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In vitro Cytotoxicity Assay: Elution Test

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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.

Elution Test

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

  1. 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
  2. United States Pharmacopeia and National Formulary (USP 41), Chapter <87> (2018). Biological Reactivity Tests, in vitro - Elution Test