2-methylpyridine

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:

1991-1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This is a GLP study undertaken by the Institute of Hygiene and Epidemiology, Division of Toxicology, in Brussels, BE, in preparation for the OECD High Production Volume chemicals programme.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Detection of DNA single strand breaks (SSB) has been developed as a short-term screening assay for the induction of DNA damage and repair in cultured mammalian cells. SSB are detected by alkaline filter elution (AFE) with fluorimetric quantification of a DNA/dye complex.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Alkaline elution of DNA single strand breaks

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpyridine

EC Number:

203-643-7

EC Name:

2-methylpyridine

Cas Number:

109-06-8

Molecular formula:

C6H7N

IUPAC Name:

2-methylpyridine

Details on test material:

98% purity

Method

Target gene:

whole chromosomes

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line was originally obtained from Flow Laboratories Inc, Catalog No. 05-566. These cells demonstrated sinsitivity to chemical mutagens, a high cloning frequency and a low spontaneous mutation frequency.

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from livers of CD rats exposed to Arochlor 1254

Test concentrations with justification for top dose:

2, 3, 4, 5 and 6 microliters/ml, undiluted.

Vehicle / solvent:

water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: ethylmethanesulfonate, 1.25 microliters/ml

Remarks:

In absence of S-9 metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

In the presence of S-9 metabolic activation

Migrated to IUCLID6: 0.01 or 0.1 microgram/ml

Details on test system and experimental conditions:

Cells are grown in either minimal media, or minimal media supplemented with 10% foetal calf serum. For treatment media, foetal calf serum is reduced to 7.5% in minimal media.
Twenty-four hours after seeding of V79 cells, the growth medium is replaced with treatment medium with or without S-9 mix (200 microliters) and cultures are treated with the test or control solutions for 4 h. This media is then removed, the cells are washed twice with phosphate buffered saline and trypsinised. The cells are counted with a Coulter Counter ZF and are layered onto a polyvinylchloride membrane filter (Nucleopore 25 mm, 2 micrometer pore size). The filtered cells are collected and lysed under inactinic red light by addition of lysis buffer (0.2 % sarkosyl, 2 M NaCl, 0.02 M EDTA, pH 10). After 10 minutes this solution is gravity filtered, and a second lysis buffer (0.5% SDS, 0.01 M EDTA, 0.01 Tris, 0.01 M NaCl, pH 8) supplemented with freshly prepared proteinase K (0.5 mg/ml) is added. After a 1 h incubation at room temperature, the lysis solution is allowed to pass through the filter by gravity. The lysis solution is removed by washing through the filter with 0.2M EDTA, pH 10. Pumping lines are connected to each funnel, elution buffer (0.06 M NaOH, 0.01 M EDTA, pH 12.6) is slowly added and pumped through the filter at 0.03 ml/min.
Fractions (4.5 ml, at 150 min/fraction) are collected. The PVC filter is removed into a vial containing 4.5 ml of elution buffer and incubated at 70o C for 1 h.
After cooling at room temperature, 0.8 ml of neutralization solution (0.6M NaOH 1 M NaH2PO4, 4 M NaCl) with Hoechst 33258 (0.5 microliter/ml) stock solution at 1 mg/ml) is added. The amount of DNA in each eluted fraction is then determined via the amount of fluorescence of the the dye/DNA complex at 430 nm (Fluorimeter: Perkin Elmer LS2B). A linear relationship exists between the intensity of the fluorescence and the DNA concentration.
Elution patterns are obtained by plotting the logarithm of the % of DNA remaining on the filter as a function of the fraction number. Such DNA elution profiles are normally straight and slope of the curve appeared proportional to the number of SSB. Slope = delta y divided by delta x. The no. of SSB/10E9 dalton = slope divided by 0.16. The method was calibrated by comparison with the classical alkaline sucrose gradient which indicated that in cells irradiated with gamma rays under identical conditions, 2.5 x 10-10 SSB/Dalton/Gy were induced in DNA. Thus, 1 SSB/10E9 dalton is induced after irradiation of cells with 4 gy with a slope of -0.16.
A preliminary dose-range finding study was performed.
After selection of doses, two independent experiments are performed.

Evaluation criteria:

A positive result (biologically significant increase in DNA SSB) is defined as a 3-fold increase in SSB or elution rate compared with the concurrent control in two consecutive doses.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DNA breakage rates ranged from 0.38 to 0.54 per 10E9 dalton. The breakage rate was not significantly different from that of the vehicle control. For the positive control, the breakage rate was 2.17 per 10E9 dalton

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that a 4 h exposure to 2-methylpyridine, in the presence and absence of metabolic activation, is not clastogenic in V79 hamster cells in vitro, under conditions of this assay. No increase in the number of DNA single strand breaks was observed, whereas in the same in vitro system, the positive control (ethylmethanesulfonate) induced a significant formation of single strand breaks.