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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Safepharm Laboratories Limited
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
-
EC Number:
481-670-5
EC Name:
-
Cas Number:
848301-66-6
IUPAC Name:
C8-C16 branched and linear hydrocarbons (full range) – Kerosine
Details on test material:
- Name of test material (as cited in study report): Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear
- Substance type: Organic
- Physical state: Liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): Information not available
- Composition of test material, percentage of components: Information not available
- Isomers composition: Information not available
- Purity test date: Information not available
- Lot/batch No.: Information not available
- Expiration date of the lot/batch: Information not available
- Stability under test conditions: Stable under normal temperature and pressure
- Storage condition of test material: Room temperature, in the dark

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
S9 cells from livers of Spraque-Dawley rats
Additional strain / cell type characteristics:
DNA polymerase A deficient
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
S9 cells from livers of Spraque-Dawley rats
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone / beta-naphtoflavone induced, rat liver S9
Test concentrations with justification for top dose:
0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
Acetone
- Justification for choice of solvent/vehicle:
Test substance not soluble in water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: further control substances: 9AA, ENNG
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Minimal / In suspension; 0.1 mL of the bacterial culture was mixed with 0.1 mL test material,
0.5 mL of phosphate buffer or S9-mix and 2 mL of molten, trace histidine or tryptophsan supplemented,
top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar.
DURATION
- Preincubation period:
Preculture period lasted 10 hours.
- Exposure duration:
48 hours at 37°C


NUMBER OF REPLICATIONS:
3


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
The plates were incubated for 48 h after an initial overnight equilibration period and then assessed for revertant colonies using a
Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
Sensitivity of the essay and the efficacy of the S9-mix were validated.
Statistics:
Statistical methods are used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
For a test material to be considered positive in this test system it should normally have induced at least a twofold increase in revertant colony frequency for TA98, TA100 and WP2uvrA- and a threefold increase in TA1535 and TA1537. The effect must be reproduced in an independant assay with evidence of a dose-related response.

Results and discussion

Test resultsopen allclose all
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: None
- Precipitation:
An oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
Range-finding test was performed (experiment 1). results are summarised in attached tables 1 and 2.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was not relevant at the concentration range tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test results: Experiment 1 (Range-finding test) – Without Metabolic activation

Test period

Number of revertants (mean number of colonies per plate)

With or without S9-Mix

Test substance concentration (µg/plate)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvtA-

TA98

TA1537

without

0

120

115

129

(121)

7.1#

26

20

27

(24)

3.8

25

18

18

(20)

4.0

25

32

32

(30)

4.0

10

9

9

(9)

0.6

without

15

98

87

104

(96)

8.6

25

19

21

(22)

3.1

22

21

30

(24)

4.9

26

14

18

(19)

6.1

11

7

10

(9)

2.1

without

50

130

104

133

(122)

15.9

33

24

24

(27)

5.2

20

25

19

(21)

3.2

29

26

23

(26)

3.0

13

13

9

(12)

2.3

without

150

74

91

110

(92)

18.0

27

23

29

(26)

3.1

36

24

21

(27)

7.9

40

22

22

(28)

10.4

13

10

9

(11)

2.1

without

500

110

106

113

(110)

3.5

38

19

33

(30)

9.8

22

22

20

(21)

1.2

23

19

18

(23)

5.5

15

11

14

(13)

2.1

without

1500

70P

77P

91P

(79)

10.7

22P

21P

21P

(21)

0.6

25P

27P

22P

(25)

2.5

26P

36P

26P

(29)

5.8

5P

4P

5P

(5)

0.6

without

5000

79P

70P

C P

(75)

6.4

19P

27P

21P

(22)

4.2

46P

27P

22P

(32)

12.7

21P

19P

22P

(21)

1.5

10P

3P

9P

(7)

3.8

Positive controls

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Without S9

Number of colonies per plate

621

662

670

(651)

26.3

278

267

227

(257)

26.8

806

839

848

(831)

22.1

189

187

132

(169)

32.3

1525

835

1057

(1139)

352.2

ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO: 4-Nitroquinoline-1-oxide; 9AA: 9-Aminoacridine;

C: Contaminated; P: Precipitate; #: Standard Deviation

 

Table 2: Test results: Experiment 1 (Range-finding test) – With Metabolic activation

Test period

Number of revertants (mean number of colonies per plate

With or without S9-Mix

Test substance concentration (µg/plate)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvtA-

TA98

TA1537

with

0

73

96

95

(88)

13.0#

13

18

18

(16)

2.9

29

25

27

(27)

2.0

23

31

31

(28)

4.6

12

21

11

(15)

5.5

with

15

81

100

77

(86)

12.3

14

8

11

(11)

3.0

23

30

33

(29)

5.1

18

34

29

(27)

8.2

12

15

22

(16)

5.1

with

50

88

81

89

(86)

4.4

27

12

18

(19)

7.5

33

34

16

(28)

10.1

C

26

27

(27)

0.7

18

14

21

(18)

3.5

with

150

75

90

65

(77)

12.6

8

11

10

(10)

1.5

29

23

23

(25)

3.5

24

26

26

(25)

1.2

14

14

7

(12)

4.0

with

500

54

74

73

(67)

11.3

22

10

5

(12)

8.7

38

33

29

(33)

4.5

32

26

24

(27)

4.2

14

14

15

(14)

0.6

with

1500

70P

74P

70P

(71)

2.3

12P

10P

13P

(12)

1.5

25P

33P

27P

(28)

4.2

29P

29P

35P

(31)

3.5

10P

21P

14P

(15)

5.6

with

5000

69P

59P

62P

(63)

5.1

11P

11P

7P

(10)

2.3

34P

31P

35P

(33)

2.1

37P

33P

31P

(34)

3.1

18P

12P

13P

(14)

3.2

Positive controls

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

 

2

With S9

Number of colonies per plate

2142

2096

2220

(2153)

62.7

240

224

252

(239)

14.0

496

464

539

(500)

37.6

212

213

203

(209)

5.5

636

615

643

(631)

14.6

2AA: 2-Aminoanthracene; BP: Bonzo(a)pyrene, C: Contaminated, P: Precipitate; #: Standard Deviation

 

Table 3: Test results: Experiment 2 (Main test) – Without Metabolic activation

Test period

Number of revertants (mean number of colonies per plate

With or without S9-Mix

Test substance concentration (µg/plate)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvtA-

TA98

TA1537

without

0

87

77

119

(94)

21.9#

17

13

17

(16)

2.3

22

23

18

(21)

2.6

29

24

22

(25)

3.6

17

20

17

(18)

1.7

without

15

79

89

80

(83)

5.5

11

20

28

(20)

8.5

32

27

25

(28)

3.6

31

19

16

(22)

7.9

9

14

11

(11)

2.5

without

50

82

90

72

(81)

9.0

18

11

25

(18)

7.0

18

21

20

(20)

1.5

14

27

20

(20)

6.5

14

11

11

(12)

1.7

without

150

75

94

70

(80)

12.7

11

17

18

(15)

3.8

34

17

19

(23)

9.3

19

27

22

(23)

4.0

11

16

11

(13)

2.9

without

500

68

85

68

(74)

9.8

17

23

19

(20)

3.1

30

20

26

(25)

5.0

28

17

17

(21)

6.4

10

15

9

(11)

3.2

without

1500

72P

74P

76P

(74)

2.0

18P

20P

10P

(16)

5.3

27P

29P

22P

(26)

3.6

25P

24P

21P

(23)

2.1

4P

18P

13P

(12)

7.1

without

5000

74P

63P

67P

(70)

3.8

16P

6P

27P

(16)

10.5

25P

24P

26P

(25)

1.0

24P

23P

20P

(22)

2.1

10P

8P

10P

(9)

1.2

Positive controls

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Without S9

Number of colonies per plate

542

479

527

(516)

32.9

245

235

276

(252)

21.4

694

800

878

(791)

92.4

214

206

211

(210)

4.0

776

772

800

(783)

15.1

ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO: 4-Nitroquinoline-1-oxide; 9AA: 9-Aminoacridine; P: Precipitate; #: Standard Deviation

 

Table 4: Test results: Experiment 2 (Main test) – With Metabolic activation

Test period

Number of revertants (mean number of colonies per plate

With or without S9-Mix

Test substance concentration (µg/plate)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvtA-

TA98

TA1537

with

0

91

112

103

(102)

10.5#

14

16

21

(17)

3.6

31

40

32

(34)

4.9

17

20

20

(19)

1.7

7

11

11

(10)

2.3

with

15

68

89

99

(85)

15.8

11

15

10

(12)

2.6

28

29

26

(28)

1.5

41

13

16

(23)

15.4

10

14

11

(12)

2.1

with

50

85

70

108

(88)

19.1

6

10

16

(11)

5.0

42

25

24

(30)

10.1

18

18

25

(20)

4.0

16

6

14

(12)

5.3

with

150

85

79

113

(92)

18.1

6

13

25

(15)

9.6

21

31

37

(30)

8.1

22

18

21

(20)

2.1

16

19

9

(15)

5.1

with

500

112

80

79

(90)

18.8

13

9

10

(11)

2.1

24

38

31

(31)

7.0

14

20

18

(17)

3.1

14

11

14

(13)

1.7

with

1500

103P

119P

108P

(110)

8.2

8P

8P

7P

(8)

0.6

23P

26P

29P

(26)

3.0

19P

33P

17P

(23)

8.7

9P

14P

25P

(16)

8.2

with

5000

79P

85P

76P

(80)

4.6

10P

11P

13P

(11)

1.5

31P

30P

24P

(28)

3.8

24P

22P

18P

(21)

3.1

14P

17P

14P

(15)

1.7

Positive controls

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

 

2

With S9

Number of colonies per plate

1985

1698

1867

(1850)

144.3

167

213

203

(194)

24.2

956

1093

1076

(1042)

74.7

170

196

165

(177)

16.6

586

650

525

(587)

62.5

2AA: 2-Aminoanthracene; BP: Bonzo(a)pyrene, P: Precipitate; #: Standard Deviation

 

Applicant's summary and conclusion

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

All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate, within expected ranges. No statistically significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the substance,
either with or without metabolic activation. The substance 'Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear' was found to be non-mutagenic under the conditions of this test.
Executive summary:
The test substance 'Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear' was investigated for possible genotoxic effects in an in-vitro study according to OECD guideline 471 as well as US-EPA TSCA OPPTS guideline 870.5100. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated at six dose levels (15 to 5000 µg/plate). The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear was considered to be non-mutagenic under the conditions of this test.