PLEASE NOTE THAT TO OBTAIN DETAILS OF THE PROTOCOL YOU SHOULD REGISTER

TO OBTAIN DETAILED PROTOCOL YOU SHOULD FIRST REGISTER AT ECVAM SIS

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

Protocol no. 31
AGAROSE OVERLAY ASSAY

This procedure describes the use of L929 mouse fibroblast cells cultured in vitro in an agarose overlay assay to assess the toxicity of test substances. The assay may be useful in assessing the irritation potential of test substances (e.g. surfactant-based products) as an alternative to the Draize rabbit eye test.

CONTACT

Ms Manoj Dixit and Ms Penny Jones
Unilever Research Colworth Laboratory
Colworth House Sharnbrook Bedford MK44 1LQ UK
Tel: England - 0234-222362 Fax: England - 0234-222000

RATIONALE

The principle of the assay involves measurement of zones of colour change of cells overlaid with agarose then treated with test material. The zones are visualised either by loss of neutral red from perviously stained cells or staining with Thiazolyl blue (MTT) after treatment.

BASIC PROCEDURE

L929 cells are plated at 2 x 106 viable cells/ml EMEM per 5-cm tissue culture dish and incubated until fully confluent. Culture medium is then replaced with a solution of 1.6% agarose in distilled water, and the resulting overlay cultures left to equilibrate at 37°C in an atmosphere of 5%CO2/95% air, for 45 minutes. For the NR assay: the cells are stained with 0.01% NR for 15 minutes at room temperature in the dark. Once the cells are loaded with neutral red, test materials are applied to the cultures. One sensitivity disk is placed in the centre of each agarose overlay onto which 25mg or 25ml sample of undiluted, or 1:10 dilution of test material is loaded. Treated cultures are then incubated at 37°C for 4 or 24 hours. At the end of the exposure period the diameter of the affected area is recorded. For the MTT assay: the test material is applied to equilibrated agarose overlays, as described above. After 4 hours the sample disks are removed from the overlays and 2ml of 0.5mg/ml solution of MTT in PBS is added to each culture. The cultures are further incubated for 1 hour or until dark blue colour has formed. The diameter of area in which no MTT reduction had occurred is recorded for each plate. Each culture is also examined by phase contrast microscopy, to assess morphological damage.

CRITICAL ASSESSMENT

The agarose overlay method along with direct contact methods and tissue culture techniques have been used extensively to screen and monitor the safety of biomaterials used in the manufacture of medical devices. The in vitro agarose overlay method using L929 mouse fibroblast cells was originally developed for testing the toxicity of plastic materials (Rosenbluth et al., 1965), and has more recently been used to assess cosmetic products (Jackson et al., 1988; Wallin et al., 1987). Rosenbluth was the first to report the use of mammalian tissue culture techniques to examine the biocompatibility of plastic materials used in medical devices. In the direct method small sections of test materials, such as plastics, were placed directly onto monolayers of a mouse fibroblast cell line, L929. After the cells were exposed to test material for 48 hours, biocompatibility was assessed by fixing and staining the monolayer and observing the cells microscopically. Nontoxic samples were observed to have no visible effect upon the cell monolayer, whilst, cytotoxic samples could be identified by zones of clearing due to the death of cells in the area beneath and surrounding the test samples. However difficulties were encountered when either low-density or high-density samples were tested e.g. low-density samples tended to float-providing poor contact with the monolayer surface, whilst high-density samples caused physical disruption of the cells due to their weight. An agar overlay modification of the direct contact method was developed to eliminated these problems. This was carried out by supplementing the liquid culture medium with 1% agar and a vital stain. Test samples could then be directly aliquoted onto the agar surface. It was observed that biocompatible samples produced no visible change in the cell monolayer whilst samples containing extractable cytotoxic components caused cell lysis. The vital stain was released and a visible zone of clearing under and around the test sample was produced. This method has been further modified by replacing the agar with agarose, since, agarose matrices allow better diffusion of aqueous solutions than agar matrices. In addition, the lower melting point of agarose provides less heat shock to the cells. The agarose overlay method can be used to test liquid samples or extracts of raw materials as well as finished products. A further modification has been that test samples or extracts are no longer applied directly to the surface of the agarose overlay but to non-cytotoxic filter paper (sensitivity) discs, which are in turn placed on the agarose surface. In the authors opinion, this test has been shown to provide a simple, rapid and inexpensive means of assessing the likely ocular irritancy of surfactant-based formulations. However, they would add that highly coloured compounds will interfere with the test. Modification of the test The reduction of thiazolyl blue (MTT) by mitochondrial dehydrogenases has also been included as another end-point besides the uptake of the vital dye, neutral red. Comparison of the end-points neutral red and MTT The results from the neutral red and MTT assays indicate little difference between the two end-points. However, the MTT assay has the advantage of greater sensitivity when dealing with samples of low toxicity. In addition the use of MTT facilitates more rapid reading of the test plates because of greater contrast between zones of colour change, which are yellow/blue for the MTT assay as compared with orange/red for neutral red. The authors strongly recommend the use of MTT instead of the neutral red end-point.

TEST STATUS

This test is currently being evaluated in-house, although it has been used extensively by the medical industry to screen materials for medical devices under investigation for irritancy.

OTHER ORGANISATIONS USING THE TEST

Noxell Corporation, Hunt, Maryland, USA.

CHEMICALS TESTED

5 Anionic formulations 8 Cationic formulations

REFERENCES

  1. Jackson, E.M., Hume, R.D. & Wallin, R.F. (1987) The agarose diffusion method for ocular irritancy screening cosmetic products, part II. J. Toxic.: Cut. Ocular Toxic., 7, 187.
  2. Milstein, S.R. & Hume, R.D. (1991) Correlating the L-929 and SIRC variants of the in vitro agarose diffusion method for the assessment of cosmetic product eye irritation potential. J. Toxic.: Cut. Ocular Toxic., 10(1/2), 3-14.
  3. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. Immun. Method, 65, 55.
  4. O'Brien, K.A.F., Jones, P.A. & Rockley, J. (1990) Evaluation of an agarose overlay assay to determine the eye irritation potential of detergent-based products. Toxic. in vitro, 4(4/5), 311-313.
  5. Rosenbluth, S.A., Weddington, G.R., Guess, L.W. & Autian, J. (1965) Tissue culture method for screening toxicity of plastics to be used in medical practice. J. Pharm. Soc., 54, 156.
  6. Wallin, R.F., Hume, R.D. & Jackson, E.M. (1987) The agarose diffusion method for ocular irritancy screening cosmetic products, part I. J. Toxic.: Cut. Ocular Toxic., 6:239.

IP-31 © November 1991