Methyl N-benzyl-N-(3-methoxy-3-oxopropyl)-β-alaninate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-12-22 (date the test subtance was receveid) to 2004-09-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

1) No data on mitogenic stimulation or the metaphase-arresting substance, 2) Only 100 metaphas e spreads scored per concentration.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Methyl N-benzyl-N-(3-methoxy-3-oxopropyl)-β-alaninate

EC Number:

212-343-5

EC Name:

Methyl N-benzyl-N-(3-methoxy-3-oxopropyl)-β-alaninate

Cas Number:

793-19-1

Molecular formula:

C15H21NO4

IUPAC Name:

methyl 3-[benzyl(3-methoxy-3-oxopropyl)amino]propanoate

Details on test material:

- Name of test material (as cited in study report): T 723
- Substance type: no data
- Physical state: pale yellow liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data
- Other: no data

Specific details on test material used for the study:

Description: Pale yellow liquid
Purity: 100 %
Date received: 2003-12-22
Storage conditions: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver homogenate metabolising system (2%)

Test concentrations with justification for top dose:

- Preliminary toxicity test: 0, 12.9, 25.8, 51.6, 103.19, 206.38, 412.75, 825.5, 1651 and 3302 μg/mL, based on a 10mM maximum dose level.

- Group 1 (4-hour without S9): 0, 87.29, 174.59, 349.18, 523.77, 698.35 and 1047.53 µg/mL (0, 174.59, 349.18 and 698.35 μg/mL were selected for metaphase analysis.)
- Group 2 (4-hour with S9): 0, 87.29, 174.59, 349.18, 523.77, 698.35 and 1047.53 µg/mL (0, 174.59, 349.18 and 698.35 μg/mL were selected for metaphase analysis.)
- Group 3 (24-hour without S9): 0, 21.82, 43.65, 87.29, 174.59, 261.89 and 349.18 µg/mL (0, 43.65, 87.29 and 261.89 μg/mL were selected for metaphase analysis.)

Vehicle / solvent:

- Vehicle used: no data
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; at 0.4 μg/ml for the 4(20) hour exposure and 0.2 μg/mL for the 24 hour continuous exposure (Groups 1 and 3 respectively)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; at 10 μg/ml for the 4(20) hour exposure (Group 2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: no data

DURATION
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time (cells in growth medium): 20 hours (Groups 1 and 2) was provided in the study report as the expression period. However, no information was given on the time of addition of a spindle inhibitor; 0 hours (Group 3)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (all groups)

SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS:
Duplicate cultures were tested. Except where there was the need to clarify an equivocal response, only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations.

NUMBER OF CELLS EVALUATED: 100 cells per evaluated culture.

DETERMINATION OF CYTOTOXICITY:
- Method: mitotic index (MI)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: investigated

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

no data

Results and discussion

Test results

Test results 1

Species / strain:

other: human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: human lymphocytes

Metabolic activation:

with

Genotoxicity:

other: dose related increases in gap-type aberrations only

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: There was a cloudy precipitate of test substance observed at and above 1396.7 µg/ml in all exposure groups.

RANGE-FINDING/SCREENING STUDIES:
- Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 698.35 µg/mL in the 4 hour pulse exposure groups and at up to 349.18 µg/mL in the 24 hour continuous exposure group.
- A dose-related increase in toxicity in all of the exposure groups was observed. Furthermore, in both 4(20)-hour pulse exposure groups there was approximately 50% mitotic inhibition at 698.35 µg/mL. Therefore, the selection of the dose range for the main test was based on toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A microscopic assessment of the slides showed that metaphase cells were present at up to 698.35 μg/ml in both the 4(20)-hour exposure groups and at up to 261.89 μg/ml in the 24-hour continuous exposure group. The test material induced mitotic inhibition in all exposure groups. In the 4 hour pulse exposures the ideal of 50% mitotic inhibition was achieved at 698.35 and 523.77 μg/ml, with and without S9 respectively. In the 24-hour exposure group there was approximately 60% mitotic inhibition at 261.89 μg/ml. Dose selection for metaphase analysis was limited by toxicity in all exposure groups.

Remarks on result:

other: Groups 1 and 3: 4 and 24-hour exposure period without S9-mix

Any other information on results incl. tables

The test substance induced statistically significant increases in the frequency of cells with aberrations at the maximum dose levels selected for scoring, in both exposure groups without S9. In the 4 -hour exposure with metabolic activation (Group 2) there were dose related increases in gap-type aberrations only. These were considered to be unusual but of less clastogenic importance than the aberration types observed in Groups 1 and 3.

The test substance did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
positive without metabolic activation

The test substance induced statistically significant increases in the frequency of cells with chromosome aberrations (without gaps) in the absence of a liver enzyme metabolizing system after a 4-hour exposure with a 20-hour recovery and a 24-hour continuous exposure. The test substance was therefore considered to be clastogenic to human lymphocytes in vitro.