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:
The experimental phase of this study was performed between 21 May 2008 and 15 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine for Salmonella.
Trytophan for E.Coli
Species / strainopen allclose all
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
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
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphthoflavone 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
Mutation Test - Experiment 1 (Range finding Test): 50, 150, 500, 1500 and 5000 µg/plate
Mutation Test - Experiment 2 (Main Test): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test material was soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
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

Migrated to IUCLID6: (ENNG)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
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

Migrated to IUCLID6: (9AA)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
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: (4NQO)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
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

Migrated to IUCLID6: (BP)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: Yes (10 hours)
- Exposure duration: 48-72 hours.
- Expression time (cells in growth medium): N/a
- Selection time (if incubation with a selection agent): N/a
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 hours


SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable.


NUMBER OF REPLICATIONS:Triplicate plating


NUMBER OF CELLS EVALUATED: Not applicable.


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


OTHER EXAMINATIONS: None



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 (5) 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
the test material did cause substantial decreases in revertant colony frequency in several strains at 5000 µg/plate, predominately in the presence of S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/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:
the test material did cause substantial decreases in revertant colony frequency in several strains at 5000 µg/plate, predominately in the presence of S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/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 soluble in sterile distilled water at 50 mg/ml.
- 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.
- Other confounding effects: None.


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). 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: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). 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

100

97

94

94

79

118

91

105

80

80

78

+

TA100

97

100

110

84

110

100

78

78

68

90

38

-

WP2uvrA-

25

28

27

22

30

20

26

29

27

18

16

+

WP2uvrA-

29

27

18

35

31

25

29

22

30

28

21

Mutation Test

Results for the negative controls (spontaneous mutation rates) are presented in Table1(see attached background material) and were considered to be acceptable.

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 (see attached backround material).

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, the test material did cause substantial decreases in revertant colony frequency in several strains at 5000 µg/plate, predominately in the presence of S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 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.

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.

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 the test material using the Ames plate incorporation method at five 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 was 50 to 5000 µg/plate. 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.

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 no visible reduction in the growth of the bacterial background lawn at any dose level. However, the test material did cause substantial decreases in revertant colony frequency in several strains at 5000 µg/plate, predominately in the presence of S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 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.