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:
October 29, 2003 - November 20, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed in accordance with OECD 471 (1987), EU Method B.13/14 (2000) and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1987)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Leuchtstoff L 165
- Physical state: white solid

Method

Target gene:
- S. typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
- Experiment 1:
Preliminary test, TA98 and TA100:
Without and with S9-mix: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Main study, TA1535, TA1537, TA102:
Without and with S9-mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment 2, all strains:
Without and with S9-mix: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: because of its solubility properties and its relative non-toxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see section "Any other information on results incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: pre-incubation

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: See section "any other information on results incl. tables"

RANGE-FINDING/SCREENING STUDIES:
- Precipitation: no data
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Only the positive control value was exceeded in strain TA1537 with S9-mix in experiment 2, only indicating the sensitivity of the strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No visible reduction of the background growth was observed up to and including the top dose of 5000 µg/plate, in both experiments
- Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in strain TA102 at 1000 µg/plate and above in experiment 1.
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate in experiment 2

Any other information on results incl. tables

The test substance precipitated in the overlay agar at the following concentrations (µg/plate):

Strain

Experiment 1

Experiment 2

 

Without S9-mix

With S9-mix

Without S9-mix

With S9-mix

TA1535

2500, 5000

5000

2500, 5000

1000 – 5000

TA1537

2500, 5000

1000 – 5000

1000 – 5000

1000 – 5000

TA98

-

-

1000 – 5000

333 – 5000

TA100

-

-

1000 – 5000

333 – 5000

TA102

1000 – 5000

1000 – 5000

333 – 5000

333 – 5000

- means: no visible precipitation observed

The undissolved particles had no influence on the scoring

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1987), EU Method B.13/14 (2000) and according to GLP principles. Based on the results of this study it is concluded that the substance is not mutagenic in the Bacterial Reverse Mutation Assay.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1987), EU Method B.13/14 (2000) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a preincubation assay, both in the absence and presence of S9-mix. Adequate negative and positive controls were included. The 5 bacterial strains tested were S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102.

In both experiments, the substance did not induce a significant increase in the number of revertant colonies in any of the 5 strains at any concentration, up to 5000 µg/plate, neither in the presence nor in the absence of metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Bacterial Reverse Mutation Assay.