Registration Dossier

Administrative data

Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
study received for publication on February 18th, 1980
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:
publication
Title:
Effect of Tobacco Smoke Compounds on the Plasma Membrane of Cultured Human Lung Fibroblasts
Author:
Thelestam M., Curvall M., Enzell C.R.
Year:
1980
Bibliographic source:
Toxicology, 15 (1980) 203-217
Report date:
1980

Materials and methods

Type of study / information:
Study of the effects of thet test substance on plasma membranes of human lung fibroblasts in vitro
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: The method used is based on the simple principle that leakage of intracellular substances indicates damage to the plasma membrane and that the molecular size of the leaked material serves as an indicator of the degree of membrane damage in terms of the size of the "holes" induced by the tes compounds. A low molecular weight marker, uridine nucleotides, and a short exposure time were employed in this study to achieve high sensitivity and avoid secondary effects arising from cytotoxic damage.

- Short description of test conditions:
Cultivation and labelling of cells: Human diploid embryonic lung fibroblasts (line MI~C-5) were cultivated in Eagle's medium [14] in polystyrene wells to a cell density of 10 s cells/cm 2 (~ 7 × l0 s cells/well). The cells in confluent monolayers were labelled with [3H]uridine to obtain a low molecular weight cytoplasmic marker, consisting of uridine nucleotides.
Eagle's minimal essential medium, new born calf serum and crude trypsin were purchased from Flow Laboratories, Ltd., Irvine, Scotland, Hank's balanced salt solution from the National Bacteriological Laboratory, Stockholm, Sweden, [5-3H]uridine and Aquasol ® Universal Cocktail from
NEN Chemicals GmbH, Frankfurt, F.R.G. and tris(hydroxymethyl)-aminomethane, analytical grade, from E. Merck, Darmstadt, F.R.G.
Testing procedure and calculation of results: Labelled cultures were washed 3 times with Hank's balanced salt solution and then incubated for 30 rain at 370C with test compounds diluted to 25 mM in Tris-buffered saline, i.e. 0.15 mM NaC1 with 0.02 M tris--HC1 (pH 7.0). Then the solution containing leaked radioactive marker was removed and centrifuged (1000 g, 10 min, 4°C) and radioactivity in 0.1-ml aliquots of the supematant was measured by liquid scintillation. A maximal release of the radioactivity was obtained by treating control
cells for 30 min with 0.06 M sodium borate buffer (pH 7.8) and scraping with a rubber policeman. This treatment ruptured the cell membranes leaving the nuclei intact. The following expression was used to calculate the relative leakage of the radioactive marker:

((experimental release - spontaneous release)/(maximal release - spontaneous release)) × 100

The spontaneous release of the nucleotide marker during incubation for 30 min at 37°C was 3--7 per cent of the maximal release.

- Parameters analysed / observed: degree of membrane damage classified as high (> 70% nucleotide release), moderate (70-45%) and nil (<15%).
GLP compliance:
no

Test material

Specific details on test material used for the study:
the test compound was tested among 464 compounds, most of them aubndant in tobacco and tobacco smoke. The test compound was checked for purity using thin-layer chromatography, gas chromatography and NMR. Compounds containing more than 3 per cent impurity were purified using liquid chromatography, recrystallization and distillation. The structures of the compounds studied were confirmed by [1H]- and [laC]-NMR, e.g. the substitution pattern of multisubstituted aromatic substances and the branching pattern of methylsubstituted long chain alkyl derivatives.

Results and discussion

Any other information on results incl. tables

81% of nucleotide release was measured for the test substance. In this study, it was classified to high degree of membrane damage.

The aim of the present study was to specifically detect primary damage to the plasma membrane caused the test substance. Since cytoplasmic leakage may arise also as a secondary effect of general cytotoxic damage on prolonged exposure, it was necessary to use a short incubation time. As a consequence of this, fairly high concentrations of the test substance (25 mM) had to be used to ensure that test item does not escape detection.

Applicant's summary and conclusion

Conclusions:
It was concluded that the test substance has a high degree of membrane damage in the experimental conditions of this study.
Executive summary:

The aim of the present study was to specifically detect primary damage to the plasma membrane caused the test substance. Since cytoplasmic leakage may arise also as a secondary effect of general cytotoxic damage on prolonged exposure, it was necessary to use a short incubation time. As a consequence of this, fairly high concentrations of the test substance (25 mM) had to be used to ensure that test item does not escape detection. 81% of nucleotide release was measured for the test substance. In this study, the test substance was classified to high degree of membrane damage.