Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 11-19, 2002
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: "Comission Directive 2000/32/EC, L1362000, Annexe 4D", dated May 19, 2000
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: 9000466124
Expiration date: April 11, 2004
Purity: 99.3 %

Method

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:
liver microsomal activation
Test concentrations with justification for top dose:
Experiment I and II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II without S9 mix: 10; 33; 100; 333; 1000; and 2500 µg/plate
Vehicle / solvent:
water deionised or DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylelene-diamine, 4-NOPD and 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
On the day of the experiment, the test item FRSKOMENTHE was dissolved in ethanol (purity > 99 %, MERCK, D-64293 Darmstad). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
No precipitation of the test item occurred up to the highest investigated dose.

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the “deep rough” (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which ena-bles substances to penetrate the cell wall more easily. A further mutation causes an inactivation of the excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named “urvB-minus”. In the strains TA 98 and TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the urvB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene) and a tetracycline resistance gene.

Regular checking of the properties if the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA98 was obtained from E. Merck (D-64293 Darmstadt). The bacterial strain 102 was ob-tained from RCC Ltd. (CH-4332 Stein).

The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO (MERCK, D-64293 Darm-stadt) in liquid nitrogen.
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
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FRESKOMENTHE at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The historical range of positive controls was exceeded in strains TA 1535, Ta 100 without metabolic activation, and in strain TA 98 with metabolic activation in experiments I and II. These effects indicate the sensitivity of the strains rather than compromising the assay. In experiment II, with metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in strain TA 102 (solvent control). Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:
Therefore, FRESKOMENTHE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
This study was performed to investigate the potential of FRESKOMENTHE to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. 
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested on triplicate.
The test item was tested at the following concentrations:
 
Experiment I and II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II without S9 mix: 10; 33; 100; 333; 1000; and 2500 µg/plate
 
Toxic effects were observed at higher concentrations with and without metabolic activation in nearly all strains used. 
Irregular background growth was observed with and without metabolic activation in nearly all strains used. 
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FRESKOMENTHE at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. 
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.