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Toxicological information

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:
The experimental phase of this study was performed between 09 September 2009 and 11 October 2009.
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:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
EC Number:
276-014-8
EC Name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
Cas Number:
71786-60-2
Molecular formula:
Not applicable
IUPAC Name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
Test material form:
liquid: viscous
Details on test material:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
EC number: 276-014-8

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.

Method

Target gene:
Histidine for Salmonella.
Tryptophan 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:
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
Experiment one: Salmonella strains (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Experiment two: Salmonella strains (absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml 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. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
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: 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

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

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
Dunnett's Linear Regression Analysis

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:
cytotoxicity
Remarks:
In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respective
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:
cytotoxicity
Remarks:
In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respective
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml 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. Dimethyl sulphoxide was therefore selected as the vehicle.
- Precipitation: A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA-) from 150 and 500 µg/plate, respectively. 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: In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 RESULTS

Preliminary Toxicity Test

The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA-) from 150 and 500 µg/plate, respectively.  The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

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

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

87

69

71

71

84

84

71

0V

0T

0T

0TP

+

TA100

73

86

95

77

80

62

83

77

0V

0T

0TP

-

WP2uvrA-

32

33

24

28

31

25

25

17

 

0V

0T

0TP

+

WP2uvrA-

30

33

28

26

27

21

20

31

15S

0T

0TP

S         Sparse bacterial background lawn
V         Very weak bacterial background lawn
T         Toxic, no bacterial background lawn

P         Precipitate

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. 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) are presented inTable 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit. A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically 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. A small, statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 5 µg/plate in Experiment 1. This response was considered not to be toxicologically significant because it was non-reproducible in three separate Experiments (including the Preliminary Toxicity Assay), the mean revertant count at 5 µg/plate was only 1.17 times the concurrent vehicle control value and individual revertant counts were within the acceptable in-house historical range for the bacterial tester strain.

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.

 

Table 1               Spontaneous Mutation Rates (Concurrent Negative Controls

Range-finding Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

123

 

11

 

22

 

26

 

10

 

98

(105)

15

(15)

18

(19)

16

(22)

12

(11)

95

 

18

 

18

 

24

 

11

 

Main Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

128

 

15

 

21

 

22

 

14

 

100

(108)

16

(16)

22

(22)

22

(22)

12

(12)

95

 

18

 

22

 

22

 

11

 

 



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

 

Test Period

From: 27 September 2009

To: 30 September 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

109

97

89

(98)

10.1#

14

14

18

(15)

2.3

25

22

21

(23)

2.1

17

22

21

(20)

2.6

10

10

8

(9)

1.2

-

0.5

96

102

85

(94)

8.6

13

21

15

(16)

4.2

N/T

22

18

16

(19)

3.1

13

13

8

(11)

2.9

-

1.5

85

99

106

(97)

10.7

15

14

14

(14)

0.6

24

19

16

(20)

4.0

17

17

18

(17)

0.6

9

12

11

(11)

1.5

-

5

114

98

106

(106)

8.0

15

15

17

(16)

1.2

19

19

22

(20)

1.7

22

20

25

(22)

2.5

9

9

10

(9)

0.6

-

15

104

92

110

(102)

9.2

17

16

12

(15)

2.6

19

19

22

(20)

1.7

22

20

19

(20)

1.5

15

9

10

(11)

3.2

-

50

136

105

90

(110)

23.5

13

19

16

(16)

3.0

19

20

21

(20)

1.0

19

18

18

(18)

0.6

7

8

7

(7)

0.6

-

150

92 V

49 V

48 V

(63)

25.1

7 V

6 V

15 V

(9)

4.9

20

17

17

(18)

1.7

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

-

500

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

-

1500

N/T

N/T

0 TP

0 TP

0 TP

(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

383

293

381

(352)

51.4

205

276

243

(241)

35.5

719

761

668

(716)

46.6

191

106

126

(141)

44.4

1011

1003

913

(976)

54.4

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

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

Test Period

From: 27 September 2009

To: 30 September 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

80

84

85

(83)

2.6#

15

11

12

(13)

2.1

22

19

20

(20)

1.5

24

24

24

(24)

0.0

8

7

11

(9)

2.1

+

1.5

82

90

91

(88)

4.9

11

10

11

(11)

0.6

21

21

20

(21)

0.6

25

24

25

(25)

0.6

13

10

13

(12)

1.7

+

5

101

101

90

$$$

(97)

6.4

10

8

12

(10)

2.0

22

19

19

(20)

1.7

21

25

18

(21)

3.5

7

12

7

(9)

2.9

+

15

82

90

81

(84)

4.9

11

7

10

(9)

2.1

20

21

24

(22)

2.1

21

17

17

(18)

2.3

10

9

9

(9)

0.6

+

50

89

88

80

(86)

4.9

8

9

8

(8)

0.6

19

19

22

(20)

1.7

24

27

22

(24)

2.5

9

8

8

(8)

0.6

+

150

80

79

80

(80)

0.6

8

9

8

(8)

0.6

17

18

20

(18)

1.5

21

20

15

(19)

3.2

12

11

10

(11)

1.0

+

500

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

12 S

9 S

11 S

(11)

1.5

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

+

1500

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1510

2079

2379

(1989)

441.4

112

156

183

(150)

35.8

371

340

348

(353)

16.1

215

251

208

(225)

23.1

313

292

314

(306)

12.4

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

$$$     p<0.005

#        Standard deviation

PLEASE SEE OVERALL REMARKS,) Tables 4 and 5 (Experiment 2)

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, TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using both the Ames plate incorporation and pre-incubation methods 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 first experiment was determined in a preliminary toxicity assay and ranged between 0.5 and 1500 µg/plate, depending on bacterial strain type and presence or absence of S9. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The test material dose range was suitably amended to allow for toxicity and ranged between 0.15 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

Results.

The vehicle (dimethyl sulphoxide) 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.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit. A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically 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. A small, statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 5 µg/plate in Experiment 1. This response was considered not to be toxicologically significant because it was non-reproducible in three separate Experiments (including the Preliminary Toxicity Assay), the mean revertant count at 5 µg/plate was only 1.17 times the concurrent vehicle control value and individual revertant counts were within the acceptable in-house historical range for the bacterial tester strain.

Conclusion.

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