Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
March 09, 2010 - April 21, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: highly viscous liquid
Details on test material:
- (Trade) Name: CapCure 3-800; C-SAT 090103

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 (Pre-Experiment), all strains:
Without and with S9-mix: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: methyl methane sulfonate, 3.0 µL/plate in deionised water for TA102
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 10 µg/plate in deionised water for TA 1535 and TA 100
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 10 µg/plate in DMSO for TA98 and 50 µg/plate in DMSO for TA1537
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2.5 µg/plate in DMSO for TA1535, TA1537, TA98 and TA100 and 10 µg/plate in DMSO for TA102
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation) and experiment 2 preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hour

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
-regular background growth in the negative and solvent control
-the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
-the positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS
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.
Statistics:
Not performed.

Results and discussion

Test results
Key result
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
Remarks:
tested up to the highest recommended dose 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
- Precipitation: Precipitation was observed at the dose level of 5000 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- In experiment I with metabolic activation, the data in the untreated control of strain TA 102 were slightly above the historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD 471 guideline and GLP principles.
Executive summary:

The genetic toxicity of C-SAT 090103 was assessed using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains with and without metabolic activation, in accordance with OECD 471 guideline and GLP principles. Precipitation was observed at the top dose of 5000 µg/plate in the presence of S9 -mix. No toxicity was observed in all strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with C-SAT 090103 up to the highest concentration of 5000 µg/plate tested, neither in the presence nor absence of metabolic activation (S9 mix) in two independently repeated experiments.

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.