2-Propen-1-one, 3-(dimethylamino)-1-(3-pyridinyl)-

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:

supporting study

Study period:

22 May 2000 - 19 September 2000

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

ST1571 Y5A

IUPAC Name:

ST1571 Y5A

Details on test material:

Identification ST1571 Y5A
Description Brown solid
Batch 0093800007
Purity >95% (treated as 100% pure)
Test substance storage In refrigerator in the dark
Stability under storage conditions Not indicated
Expiry date 08 May 2001 (allocated by NOTOX, 1 year after

Method

Species / strain

Species / strain / cell type:

lymphocytes: humane peripheral

Details on mammalian cell type (if applicable):

Blood samples :

Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25°C. Within 4 h after blood collection, lymphocyte cultures were started.

F10 complete culture medium :

F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/l) and 30 U/ml heparin.

Cell culture conditions:

Whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). Per culture (in the absence of S9-mix 5 ml F10 complete culture medium and in the presence of S9-mix 4.8 ml F10 complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added.

Environmental conditions:

All incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% CO2 in air in the dark at 37 ± 1°C. The temperature, humidity and CO2-percentage were monitored during the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Araclor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

In the dose range finding test:
100, 333, 1000, 3330 and 5000 ug ST1571 Y5A /ml culture medium with and without S9-mix.
Experiment 1:
With and without S9-mix: 1000, 3330 and 5000 ug ST1571 Y5A/ml culture medium (3 h treatment time, 24 h fixation time).
Experiment 2:
Without S9-mix: 180, 420 and 750 ug ST1571 Y5A/ml culture medium (24 h treatment time, 24 h fixation time)
333, 420 and 560 ug ST1571 Y5A/ml culture medium (48 h treatment time, 48 h fixation time)

With S9-mix: 1000, 3330 and 5000 ug ST1571 Y5A/ml culture medium (3 h treatment time, 48 h fixation time)

Vehicle / solvent:

dimethyl sulfoxide
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide.
- Justification for choice of solvent/vehicle: A solution could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

the vehicle of the test article, being dimethyl sulfoxide.

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Migrated to IUCLID6: Hank's Balanced Salt Solution: 0.5 µg/ml (solvent: HBSS) for a 3 h treatment period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure time)

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: Hank's Balanced Salt Solution:15 µg/ml (solvent: HBSS) for a 3 h treatment period (24 h fixation time).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

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

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the
number of cells with chromosome aberrations.
b) a statistically significant increase in the frequencies of the number of cells with chromosome
aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Additional information on results:

The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {min=0, max=5 (mean=0.8, standard deviation=1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min=0, max=5 (mean=0.8, standard deviation=0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded}.

The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

ST1571 Y5A did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

It is concluded that ST1571 Y5A is not clastogenic in human lymphocytes under the experimental conditions described in this report.