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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
Between 31 July 2007 and 27 August 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 30 August 2005 Date of Signature: 21 November 2005
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Not reported.
- Storage condition of test material: room temperature in the dark
- Other: Not reported.

Method

Target gene:
Histidine encoding gene (his) for Salmonella.
Tryptophan encoding gene (trp) for E.Coli.
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):
Not applicable.
Additional strain / cell type characteristics:
other: Including a deletion through the excision repair gene (uvrB-) which renders the capability of DNA exision repair and deep rough mutation (rfa)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
other: Including a deletion through the excision repair gene (uvrA-)
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
Range-finding Test: 0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main Test: 0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
concurrent untreated control
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
concurrent untreated control
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
concurrent untreated control
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix

Migrated to IUCLID6: 4-nitroquinoline-1-oxide
Untreated negative controls:
yes
Remarks:
concurrent untreated control
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
concurrent untreated control
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
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 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 hrs

SELECTION AGENT (mutation assays): Not applicable.

NUMBER OF REPLICATIONS: Triplicate plating.

NUMBER OF CELLS EVALUATED: Not applicable.

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

OTHER EXAMINATIONS
- Other:
Solubility: Test material precipitation was examined on the plates.
Sterlility: (Preliminary study only) The aliquot of 0.1 ml of maximum concentration of the test material (5000 µg/plate) and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material.



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:
UKEMS

Results and discussion

Test resultsopen allclose all
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: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in–house.
- 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 toxic initially at 150 µg/plate and 500 µg/plate to TA100 and WP2uvrA- respectively (Table 1 below). 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). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
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: The test material caused a visible reduction in the growth of the bacterial background lawns to all of the strains initially at 50 µg/plate (Table 2 to 4 below and Table 5 attached).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Preliminary Toxicity Test

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

70

74

75

76

86

80

80

0*

0*

0*

0*

+

TA100

77

87

84

87

73

62

62

37

0*

0*

0*

-

WP2uvrA-

24

30

28

20

32

18

31

21

22*

9*

0*

+

WP2uvrA-

30

34

36

37

34

24

29

23

18

23*

0*

* Partial or total absence of bacterial background lawn.

Table 2 Test Results: Range-Finding Test– Without Metabolic Activation

Test Period

From:09 August 2007

To: 12 August 2007

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

113

98

97

(103)

9.0#

13

28

9

(17)

10.0

24

24

23

(24)

0.6

18

16

13

(16)

2.5

15

2

5

(7)

6.8

 

0.5

105

98

108

(104)

5.1

16

15

15

(15)

0.6

N/T

10

10

17

(12)

4.0

5

3

6

(5)

1.5

 

1.5

112

112

107

(110)

2.9

16

20

19

(18)

2.1

N/T

20

15

18

(18)

2.5

14

7

7

(9)

4.0

-

5

97

105

95

(99)

5.3

23

13

21

(19)

5.3

27

23

22

(24)

2.6

9

15

11

(12)

3.1

9

8

9

(9)

0.6

-

15

95

100

126

(107)

16.6

18

25

20

(21)

3.6

19

21

15

(18)

3.1

20

14

15

(16)

3.2

2

16

8

(9)

7.0

-

50

74 *

72 *

75 *

(74)

1.5

10 *

6 *

7 *

(8)

2.1

23

23

22

(23)

0.6

11

9

11

(10)

1.2

3 *

3 *

4 *

(3)

0.6

-

150

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

14

16

11

(14)

2.5

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

-

500

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

22

19

15

(19)

3.5

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

-

1500

N/T

N/T

8 *

6 *

6 *

(7)

1.2

N/T

N/T

-

5000

N/T

N/T

0 *

0 *

0 *

(0)

0.0

N/T

N/T

Positive

controls

S9-Mix

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

457

449

445

(450)

6.1

272

259

274

(268)

8.1

427

456

404

(429)

26.1

97

100

92

(96)

4.0

2033

1887

2043

(1988)

87.3

Table 3 Test Results: Range-Finding Test– With Metabolic Activation

Test Period

From:09 August 2007

To: 12 August 2007

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

112

103

100

(105)

6.2#

8

7

11

(9)

2.1

28

34

21

(28)

6.5

21

14

27

(21)

6.5

11

2

8

(7)

4.6

+

0.5

103

90

91

(95)

7.2

7

7

5

(6)

1.2

N/T

14

13

32

(20)

10.7

3

7

2

(4)

2.6

+

1.5

99

104

103

(102)

2.6

13

13

9

(12)

2.3

N/T

21

19

24

(21)

2.5

4

5

11

(7)

3.8

+

5

98

97

92

(96)

3.2

7

5

7

(6)

1.2

32

19

25

(25)

6.5

23

21

15

(20)

4.2

8

6

4

(6)

2.0

+

15

91

102

90

(94)

6.7

5

5

4

(5)

0.6

13

26

15

(18)

7.0

15

14

20

(16)

3.2

11

15

5

(10)

5.0

+

50

75

87

90

(84)

7.9

5

10

5

(7)

2.9

25

21

21

(22)

2.3

20

18

16

(18)

2.0

4

7

6

(6)

1.5

+

150

61

54

65

(60)

5.6

0 *

0 *

0 *

(0)

0.0

27

21

23

(24)

3.1

19

19

8

(15)

6.4

4 *

1 *

3 *

(3)

1.5

+

500

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

15

20

11

(15)

4.5

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

+

1500

N/T

N/T

14 *

9 *

8 *

(10)

3.2

N/T

N/T

+

5000

N/T

N/T

0 *

0 *

0 *

(0)

0.0

N/T

N/T

Positive

controls

S9-Mix

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1159

1102

1069

(1110)

45.5

190

196

195

(194)

3.2

206

134

359

(233)

114.9

278

278

337

(298)

34.1

387

329

236

(317)

76.2

Table 4 Results: Main Test– Without Metabolic Activation

Test Period

From: 20 August 2007

From:24 August 2007†

To: 23 August 2007

To: 27 August 2007†

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

98

103

91

(97)

6.0#

8

10

10

(9)

1.2

24

23

22

(23)

1.0

11

10

12

(11)

1.0

9

12

11

(11)

1.5

-

0.5

92

107

85

(95)

11.2

3

19

4

(9)

9.0

N/T

10

12

9

(10)

1.5

12

25

18

(18)

6.5

-

1.5

93

92

93

(93)

0.6

5

5

9

(6)

2.3

N/T

12

11

12

(12)

0.6

16

11

10

(12)

3.2

-

5

96

88

90

(91)

4.2

2

4

7

(4)

2.5

25

21

19

(22)

3.1

9

18

13

(13)

4.5

16

11

23

(17)

6.0

-

15

102

81

102

(95)

12.1

3

1

7

(4)

3.1

16

31

22

(23)

7.5

16

7

15

(13)

4.9

8

21

12

(14)

6.7

-

50

86

84

77

(82)

4.7

7

2

4

(4)

2.5

22

24

15

(20)

4.7

10

12

8

(10)

2.0

0 *

0 *

0 *

(0)

0.0

-

150

0 *

0 *

0 *

(0)

0.0

1 *

4 *

8 *

(4)

3.5

15

27

22

(21)

6.0

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

-

500

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

13

14

14

(14)

0.6

0 *

0 *

0 *

(0)

0.0

0 *

0 *

0 *

(0)

0.0

-

1500

N/T

N/T

15 *

8 *

13 *

(12)

3.6

N/T

N/T

-

5000

N/T

N/T

0 *

0 *

0 *

(0)

0.0

N/T

N/T

Positive

controls

S9-Mix

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

429

438

415

(427)

11.6

231

264

331

(275)

51.0

236

240

239

(238)

2.1

134

141

114

(130)

14.0

589

1111

841

(847)

261.1

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

4NQO   4-Nitroquinoline-1-oxide

9AA     9-Aminoacridine

N/T       Not tested at this dose level

*   Partial or total absence of bacterial background lawn

†  Experimental procedure repeated at a later date due to excessive contamination (TA100) and toxicity (WP2uvrA-)

#   Standard deviation

Applicant's summary and conclusion

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. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with solutions of the test material using the Ames plate incorporation method at seven 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 for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.5 and 5000 µg/plate depending on strain type. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels (0.5, 1.5, 5 and 15 µg/plate) were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Results. The vehicle (sterile distilled water) 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 a visible reduction in the growth of the bacterial background lawns to all of the strains initially at 50 µg/plate. The test material was, therefore, either tested up to the maximum recommended dose level or its toxic limit, depending on strain type. 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.