Registration Dossier

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:
7 March 2011 - 15 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP 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:
2012
Report Date:
2012

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
Qualifier:
according to
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Regulatory Authorities including METI, MHLW and MAFF
Deviations:
no
Principles of method if other than guideline:
There was one exception to GLP. No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within four hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
S. typhimurium histidine locus.
E. coli Tryptophan locus.
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):
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Additional strain / cell type characteristics:
other: TA 100 and TA1535 are sensitive to agents inducing base pair substitution. TA 1537 & TA98 are sensitive to agents inducing frame-shift mutations.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Additional strain / cell type characteristics:
other: WP2uvrA is sensitive to agents inducing base pair substitution.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Mutation Test - Experiment 1 (Range-finding Test)
Salmonella strains: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
E.coli strain WP2uvrA: 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

Mutation Test - Experiment 2 (Main Test)
Salmonella strains (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
E.coli strain WP2uvrA: 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Vehicle / solvent:
DMSO.

The test item was insoluble in distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: used as positive control for WP2uvrA, TA100 and TA1535 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: used as positive control for TA1537 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: positive control for TA98 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene - positive control for WP2uvrA, TA100, TA1535 and TA1537 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: positive control for TA98 with activation
Details on test system and experimental conditions:
PREPARATION OF TEST AND REFERENCE ITEMS
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (10.5%) of the test item (except in the Preliminary Toxicity Test). All formulations were used within four hours of preparation and were assumed to be stable for this period.
Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.

A solvent treatment group was used as the vehicle control and the positive controls used in the series of plates without S9-mix were as follows:
ENNG: 2 µg/plate for WP2uvrA
ENNG: 3 µg/plate for TA100
ENNG: 5 µg/plate for TA1535
9AA: 80 µg/plate for TA1537
4NQO: 0.2 µg/plate for TA98

The positive controls used in the series of plates with S9-mix were as follows:
2AA: 1 µg/plate for TA100
2AA: 2 µg/plate for TA1535 and TA1537
2AA: 10 µg/plate for WP2uvrA
BP: 5 µg/plate for TA98

MICROSOMAL ENZYME FRACTION
The S9 Microsomal fraction was prepared in-house from rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 x gravity. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196°C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

S9-MIX AND AGAR
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.

S9 5.0 mL
1.65 M KCI/0.4 M MgCI2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.

TEST PROCEDURE

PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The assay was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 0.1 mL of test item formulation, 0.5 mL of S9-mix or phosphate buffer and 2 mL of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test item and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test item 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 item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

MUTATION TEST - EXPERIMENT 1 (RANGE-FINDING TEST)
Seven concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Additional dose levels and an expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test item.

Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test item formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.

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

MUTATION TEST - EXPERIMENT 2 (MAIN TEST)
The second experiment (main test) was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was amended slightly, based on the results of the range-finding test.

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

As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:

Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The reference items (positive and untreated controls) were dosed using the standard plate incorporation method described in the range-finding test above.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Evaluation criteria:
ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:

-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

EVALUATION CRITERIA
There are several criteria for determining a positive result. Any of the following can be used to determine the overall result of the study:

-A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
-A reproducible increase at one or more concentrations.
-Biological relevance against in-house historical control ranges.
-Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
-Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item 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 making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
UKEMS (Mahon et al (1989))

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:
cytotoxicity
Remarks:
from 50 µg/plate in the absence and 150 µg/plate in the presence of S9 fraction.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 50 µg/plate in the absence and 150 µg/plate in the presence of S9 fraction.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
The test item was initially toxic at 50 µg/plate to TA100 and 5000 µg/plate to WP2uvrA. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

MUTATION TEST
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. 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 item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 50 and 150 µg/plate in the absence and presence of S9-mix, respectively. In the range-finding test (plate incorporation) Escherichia coil strain WP2uvrA showed substantial reductions in revertant colony frequency at 5000 µg/plate without a weakening of the bacterial background lawns. In the main test (pre-incubation methodology) the test item caused a visible reduction in the growth of the WP2uvrA bacterial background lawns at 5000 µg/plate in the absence of S9-mix and was non-toxic in the presence of S9-mix. The test item was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. A test item precipitate (powdery in appearance) was observed at 5000 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 item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9-mix in the main test at 5 µg/plate. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose level (5 µg/plate) were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.48 times the concurrent vehicle control.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Range-finding Test Without Metabolic Activation

Test Substance concentration (µg/plate)

Mean Number of Colonies Per Plate and Standard Deviation

Base-pair Substitution Type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

94

6.1

24

2.6

35

0.6

25

2.1

12

1.0

0.5

87

6.8

21

2.3

-

23

1.5

11

2.1

1.5

84

3.5

23

2.3

-

23

2.5

13

4.0

5

90

4.6

25

2.3

35

1.5

20

5.3

14

2.1

15

85

9.5

23

1.7

34

3.1

18

4.9

15

0.6

50

79*

6.4

23*

3.5

34

3.8

18*

1.5

11*

2.5

150

49*

6.5

24*

1.2

30

0.6

15*

2.5

7*

2.1

500

23*

3.2

16*

2.1

33

3.6

9*

3.6

4*

1.2

1500

-

-

24

4.9

-

-

5000

-

-

12

3.5

-

-

Positive Control

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

No. Colonies per plate + SD

530

17.1

183

14.7

843

32.7

185

5.0

429

65.1

*Partial absence of bacterial background lawn

Precipitate

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

 4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

SD = Standard deviation

 

Table 2: Range-finding Test With Metabolic Activation

Test Substance concentration (µg/plate)

Mean Number of Colonies Per Plate and Standard Deviation

Base-pair Substitution Type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

92

4.7

11

0.6

37

2.6

24

3.2

13

1.0

0.5

90

3.6

12

2.1

-

20

2.1

14

2.5

1.5

90

2.1

11

1.2

-

23

2.9

11

1.0

5

95

4.4

9

1.0

41

6.2

25

1.5

9

1.7

15

94

11.5

9

1.0

34

5.3

24

0.6

12

3.6

50

77

4.9

9

3.2

34

2.1

23

1.2

10

1.5

150

66*

11.5

8*

3.1

35

6.1

22*

2.9

13*

2.6

500

55*

13.7

8*

1.5

34

9.3

17*

1.7

9*

5.0

1500

-

-

26

3.6

-

-

5000

-

-

15

5.0

-

-

Positive Control

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. Colonies per plate + SD

1131

109.0

240

16.1

264

23.9

275

5.2

294

21.5

* Partial absence of bacterial background lawn

Precipitate

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

SD = Standard deviation

 

 

Table 3: Main Test Without Metabolic Activation

Test Substance concentration (µg/plate)

Mean Number of Colonies Per Plate and Standard Deviation

Base-pair Substitution Type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

83

12.3

18

2.6

17

6.2

15

1.0

12

3.1

0.15

67

5.1

18

7.4

-

19

6.1

16

1.7

0.5

72

13.9

18

4.4

-

17

6.1

11

5.8

1.5

68

3.1

19

8.5

-

20

2.1

11

6.5

5

57

6.0

22

2.6

19

3.2

16

7.4

12

3.1

15

57

7.2

18

0.6

19

5.7

14

1.5

9

1.2

50

0*

0.0

17*

3.8

16

3.1

12

1.5

14

4.6

150

0*

0.0

0*

0.0

18

5.5

18*

1.7

8*

2.5

500

-

-

13

1.5

-

-

1500

-

-

18*

11.5

-

-

5000

-

-

10

6.8

-

-

Positive Control

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

No. Colonies per plate + SD

395

39.5

176

41.6

780

21.4

173

28.1

561

54.7

*Partial absence of bacterial background lawn

Precipitate

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

 4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

SD = Standard deviation

 

Table 4: Main Test Without Metabolic Activation

Test Substance concentration (µg/plate)

Mean Number of Colonies Per Plate and Standard Deviation

Base-pair Substitution Type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

118

22.1

18

4.6

29

4.4

19

1.2

8

1.5

0.5

104

23.9

13

4.0

-

16

6.2

11

3.2

1.5

86

12.5

24

8.1

-

16

4.7

11

1.5

5

96

5.8

20

2.3

32

7.2

19

4.2

12

1.2

15

93

19.5

19

4.0

35

5.5

16

7.2

10

1.0

50

71

12.9

16

8.0

33

2.9

19

4.9

10

1.5

150

68

14.2

19

6.1

26

4.6

16

3.1

1*

0.6

500

70*

0.6

4*

1.2

22

8.5

18*

2.5

0*

0.0

1500

-

-

18

2.3

-

-

5000

-

-

22

6.4

-

-

Positive Control

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. Colonies per plate + SD

887

58.1

195

5.5

137

2.5

268

21.4

266

6.0

* Partial absence of bacterial background lawn

Precipitate

p0.05

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

SD = Standard deviation

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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

The test item was considered to be non-mutagenic under the conditions of this test. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, 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.