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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:
The experimental phase of this study was performed between 22 December 2009 and 07 February 2010.
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:
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:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
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. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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
main test:
Experiment one: 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: All Salmonella strains without S9-mix: 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
All Salmonella strains with S9-mix: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate and Escherichia coli strain WP2uvrA- with and without S9-mix: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was fully soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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:
DMSO
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:
DMSO
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:
DMSO
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:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) Experiment 1

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.

METHOD OF APPLICATION: in agar (pre-incubation) Experiment 2

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): 20 minutes at 37 degrees C

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

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:
The test material was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type, presence or absence of S9-mix and Experiment number.
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:
The test material was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type, presence or absence of S9-mix and Experiment number.
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 insoluble in sterile distilled water at 50 mg/ml but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
- 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 to TA100 and WP2uvrA- (absence and presence of S9-mix, respectively) at 5000 µg/plate and non-toxic to TA100 and WP2uvrA- (presence and absence of S9-mix, 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 range-finding test (Experiment 1 – plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of TA100 and TA1535 from 1500 µg/plate in the absence of S9-mix and TA100, TA1535 and TA1537 at 5000 µg/plate in the presence of S9-mix. A much greater toxic response was noted in the main test (Experiment 2 – pre-incubation methodology) with weakened bacterial background lawns noted for all of the bacterial tester strains. Toxicity was noted to all of the Salmonella strains in the absence of S9-mix from 50 µg/plate and initially from 1500 µg/plate in the presence of S9-mix. Weakened bacterial background lawns were also noted for Escherichia coli strain WP2uvrA- initially from 1500 and at 5000 µg/plate in the absence and presence of S9-mix, respectively. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type, presence or absence of S9-mix and Experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Any other information on results incl. tables

RESULTS

Preliminary Toxicity Test

The test material was toxic to TA100 and WP2uvrA-(absence and presence of S9-mix, respectively) at 5000 µg/plate and non-toxic to TA100 and WP2uvrA-(presence and absence of S9-mix, 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

105

108

107

89

75

99

92

119

114

98

114*

+

TA100

86

89

85

85

75

87

79

81

88

77

72

-

WP2uvrA-

28

27

18

25

18

25

27

28

21

27

41

+

WP2uvrA-

27

34

27

17

22

34

29

33

33

40

0*

*          Partial absence of bacterial background lawn

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 in Table 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 with the results also expressed graphically in Figure 1 to Figure 4.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1.

In the range-finding test (Experiment 1 – plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of TA100 and TA1535 from 1500 µg/plate in the absence of S9-mix and TA100, TA1535 and TA1537 at 5000 µg/plate in the presence of S9-mix. A much greater toxic response was noted in the main test (Experiment 2 – pre-incubation methodology) with weakened bacterial background lawns noted for all of the bacterial tester strains. Toxicity was noted to all of theSalmonellastrains in the absence of S9-mix from 50 µg/plate and initially from 1500 µg/plate in the presence of S9-mix. Weakened bacterial background lawns were also noted for Escherichia coli strain WP2uvrA-initially from 1500 and at 5000 µg/plate in the absence and presence of S9-mix, respectively. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type, presence or absence of S9-mix and Experiment number. 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 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, at any dose level either with or without metabolic activation or exposure method.

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.

Table1               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

93

 

14

 

43

 

13

 

11

 

120

(110)

13

(16)

24

(33)

23

(19)

11

(12)

118

 

22

 

33

 

20

 

14

 

Main Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

104

 

21

 

19

 

19

 

9

 

104

(101)

20

(24)

21

(18)

9

(16)

13

(13)

95

 

32

 

13

 

21

 

16

 

104

 

20

 

 

22

 

12

 

102

(101)†

28

(22)†

16

(19)†

11

(11)†

98

 

19

 

19

 

10

 

         Experintal procedure perford at a later date (without S9-mix only) due to excessive toxicity in the original test 

PLEASE SEE ATTACHED

1) Tables of results (pages 18 -21)

2) Figures1 -4 Dose-Response Curves (pages 22 -25)

3) Pages 26 -31 (Appendix 1 Report of Results in Mutagenicity Test using Micro-organisms)

4) Composition of the test material (page 32)

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

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

Introduction.

The method was designed to meet the requirements 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. It also conforms to the, EPA (TSCA) OPPTS harmonised guidelines and the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to 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 was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the range-finding test. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range (based on the results of the range finding test), fresh cultures of the bacterial strains and fresh test material formulations. The dose levels used ranged between 0.5 and 5000 µg/plate, depending on bacterial tester strain type and exposures in the absence or presence of S9-mix.

Additional dose levels and an expanded dose range were selected 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 range-finding test (Experiment 1 – plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of TA100 and TA1535 from 1500 µg/plate in the absence of S9-mix and TA100, TA1535 and TA1537 at 5000 µg/plate in the presence of S9-mix. A much greater toxic response was noted in the main test (Experiment 2 – pre-incubation methodology) with weakened bacterial background lawns noted for all of the bacterial tester strains. Toxicity was noted to all of the Salmonella strains in the absence of S9-mix from 50 µg/plate and initially from 1500 µg/plate in the presence of S9-mix. Weakened bacterial background lawns were also noted for Escherichia coli strain WP2uvrA- initially from 1500 and at 5000 µg/plate in the absence and presence of S9-mix, respectively. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type, presence or absence of S9-mix and Experiment number. 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 or exposure method.

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