Introduction

Commercially exploitable compounds are being produced using modern biotechnology for use as food additives, chemotherapeutic agents, and pesticides. Traditionally, animal testing has always played an important role in the safety evaluation of such agents. Financial and ethical considerations together with an increased awareness of the limitations of animal models in relation to human metabolism now warrant the development of alternative testing methods. The ultimate aim of invitro toxicity testing is the replacement of animals in testing protocols, but in the short-term, procedures are refined to reduce the number of animals required. This three R’s philosophy of Reduction, Refinement, and Replacement was first proposed by Rusell and Burch as early as 1959.

Causes and effects of toxicity testing

Toxicity testing encompasses a wide range of causes and effects such as mutagenicity, carcinogenicity, teratogenicity and acute and chronic Cytotoxicity. The fundamental requirements of invitro toxicity testing are:

1. An assay system should give a reproducible dose-response curve over a concentration range that includes the in vivo dose.

2. There should be a linear relationship between cell number and assay response, and the resultant dose response curve should relate predictively to the invivo effect of the same compound.

Toxicity studies

Designing toxicity studies involves knowledge on A. Culture methods B. Assay design and C. Choice of cell type.

A. Culture method

Five types of culture methods are commonly employed in toxicity studies.

1. Primary cells and organ tissues.

2. Spheroid cultures-for penetration assays and solid tumor modeling.

3. Suspension cultures can be used in long and short term assays for drug sensitivity, but are mainly applied to systems using tumor biopsy material.

4. Clonogenic growth in soft agar is also in the growth of primary tumor cells. These assays have the advantage of minimizing the growth of anchor-dependent stromal cells.

5. Monolayer culture is the most widely used method in Cytotoxicity testing.

B. Assay design

Assay design involves Exposure to study compound, recovery method,, use of controls.

Exposure to study compound:

The concentration of the test compound should be dictated by the exposure level experienced in vivo. Peak plasma concentrations and plasma clearance curves can be used to estimate the most appropriate concentration range for testing, which must be adjusted to give a dose-response curve.

Recovery period:

A recovery period must be instituted when metabolic parameters are used as an index of the test compound’s effect, as this allows recovery from any metabolic dysfunction unrelated to cell death.

Us e of controls appropriate to the mode toxin of application:

Many potential toxins are highly lipophilic and therefore can only be added to culture medium in an organic solution. However these routes of application may affect the resultant data. Therefore the use of blank controls, in which the cells are treated with media containing solvent alone, are vitally important.

C. Choice of cell type

The choice of cell type will be dictated by the type of compound to be tested. For general toxicity screening against a reference compound, fast growing robust fibroblast or epithelial lines are often employed. Cell lines commonly used for toxicity studies are commercially available from commercial cell culture suppliers. » Read more: Cytotoxicity Testing Using Cell Lines