In vitro Cytotoxicity Assay: Elution Test
In vitro Cytotoxicity Assay: Elution Test
(mouse cell line L929)
This in vitro method analyzes the cytotoxic potential of the test item microscopically according to USP 41, Chapter <87>.
Pre-seeded cells are cultured with an extract of the test material and thereafter the biological reactivity of the cells is examined after a treatment period of 48 h.
Assessment of growth inhibition of cells by extraction method and microscopic evaluation
- The test item is extracted under agitation for 24 h in MEM supplemented with 10% FBS at 37 ± 1°C.
- At the end of the incubation period, the cells are examined microscopically for morphological changes. The biological reactivity (cellular degeneration and malformation) is described and rated on a scale of 0 to 4 (see Table). The responses of the cell cultures treated with the extract of the test item and the controls are evaluated.
- The test item meets the requirements of the test, if the response to the test item extract is not greater than grade 2 (mildly reactive).
|
Grade |
Reactivity |
Conditions of all Cultures |
|
0 |
None |
Discrete intracytoplasmic granules; no cell lysis |
|
1 |
Slight |
Not more than 20% of the cells are round, loosely attached, and without intracytoplasmic granules; occasional lysed cells are present |
|
2 |
Mild |
Not more than 50% of the cells are round and devoid of intracytoplasmic granules; no extensive cell lysis and empty areas between cells |
|
3 |
Moderate |
Not more than 70% of the cell layers contain rounded cells or are lysed |
|
4 |
Severe |
Nearly complete destruction of the cell layers |
|
Protocol |
|
|
Cell line |
L929 cells (ATCC No. CCL1, NCTC clone 929 (connective tissue mouse), clone of strain L (DSMZ)) |
|
Analysis |
Microscopic evaluation |
|
Extraction time |
24 h |
|
Incubation time |
48 h |
|
Quality controls |
Solvent Control: MEM 10% FBS Negative control: high-density polyethylene material (HDPE) Positive control: 0.1% Zinc-diethyldithiocarbamate (ZDEC), 0.25% Zinc-dibutyldithiocarbamate (ZDBC) |
|
Data delivery |
Biological Reactivity of test material according to USP 41, Chapter <87> |
|
Positive prediction |
The test item meets the requirements of the test, if the response to the test item extract is not greater than grade 2 (mildly reactive) |
References
- Guess, W.L.; Autian, J. (1966). Toxicity Evaluation of Lexan, Kyonar, Rilsan, Short-Term Studies. Journal of Oral Therapeutics and Pharmacology Vol 3, No.2, pp 116 – 123
- United States Pharmacopeia and National Formulary (USP 41), Chapter <87> (2018). Biological Reactivity Tests, in vitro - Elution Test
