Assessment of cytotoxicity by direct contact

Evaluation of cellular responses after direct exposure to the test material.

The direct cell contact test enables the qualitative assessment of the cytotoxic potential of a test item in accordance with ISO 10993-5. The test is performed by placing the test item directly onto a monolayer of L929 cells.

Cytotoxic substances released from the test item may cause deformation or lysis of cells beneath or surrounding the test item. Cytotoxic effects are evaluated microscopically after direct cell contact.

Assessment of cytotoxicity by microscopic evaluation after direct cell contact

  • Test and control samples are placed on a pre-seeded cell monolayer without disrupting the integrity of the cell monolayer and incubated at 37 ± 1°C for at least 24 hours. 
  • Cell malformation, degeneration, detachment and lysis are used as indicators of cytotoxicity. Biological reactivity is described and rated on a scale from 0 to 4 (see table). 
  • The test item meets the acceptance criteria if the response to the test item does not exceed Grade 2 (mildly reactive).

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Biological reactivity grading

Grade

Reactivity

Conditions of all cultures

0

None

No detectable zone around or under specimen

1

Slight

Some malformed or degenerated cells under specimen

2

Mild

Zone limited to area under specimen

3

Moderate

Zone extending specimen size up to 1,0 cm

4

Severe

Zone extending farther than 1,0 cm beyond specimen

 

Direct cell contact test protocol

Protocol

Cell line

L929 cells (ATCC No. CCL1, NCTC clone 929; connective tissue mouse cell line, clone of strain L, DSMZ)

Analysis

Microscopic evaluation

Incubation time

24 h at 37 ± 1°C

Quality controls

Solvent Control: DMEM 10% FBS

Negative control: High-density polyethylene material (HDPE)

Positive control: 0.1% Zinc-diethyldithiocarbamate (ZDEC)

Data delivery

Cytotoxicity assessment according to biological reactivity grading scores

Positive prediction

The test item meets the acceptance criteria if the response to the test item does not exceed Grade 2 (mildly reactive).

 

References

  1. ISO 10993-5: 2009 “Tests for in vitro cytotoxicity”
  2. Autian, J. (1977). Toxicological evaluation of biomaterials: Primary acute toxicity screening program. Artif. Organs Vol 1, pp 53 – 60