Benzoic acid, 2-ethoxy-4-[2-[[(1S)-3-methyl-1-[2-(1-piperidinyl)phenyl]butyl]amino]-2-oxoethyl]-, ethyl ester

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:

June 25 - July 4, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Ethoxy-4-{3-[(S)-3-methy-1-(2-piperidin-1-yl-phenyl)-butylamino]-2-oxo-propyl}-benzoic acid ethyl ester

EC Number:

604-604-0

Cas Number:

147770-06-7

Molecular formula:

C29 H40 N2 O4

IUPAC Name:

2-Ethoxy-4-{3-[(S)-3-methy-1-(2-piperidin-1-yl-phenyl)-butylamino]-2-oxo-propyl}-benzoic acid ethyl ester

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 RAT, AROCLOR 1254

Test concentrations with justification for top dose:

20, 100, 500, 1000, 2500 µg/plate
Five concentration levels of the test article were tested along with the appropriate negative
(vehicle) and positive controls. The concentration levels tested were based on the results of a
pretest (raw data). According to relevant guidelines, not more than 5000 μg/plate should be
investigated.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Negative Control
The water-unsoluble test article was dissolved in dimethylsulfoxide (DMSO). The solvent
medium was used as negative control. The solutions were prepared freshly for each
experiment.

Test Concentrations
Five concentration levels of the test article were tested along with the appropriate negative
(vehicle) and positive controls. The concentration levels tested were based on the results of a
pretest (raw data). According to relevant guidelines, not more than 5000 μg/plate should be
investigated.

Positive Controls
To confirm the sensitivity of the tester strains and the metabolic capacity of the S9 fractions
following known diagnostic mutagens were used

Confirmation of Tester Strain Genotypes
Tester strain cultures were checked for their typical genetic markers when frozen permanents
were prepared or when the number of spontaneous revertants fell outside the normal range.
The R-factor strains (TA 98 and TA 100) were routinely tested for the ampicillin resistance
factor because the plasmid is somewhat unstable and can be lost from the bacteria.
Technics
Plate Incorporation
0.1 ml of a bacterial shaking culture (6 hr, exponential phase), 0.1 ml vehicle or test article solution
and 0.5 ml phosphate buffer or S9-mix were added to 2 ml soft agar. After vortexing,
these mixtures were overlaid immediately onto minimal medium plates (Vogel and Bonner,
1956) in triplicate (control: 6 plates).
All tests were performed in the presence or absence of rat liver microsomal enzymes.
Finally, the titer of the initial bacterial suspension was determined with histidine-enriched
medium (5 replicates).
The results were confirmed in an independent experiment.

Rationale for test conditions:

According to the recommendations of De Serres and Shelby (1979) a reproducible, concentration-
dependent increase in the number of revertants of at least one tester strain over the vehicle
control value is indicative of genotoxic activity.
Since the results were unequivocal, no detailed statistical evaluation was performed.

Evaluation criteria:

The assay was considered valid since the following criteria were met:
All tester strains exhibited a characteristic number of spontaneous revertants per plate. The acceptable
ranges were:
TA 1537: 4-31
TA 98: 15-60
TA '100: 75-200
TA 1535: 3-37
In addition, the reference mutagens induced a distinct increase in the number of revertants, reflecting
also the activity of the metabolizing system.

Statistics:

Revertant his+ colonies were counted using the automatic colony counter IPI Analyser 982 B
after incubation at 37°C for 2 days. For all replicate platings, the mean number of revertants
per test concentration was calculated.
The condition of the background bacterial lawn (residual growth by histidine residues) was
evaluated macroscopically for evidence of bacteriotoxicity due to the test article. If extreme
thinning or complete lack of the microcolony lawn compared to the-vehicle control plates was
observed, no revertants were counted. Evidence of macroscopic test article precipitate in the
agar overlay was recorded.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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 applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on these results it is concluded, that AG-EE 623 amide ester, when tested up to
maximal soluble concentrations, caused neither base-pair substitution nor frameshift
mutations in bacteria. No evidence of genotoxic activity was observed in S. typhimurium
in the absence and presence of metabolic activation. The test compound is, therefore,
classified as "Ames negative".

Executive summary:

As part of the classification of new chemicals, the chemical intermediate AG-EE 623 amide
ester of AG-EE 623 ZW-synthesis was investigated in the bacterial mutagenicity test described
by Ames et al. (1975). This bacterial assay is highly predictive for mammalian mutagens/carcinogens.
The experiments were conducted with and without addition of microsomal liver enzymes
from rats using the plate incorporation method. The bacterial tester strains used were
histidine-auxotrophic S. typhimurium TA 1535, TA 100, (sensitive to base-pair substitution),
TA 1537 and TA 98 (susceptible to frameshift mutagens).
Solubility and Toxicity
AG-EE 623 amide ester was tested in triplicate over a range of 20 to 2500 μg/plate. Higher
concentration levels could not be tested adequately due to limited solubility as indicated by microcrystalline precipitation in the soft agar. There was no bacteriotoxicity as seen by a
reduced·background lawn and/or a decrease of absolute revertants.
Mutagenicity
Results showed that AG-EE 623 amide ester did not increase the number of revertants in
different tester strains of S. typhimurium compared to the negative control. Furthermore, metabolic
activation by hepatic microsomal enzymes from rats did not alter the mutation frequency
of these bacterial strains. The results were qualitatively confirmed in a repeat experiment. As
expected, the positive control 2-aminoanthracene (2-AA) showed a marked mutagenic
response after metabolic activation. In contrast sodium azide (NaN3) and 2-nitrofluorene
(2-NF) proved to be directly acting mutagens (-S9).
Based on these results it is concluded, that AG-EE 623 amide ester, when tested up to
maximal soluble concentrations, caused neither base-pair substitution nor frameshift
mutations in bacteria. No evidence of genotoxic activity was observed in S. typhimurium
in the absence and presence of metabolic activation. The test compound is, therefore,
classified as "Ames negative".