Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 25 to July 29, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on December 02, 2002/ signed on February 13, 2003
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Physical state: Colourless slightly viscous liquid
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen

Method

Target gene:
Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: TA 100, TA1535 and E Coli WP2 sensitive to agents inducing base pair mutation. TA1537, TA1538 and TA98 sensitive to agents inducing frame-shift mutations.
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix; S9 from liver of male Sprague- Dawley rats induced with phenobarbitone/β-naphthoflavone (oral)
Test concentrations with justification for top dose:
Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 100 and WP2 uvr A strains, with or without S9-mix using the direct plate incorporation method.
Range finding study: 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvr A, with and without S9-mix using the direct plate incorporation method (up to maximum recommended concentration).
Main study: 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvr A, with and without S9-mix using the direct plate incorporation method (up to maximum recommended concentration).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Preparation of test materials: The test material was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (3 %) of the test material. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) i.e., 2 mm pellets with a nominal pore diameter of 4 x 10^-4 microns.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley and Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Range finding study and mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

OTHERS:
- After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter.
Rationale for test conditions:
Preliminary toxicity study: Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Range finding study: Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Main study: Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett' s method of linear regression

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: None
- Precipitation: An oily precipitate was observed at and above 1500 μg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: None

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

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutation test: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
- The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

Any other information on results incl. tables

Table 7.6.1/2: Preliminary toxicity study

S9 mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

- S9

TA 100

131

114

111

124

122

125

132

109

123

128 P

97 P

+ S9

TA 100

123

126

98

109

117

121

109

110

90

89 P

90 P

- S9

WP2 uvr A

34

19

16

22

12

18

15

18

19

26 P

22 P

+ S9

WP2 uvr A

24

28

18

30

29

26

29

26

12

17 P

20 P

                             

P: Precipitate

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. 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. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

The vehicle 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. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. An oily precipitate was observed at and above 1500 μg/plate, this did not prevent the scoring of revertant colonies. 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.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.