Endo-(±)-8-aza-8-isopropylbicyclo[3.2.1]oct-3-yl (hydroxymethyl)phenylacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September - 26 September 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Guideline:

other: Ames II Test

Principles of method if other than guideline:

Ames II Test

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Endo-(±)-8-aza-8-isopropylbicyclo[3.2.1]oct-3-yl (hydroxymethyl)phenylacetate

EC Number:

244-859-1

EC Name:

Endo-(±)-8-aza-8-isopropylbicyclo[3.2.1]oct-3-yl (hydroxymethyl)phenylacetate

Cas Number:

22235-81-0

Molecular formula:

C19H27NO3

IUPAC Name:

Endo-(±)-8-aza-8-isopropylbicyclo[3.2.1]oct-3-yl (hydroxymethyl)phenylacetate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005 and TA 7006)

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

0.2 mL strain mixture and 0.04 mL S9-mix (30%)

Test concentrations with justification for top dose:

Seven concentration levels (1, 4, 20, 100, 500, 2500 and 5000 μg/mL) of the test article SCH 1000 Tropaester NV were tested along with the appropriate negative (vehicle) and positive controls.

Vehicle / solvent:

Dimethylsulfoxide DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

2-nitrofluorene/2-NF, 4-nitroquinoline-N-oxide/4NQO and 2-aminoanthracene/2-AA

Details on test system and experimental conditions:

The assay was performed according to the instruction manual for the Ames II (Xenometrix, Boulder/USA). 0.01 mL of the vehicle, test article or positive control were incubated with 0.24 mL bacterial overnight culture (ca 10^7/mL)/exposure medium in 24-well plates for 90 mins at 37 degC, 250rpm. With metabolic activation 0.2mL strain mixture and 0.04mL S9-mix (30%) were used/ After 90mins, the exposed cultures were diluted with pH indicator medium lacking histidine and aliquoted into 48 wells of a 384-well plate (3 replicates) using a 8-channel pipettor. The plates were incubated for 48hrs at 37degC.

To confirm the sensitivity of the tester strains and metabolic capacity of the S9 fractions known diagnostic mutagens were used (2-NF, 4-NQO and 2-AA respectively).

Rationale for test conditions:

The experiment is regarded valid, if the vehicle control shows the normal spontaneous revertant and the diagnostic mutagens cause the expected increase in the mutation rate.

Evaluation criteria:

The experiment is regarded valid, if the vehicle control shows the normal spontaneous revertant and the diagnostic mutagens cause the expected increase in the mutation rate. The test chemical was classified according to the following criteria:
- Negative: ≤8/48 wells
- Equivocal: 9-12/48 wells
- Positive: ≥13/48 wells

The historical control range experienced in this laboratory covering more than 100 experiments is 0-7/48 wells.

A concentration-dependent increase of revertant wells (mean of triplicate) over the vehicle
control is indicative for a genotoxic activity of the test substance.

Statistics:

The pH indicator bromocresol purple turns yellow as the pH drops due to the accumulation of catabolites from the metabolic activity of revertant cells. The number of positive wells (yellow) out of a total of 48 wells is indicative of the reversion frequency of reversion per replicate per concentration and was compared to the number of spontaneous revertant wells of the solvent control.

Each test point contains 48 wells of a 384-well plate. In each 48-well section, the wells were scored for the number of revertant wells (yellow) and the mean value of the triplicates was calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Slight cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Negative

Based on the described results it is concluded, that SCH 1000 Tropaester NV, when
tested up to slightly toxic concentrations, caused neither base-pair substitution nor
frameshift mutations in bacteria. No evidence of genotoxic activity was observed in a
series of S. typhimurium tester strains (TA Mix and TA 98) in the absence and presence
of metabolic activation. The test compound is, therefore, classified as "Ames II negative."