mitotic indices of cultured human lymphocytes treated with Oxooil LS9 are shown in Table 2 (attachment below)

In the absence of S9 mix following three hour treatment, Oxooil LS9 caused a reduction in the mitotic index to 54% of the solvent control value at 45 μg/mL. The concentrations selected for the metaphase analysis were 35, 40 and 45 μg/mL. In the presence of S9 mix (2% v/v final concentration) following three hour treatment, Oxooil LS9 caused a reduction in the mitotic index to 49% of the solvent control value at 48.5 μg/mL. The concentrations selected for the metaphase analysis were 17.5, 29.1 and 48.5 μg/mL.

Metaphase analysis

 

 

The effects of Oxooil LS9 on the chromosomes of cultured human lymphocytes are summarised in Table1 (attachment below).

In the absence of S9 mix, Oxooil LS9 caused statistically significant increases in the proportion of cells with chromosomal aberrations at 35 μg/mL (P<0.01; including and excluding gaps) and 40 μg/mL (P<0.0001: including gaps and P<0.01: excluding gaps), when compared with the solvent control. The mean values for the vehicle control (ethanol) were within the historical control range for this laboratory, when taken at the 99% confidence limit. The mean values for Oxooil LS9 treatment concentrations at 35 μg/mL and 40 μg/mL exceeded the historical control range for this laboratory. No statistical significance was observed at the highest tested concentration of 45 μg/mL, however, the mean value (excluding gaps only), exceeded the historical control range for this laboratory.

In the presence of S9 mix, Oxooil LS9 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control. All mean values for the vehicle control (ethanol), and all Oxooil LS9 treatment concentrations were within the historical control range for this laboratory, when taken at the 99% confidence limit.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system

 

Second test

Toxicity test

Mitotic indices of cultured human lymphocytes treated with Oxooil LS9 are shown in Table 4 (attachment below).

In the absence of S9 mix following 21 hour continuous treatment, Oxooil LS9 caused a reduction in the mitotic index to 46% of the solvent control value at 50mg/mL. The concentrations selected for the metaphase analysis were 20, 40 and 50mg/mL. In the presence of S9 mix (5% v/v final concentration) following three hour treatment, Oxooil LS9 caused a reduction in the mitotic index to 49% of the solvent control value at 140mg/mL. The concentrations selected for the metaphase analysis were 132.5, 137.5 and 140 mg/mL.

        

Metaphase analysis

       

The effects of Oxoil LS9 on the chromosomes of cultured human lymphocytes are summarised in Table1 (attachment below)

.

In the absence of S9 mix, Oxooil LS9 caused statistically significant increases in the proportion of cells with chromosomal aberrations at 40 μg/mL (P<0.01; excluding gaps) and 50 μg/mL (P<0.001; including and excluding gaps), when compared with the solvent control. No statistical significance was observed at the lowest tested concentration of 20 μg/mL. The mean values for the vehicle control (ethanol), were within the historical control range for this laboratory, when taken at the 99% confidence limit. However, all mean values for Oxooil LS9 treatment concentrations exceeded the historical control range for this laboratory.

In the presence of S9 mix, Oxooil LS9 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control. All mean values for the vehicle control (ethanol), and all Oxooil LS9 treatment concentrations were within or close to the historical control range for this laboratory.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploid anaylysis

In the first test only, statistically significant increases in polyploid metaphases were observed during metaphase analysis at 40 and 45 μg/mL, in the absence of S9 mix only. As a statistical significance was observed, a quantitative analysis for polyploidy using 500 metaphase cells was made for all cultures used in chromosomal analysis in the absence S9 mix only. No statistically significant increases in the proportion of polyploid cells were seen when compared to the solvent control.

Conclusions:
It is concluded that the test substance Oxooil LS9 has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix only, in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

A study was performed at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH,, to investigate the possible clastogenic properties of the test substance Oxooil LS9. The study investigated the ability of the test substance to induce chromosomal aberrations in cultured human lymphocytes from healthy donors in two independent experiments. The investigations were performed with and without rat liver S9 fraction as metabolizing system. The study was conducted to GLP and according to OECD guidelines 473 and EU method B.10.

In order to determine the toxicity of Oxooil LS9 to cultured human lymphocytes, the mitotic index was assessed for all cultures treated with the test substance and the solvent control, ethanol. Justification for concentration selection was based on cytotoxicity. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test

In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 35, 40 and 45  μg/mL.

In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 17.5, 29.1 and 48.5 μg/mL.

Second test

In the absence of S9 mix - 21 hour continuous treatment: 20, 40 and 50 μg/mL.

In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 132.5, 137.5 and 140 μg/mL

.

First test

In the absence of S9 mix, Oxooil LS9 caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at 35 μg/mL (P<0.01; including and excluding gaps) and 40 μg/mL (P<0.001; including gaps and P<0.01; excluding gaps), when compared with the solvent control. No statistical significance was observed at the highest tested concentration of 45 μg/mL.

Statistically significant increases in the proportion of polyploid cells were observed during metaphases analysis at 40 μg/mL and 45 μg/mL only. A quantitative analysis of polyploidy from all cultures used in chromosomal analysis in the absence of S9 mix showed no statistically significant increase in the proportion of polyploid cells, compared to the solvent control. 

In the presence of S9 mix, Oxooil LS9 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control. No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis.

 

Second Test

In the absence of S9 mix, Oxooil LS9 caused no statistical significant increases in the proportion of metaphase figures containing chromosomal aberrations at the lowest tested concentration of 20 μg/mL. However, statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations were observed at 40 μg/mL (P<0.01; excluding gaps) and 50 μg/mL (P<0.001; including and excluding gaps), when compared with the solvent control. 

In the presence of S9 mix, Oxooil LS9 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control.

No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis.

Conclusion

It is concluded that Oxooil LS9 showed evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix only. 

 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August 2009 to 19 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate OECD, EU & EPA Test Guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Monitoring Authority (MHRA)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus (TK+/-)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The following media, obtained from a suitable supplier, were used:
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with
2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and
HiDHS at 10% v/v.
R30p R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and
HiDHS at 30% v/v.
R10p medium was used for cell culture unless otherwise specified.
R20p medium was used for the cloning efficiency plating. This was prepared by mixing
equal volumes of R10p and R30p.
Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 2, 4.1, 8.1, 16.3, 32.5, 65, 130, 260, 520 and 1040 μg/mL
Mutation tests:
-S9 mix Test 1 (3 hours): 10, 20, 30, 35, 40, 45, 50, 55, 60, 65 and 70 μg/mL
+S9 mix Test 1 (3 hours): 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 and 130 μg/mL
-S9 mix Test 2 (24 hours): 5, 10, 20, 30, 35, 40, 45, 50, 55, 60, 65 and 70 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Oxooil LS9 was found to be soluble at approximately 104 mg/mL in ethanol.A solution of
approximately 104 mg/mL, dosed at 1% in medium, showed no precipitate in the culture
medium. The osmolality of the test substance in medium was tested at concentrations of
1040 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were
observed compared with the solvent (vehicle) control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: in the absence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 3 hours, washed once and incubated for a further 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):



NUMBER OF REPLICATIONS: 2 plates per culture


NUMBER OF CELLS EVALUATED: cultures with a density of more than 1 x 10E5 cells/mL were assessed for cloning
efficiency (viability) and mutant potential


DETERMINATION OF CYTOTOXICITY
- Method: On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies,
spontaneous and positive control MF, and percent small colonies in positive control cultures.

Evaluation criteria:
The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6 (Moore et al., 2006).
The assay was considered valid in accordance with the assay acceptance criteria.
The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent solvent control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear
trend test was applied: If the linear trend test was negative, the result was regarded as negative. If the linear trend test was positive, this indicated a
positive, biologically relevant response. Where appropriate, other factors were considered in the interpretation of the results, for example, the
reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any
concentration-related effect(s). Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods
described by Robinson et al. (1989).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in pH were observed of more than 1.0 unit at 1040 μg/mL compared with the solvent control.
- Effects of osmolality: The osmolality of the test substance in medium was tested at concentrations of 1040 μg/mL; no fluctuations in osmolality of
the medium of more than 50 mOsm/kg were observed compared with the solvent (vehicle) control.
- Evaporation from medium: No data
- Water solubility: N/A
- Precipitation: None found
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
Precipitate (observed by eye at the end of treatment) was observed at concentrations of 130 μg/mL and greater and 260 μg/mL and greater in the absence and presence of S9 mix, respectively, following a 3 hour exposure. Exposure to Oxooil LS9 at concentrations from 2 to 1040 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 114 to 0% and from 101 to 0%
respectively. Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any
concentrations. Exposure to concentrations from 2 to 1040 μg/mL resulted in RSG values from 105 to 0%. Concentrations used in the main test
were based upon these data.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: 1-3 hour treatment

Preliminary toxicity test

Precipitate (observed by eye at the end of treatment) was observed at concentrations of 130 μg/mL and greater and 260 μg/mL and greater in the absence and presence of S9 mix, respectively, following a 3 hour exposure. Exposure to Oxooil LS9 at concentrations from 2 to 1040 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 114 to 0% and from 101 to 0% respectively. Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentrations. Exposure to concentrations from 2 to 1040 μg/mL resulted in RSG values from 105 to 0%. Concentrations used in the main test were based upon these data.

Results are shown in Table 1 attached (pdf)

Main mutation test 1 - 3 hour treatment in the absence of S9 mix

There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Results are shown in Tables 2 & 3 attached (pdf)

Main mutation test 1 - 3 hour treatment in the presence of S9 mix

There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, within acceptable levels of toxicity.

Results are shown in Tables 4 & 5 attached (pdf)

Main mutation test 2 - 24 hour treatment in the absence of S9 mix

There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Results are shown in Tables 6 & 7 attached (pdf)

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It was concluded that Oxooil LS9 did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions
described.
Executive summary:

A study was performed at the Laboratories of Huntingdon Life Sciences, Eye, Suffolk, UK on behalf of Evonik Oxeno GmbH, Germany, to assess the mutagenic potential of the test substance Oxooil LS9 in an in vitro mammalian cell mutation assay. The study was conducted to GLP and in accordance with OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300. The test system was based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and two main tests (which were based on the results of the preliminary test) and comprised of three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Following 3 hour treatment in the absence and presence of S9 mix, in the main test, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the global evaluation factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 60 and 90 μg/mL respectively. In the absence and presence of S9 mix the relative total growth (RTG) was reduced to 6 and 11% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 55 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF was observed at concentrations up to 55 μg/mL, where RTG was reduced to 18%. In all tests the concurrent solvent and positive control were within acceptable ranges. It was concluded that Oxooil LS9 did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: crl:CD(SD)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld. Germany
- Age at study initiation: Main Study: 52 - 53 days
Satellite animals: 62 days

- Weight at study initiation: Main Study: 160 - 196 g
Satellite animals: 198 - 227 g

- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C */- 3°C
- Humidity (%): 55% */- 15%
- Photoperiod (hrs dark / hrs light): The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each

IN-LIFE DATES: From: July 05 To: July 14, 2016
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 4 mL/kg b.w.
Frequency of treatment:
single dose
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
positive reference: cyclophosphamide (CPA)
- Doses / concentrations: 27 mg/kg b.w., i.p.
Tissues and cell types examined:
Bone marrow cells and plasma cells for toxicokinetics
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels for the study had been selected in agreement with the Sponsor based on the results of a preliminary dose-range-finding study in rats.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Bone marrow smears were prepared from all treatment groups 24 hours post administration. Additional samples were prepared from the vehicle control and the high dose group 48 hours post administration.

DETAILS OF SLIDE PREPARATION:
Two smears from the bone marrow of 30 to 60 mm length were prepared.
After air drying, the preparations were immediately fixed in methanol for 5 minutes. Immediately after fixation, the cells were stained for 6 minutes using filtered Mayers Haemaleum. The slides were rinsed with cold tap water for 5 minutes and then further stained in 0.5% w/v ethanolic eosin solution for 1 minute. The slides were rinsed again and then left to air-dry before being dipped in xylene and mounted.


METHOD OF ANALYSIS:
Four thousand (4000) polychromatic erythrocytes per animal were scored for the incidence of micronuclei, and the ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 500 erythrocytes.
Evaluation criteria:
After completion of scoring and decoding of slides, the ratio of PCE/NCE for each animal and the mean for each group was calculated. The individual and group mean frequencies of micronucleated PCE/1000 were also determined.

The test chemical was considered as clearly positive in this assay if
i) - a statistically significant increase in the frequency of micronucleated PCE occurred for at least one dose at one kill time
ii) - the frequency of micronucleated PCE at such a point exceeded the historical control range
iii) - corroborating evidence was obtained, for example, increased but statistically insignificant frequencies or micronucleated PCE at other doses or kill times, or dose response profiles.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No test item-related increase of micronucleated polychromatic erythrocytes was observed at the two sampling times
- Ratio of PCE/NCE (for Micronucleus assay): Neither the PCE/NCE ratios nor the numbers of micronucleated PCE were influenced up to highest reasonable dose level 2000 mg/kg b.w. The numbers of micronucleated PCE were also similar to those seen in historical controls.
-Toxicokinetik evaluation: The results of the toxicokinetic evaluation confirmed a pronounced exposure of the plasma and in particular of the bone marrow to the test item, the bone marrow being the endpoint in this assay.
- Statistical evaluation: No statistical significance (at p ≤ 0.05) was reached after statistical analysis by chi2 test. In all of the cyclophosphamide-treated rats, the numbers of micronucleated PCE significantly exceeded those seen in the vehicle control groups, such that the group mean frequency (3.4/1000) was approximately 22 times higher than the group mean frequency seen in the concurrent vehicle control.
Conclusions:
In conclusion, under the present test conditions, Oxooil LS9 tested up to the highest reasonable dose level of 2000 mg/kg b.w. following single oral administration showed no genotoxic properties in the rat bone marrow micronucleus test at the two tested sampling times of 24 hours and 48 hours.
The results of the toxicokinetic evaluation confirmed a pronounced exposure of the plasma and in particular of the bone marrow to the test item, the bone marrow being the endpoint in this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The ability of Oxooil LS9 to induce chromosomal aberrations in cultured human lymphocytes from healthy donors was investigated in an in vitro cytogenetics test (OECD 473). Two independent experiments were performed with and without rat liver S9 fraction as metabolizing system. 

In the absence of S9 mix in the first test, Oxooil LS9 caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at 35 µg/mL (P<0.01; including and excluding gaps) and 40 µg/mL (P<0.001; including gaps and P<0.01; excluding gaps), when compared with the solvent control. No statistical significance was observed at the highest tested concentration of 45 µg/mL. In the second test, in the absence of S9 mix, statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations were observed at 40 µg/mL (P<0.01; excluding gaps) and 50 µg/mL (P<0.001; including and excluding gaps), when compared with the solvent control. 

In the presence of S9 mix, Oxooil LS9 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent controls.

It was concluded that Oxooil LS9 showed in vitro evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix only.

 

No in vitro evidence of mutagenicity was observed in an Ames test (OECD 471) or a mouse lymphoma gene mutation assays (OECD 476).

Since a positive result was obtained for clastogenicity in one of the three in vitro genetic toxicity tests undertaken, and in accordance with section 8.4 of Annex VIII of the REACH regulation, it is proposed to undertake an in vivo mammalian erythrocyte micronucleus test (OECD 474).


Short description of key information:
Oxooil LS9 showed evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix only, in an in vitro human lymphocyte cytogenetic test (OECD 473). No in vitro evidence of mutagenicity in an Ames test (OECD 471) or a mouse lymphoma gene mutation assays (OECD 476). An in vivo mammalian erythrocyte micronucleus test (OECD 474) was conducted and revealed no genotoxic properties in the rat bone marrow micronucleus test at the two tested sampling times of 24 hours and 48 hours.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

No classification for genetic toxicity is indicated according to the general classification and labelling requirements for dangerous substances and preparations (Directive 67-548-EEC) or the classification, labelling and packaging (CLP) regulation (EC) No 1272/2008.