Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Specific details on test material used for the study:
Name: Irone Alpha
Batch No.: 9000372727
Aggregate state at Room Temperature: liquid
Colour: pale yellow
Purity: 95% sum of isomers
Storage: room temperature
Expiration date: February 17, 2002

Method

Target gene:
Histidine
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:
S9
Test concentrations with justification for top dose:
In the pre-sexeriment the concentration range of the test item was 3 - 5000 ‎μg/plate. The pre-experiment is reported as part of experiment I since no relevant toxic effects were observed and 5000 ‎μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested:
33; 100; 333; 1000; 2500; and 5000 ‎μg/plate
Vehicle / solvent:
Ethanol. The solvent was chosen becuase of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (TA 1437, TA 98 without metabolic activation), 2-aminoanthracene (TA 1535, TA 1537, TA 98, TA 100, TA 102 with metabolic activation)
Details on test system and experimental conditions:
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 posess a faulty lipopolysaccharide envelope which enables 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 'uvrB-minus'. In the strains TA 98 and TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The train TA 102 does not contain the uvrB-mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG hene) and a tetracycline resistance gene.
Regular checking of the properties of 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.. In this was it was ensured that the experimental consitions set down by Ames were fulfilled.
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO in liquid nitrogen.
Evaluation criteria:
A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
A test item is considered mitagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100 and TA 102 or thrice in strains TA 1535 and TA 1537 (3, 4).
Also, a dose-dependant and reproducible increase in the number of revertants is regarded as an indication of possibly exisitng mutagenic potential of the test item regardless whether the highest dose induced the criteria described above or not.

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 our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The plates incubated with the tset item showed irregular background growth at 333 ‎μg/plate and above with and without S9 mix in nearly all strains used.
No relevant increase in revertant colony numbers of any of the five tester strains was observed following treatment with Irone Alpha at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). In experiment II there was an increase in revertant colony exceeding the threshold of thrice the number of the corresponding solvent control at 2500 nad 5000 ‎μg/plate in strain 1535 with metabolic activation. Most likely, this increase is cuased by toxic effects of the test item, resulting in lower numbers of surviving bacteria on the corresponsing plates. A low number of bacteria competing for the traces of histidine introduced with the top agar leads to the bacterial colony grouwth intil the histidine is depleted. Such colonies were mistaken for mutant colonies although they tend to be rather small.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
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.
Therefore, Irone Alpha is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.