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:
November 23 to december 03, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD guideline and national and international GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Remarks:
German GLP Regulations (§19a ChemG [German Chemical Law], Annex 1) and OECD GLP Regulations (OECD principles on GLP [as revised in 1997], ENV/MC/CHEM [98]17) and the Standard Operating Procedures (SOPs).
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): Tritil-olmesartan-medoxomil
- Physical state: white to pale yellowish white powder
- Analytical purity: 99.7 %
- Lot/batch No.: 2U003
- Expiration date of the lot/batch: Apr-2013
- Storage condition of test material: in a refrigerator

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: A deletion in the uvrB gene (except for TA102) and a mutation in the rfa gene. TA98, TA100 and TA102 carry the pKM101 plasmid and TA102 carries the pAQ1 plasmid.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared from livers of male rats (Sprague Dawley strain) treated with Aroclor 1254 (500 mg/kg)
Test concentrations with justification for top dose:
First repeat of mutagenicity experiment using the plate incorporation method (TA1537):
With S9-mix: 50, 100, 200, 400, 800, 1200, and 1600 µg/plate
Without S9-mix: 5, 16, 50, 160, 500, 1600, and 5000 µg/plate
Mutagenicity experiment using the plate incorporation method:
With and without S9-mix: 5, 16, 50, 160, 500, 1600, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 and TA 100 without S-9 mix
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
TA 102 without S-9 mix
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without S-9 mix
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 6-Chloro-9-(3-[2-chloroethylamino]propylamino)- 2-methoxyacridine] dihydrochloride (ICR191)
Remarks:
TA 1537 without S-9 mix
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100 and TA 102 with S-9 mix
Details on test system and experimental conditions:
Tritil-olmesartan-medoxomil was dissolved and diluted in DMSO. Bacteria were treated in two mutagenicity experiments using the plate incorporation method with and without S9-mix. Three plates were used for each dose level.
The number of revertant colonies grown on minimum selective agar medium was determined 48 hours after treatment. The toxicity of the compound to bacteria was evaluated by the inhibition of the background growth and/or the decrease in the number of revertant colonies. The negative controls consisted of a solvent control (DMSO), and the positive controls consisted of several reference compounds, depending on the type of strain and on the presence of S9-mix.
Evaluation criteria:
Experiment acceptance criteria:
The experiment is considered valid only when:
1. the S9-mix, the phosphate buffer and the highest treatment solution are proven sterile;
2. the bacterial cultures used for the mutagenicity experiment have grown properly during the overnight culture (between 5x10 8 and 5x10 9 bacteria/mL);
3. the mean number of spontaneous revertant colonies for the three negative control plates remains consistent with the range of the Test Facility historical control data;
4. the mean number of revertant colonies for the three positive control plates represents at least a two fold increase (strains TA98, TA100 and TA102) or a three-fold increase (strains TA1535 and TA1537) when compared to the mean number of spontaneous revertant colonies in the negative control plates;
5. at least five dose levels for the test article are analyzable (ie, number of revertant colonies can be determined), for each strain;
6. the highest dose level fulfills the rationale for the highest dose level selection;
7. at least three non-bacteria toxic dose levels and at least three dose levels without precipitates are observed.

Test evaluation criteria:
A test article is considered to induce a positive response in the bacterial reverse mutation test when at least two of the following criteria are fulfilled:
1. the test article induces at least a two-fold increase in the number of revertant colonies for one or more dose levels for Salmonella typhimurium strains TA98, TA100 and TA102 or at least a three-fold increase in the number of revertant colonies for one or more dose levels for Salmonella typhimurium strains TA1535 and TA1537;
2. the increase is dose-related;
3. the increase number of revertant colonies is reproducible;
4. the number of revertant colonies exceeds the upper value of the range of historical negative control data.

Moreover, biological significance needs to be discussed, taking into consideration the criteria mentioned above.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 160 µg/plate for TA 100 and TA 1535, 500 µg/plate for TA 98 and TA 102 and none for TA 1537.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 500 µg/plate for TA 100, TA 1535 and TA 102, 1200 µg/plate for TA 1537 and 1600 µg/plate for TA 98.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Tritil-olmesartan-medoxomil precipitated on the plates at dose levels ≥ 500 μg/plate without metabolic activation and ≥ 1600 μg/plate with metabolic activation.
Using the plate incorporation method, dose-related bacterial toxicity, ie, inhibition of the background growth and/or decrease in the number of revertant colonies, was observed in the presence of metabolic activation in the dose range ≥ 500 to ≥ 1600 μg/plate and in the absence of metabolic activation in the dose range ≥ 160 to ≥ 1600 μg/plate depending on the strain.
In the presence or absence of the metabolic activation, Tritil-olmesartan-medoxomil did not cause biological relevant increases in the number of revertant colonies in any of the bacterial strains.
All acceptance criteria were fulfilled. In particular, the positive controls with and without S9-mix induced a clear increase in the number of revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: The substance precipitated on the plates at dose levels ≥ 500 µg/plate without metabolic activation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions of the study, Tritil-olmesartan-medoxomil was found negative in the bacterial reverse mutation test conducted on the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of metabolic activation, testing up to 5000 µg/plate.