2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

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:

22nd January 1999 - 25th January 1999

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

EC Number:

246-014-2

EC Name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Cas Number:

24085-08-3

Molecular formula:

C20H25NO3.HCl

IUPAC Name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Test material form:

other: Solid

Details on test material:

- Name of test material (as cited in study report): Benzylsalbutamon HCL
- Molecular formula (if other than submission substance): C20-H25-N-O3.H-Cl
- Molecular weight (if other than submission substance): 363.87
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:
- Physical state: Solid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.:
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material: In the dark at 37°C
- Other:

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the two strains were stored in liquid nitrogen (-198°C).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no.2) and incubated in a shaking incubator (37°C, 150spm), until the cultures reached an O.D. of 0.4 at 700 nm (10 to the power 9 cells/ml). Freshly grown cultures of each strain were used for each test.

All incubations were carried out in the dark at 37°C. The temperature was monitored during the experiment.

Vehicle / solvent:

Dimethylsulphoxide

Controls

Untreated negative controls:

yes

Remarks:

Dimethylsulphoxide

Positive controls:

yes

Remarks:

Saline (physiological saline) and DMSO (Dimethylsuphoxide)

Details on test system and experimental conditions:

Preparation of bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no.2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an O.D. of 0.4 at 700nm (10 to the power 9 cells/ml). Freshly grown cultures of each strain were used for each test.

Agar plates:
Agar plates (Ø 9cm) contained 25ml glucose agar medium. Glucose agar medium contained per liter: 18g purified agar (Oxoid, code L28) in Vogel-Bonner Medium E, 10g glucose, 0.5mg biotin and 0.6mg histidine.

Top agar:
Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3ml top agar were transferred into 10ml glass tubes with metal caps. Top agar tubes were autoclaved for 20min at 121°C.

Environmental conditions:
All incubations were carried out in the dark at 37°C. The temperature was monitored during the experiment.

Mutation assay:
Eight different doses (with approximately half-log steps) of the test substance were tested in tripicalte in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3ml molten top agar: 0.1ml of a fresh bacterial culture (10 to the power 9 cells/ml) of one of the tester strains, 0.1ml of a dilution of the test substance in dimethylsuylphoxide and either 0.5ml S9-mix (in case of activation assays) or 0.5ml 0.1M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48h. After this period of revertant (histidine independent) colonies were counted.

Colony counting:
The revertant colonies (histine independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were prescent.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
- The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.

A test substance is considered postivie (mutagenic) in the test if:
-It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.

The preceding criteria were not absolute any other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Salmonella typhimurium

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Benzylsalbutamon HCL was tested in the tester strains TA98 and TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate in the absence and presence of the S9 -mix.

Precipitate:

Benzylsalbutamon HCL did not precipitate in the top agar. Precipitation of Benzylsalbutamon HCL was not observed on the plates at the start or at the end of the incubation period in both tester strains.

Toxicity of the test substance:

The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

Number of revertants:

Tester strain TA98 showed negative responses over the entire dose range, i.e no dose-related, two-fold, increase in the number of revertants. In tester strain TA100, Benzylsalbutamon HCL induced up to 4.5- and 4.0- fold, dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9 -mix, respectively.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Based on the results of this study it is concluded that Benxylsalbutamon HCL is mutagenic in the Salmonella typhimurium reverse mutation assay in strain TA100.

Executive summary:

Benzylsalbutamon HCL was tested in the Salmonella typhirmurium reverse mutation assay with two histidine-requiring strains of Salmonella typhimurium (TA100 and TA98).

Benzylsalbutamon HCL was tested up to concentrations of 5000µg/plate in the absence and presence of S9 -mix. Benzylsalbutamon HCL did not precipitate on the plates at this dose level and no toxicity was observed.

Tester strain TA98 showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants. In tester strain TA100, Benzylsalbutamon HCL induced up to 4.5- and 4.0 -fold, dose related, increases in the number of revertant colonies compated to the solvent control in the absence and presence of S9 -miox, respectively.

Based on the results of this study it is concluded that Benzylsalbutamon HCL is mutagenic in the Salmonella typhimurium reverse mutation assay in strain TA100.