Elution Test - Eurofins Medical Device Testing

This in vitro method analyses 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

In vitro Cytotoxicity Assay: Elution Test (mouse cell line L929)

Assessment of growth inhibition of cells by extraction method and microscopic evaluation