1H-Imidazole-5-carboxylic acid, 4-(1-hydroxy-1-methylethyl)-2-propyl-1-[[2'-[1-(triphenylmethyl)-1H-tetrazol-5-yl][1,1'-biphenyl]-4-yl]methyl]-, (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl ester

Genetic toxicity: in vitro

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

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

Test material information

Constituent 1

Reference substance name:

(5-methyl-2-oxo-2H-1,3-dioxol-4-yl)methyl 4-(2-hydroxypropan-2-yl)-2-propyl-1-({2'-[1-(triphenylmethyl)-1H-1,2,3,4-tetrazol-5-yl]-[1,1'-biphenyl]-4-yl}methyl)-1H-imidazole-5-carboxylate

EC Number:

604-437-3

Cas Number:

144690-92-6

Molecular formula:

C48H44N6O6

IUPAC Name:

(5-methyl-2-oxo-2H-1,3-dioxol-4-yl)methyl 4-(2-hydroxypropan-2-yl)-2-propyl-1-({2'-[1-(triphenylmethyl)-1H-1,2,3,4-tetrazol-5-yl]-[1,1'-biphenyl]-4-yl}methyl)-1H-imidazole-5-carboxylate

Constituent 2

Reference substance name:

Trityl Olmesartan Medoxomyl

IUPAC Name:

Trityl Olmesartan Medoxomyl

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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 results

Test results 1

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

Test results 2

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.