## 4.3.9. Human toxicity testing - III. Carcinogenicity assays

(Draft)

Author: Jan-Pieter Ploem

Reviewers: Frank van Belleghem

Learning objectives:

You should be able to

- explain the different approaches used for carcinogen testing.
- list some advantages and disadvantages of the different methods
- understand the difference between GTX and NGTX compounds, and its consequence regarding toxicity testing

Introduction

The term “carcinogenicity” refers to the property of a substance to induce or increase the incidence of cancer after inhalation, ingestion, injection or dermal application.

Traditionally, carcinogens have been classified according to their mode of action (MoA). Compounds directly interacting with DNA, resulting in DNA-damage or chromosomal aberrations are classified as genotoxic (GTX) carcinogens. Non-genotoxic (NGTX) compounds do not directly affect DNA and are believed to affect gene expression, signal transduction, disrupt cellular structures and/or alter cell cycle regulation.

The difference in mechanism of action between GTX and NGTX require a different approach in many cases.

Genotoxic carcinogens

Genotoxicity itself is considered to be an endpoint in its own right. The occurrence of DNA-damage can be observed/determined quit easily by a variety of methods based on both bacterial and mammalian cells. Often a tiered testing method is used to evaluate both heritable germ cell line damage and carcinogenicity.

Currently eight in vitro assays have been granted OECD guidelines, four of which are commonly used.

- The Ames test

The gold standard for genotoxicity testing is the Ames test, a test that has been developed in the early seventies. The test evaluates the potential of a chemical to induce mutations (base pair substitutions, frame shift induction, oxidative stress, etc.) in Salmonella typhimurium. During the safety assessment process, it is the first test performed unless deemed unsuitable for specific reasons (e.g. during testing for antibacterial substances). With a sensitivity of 70-90% it is a relatively good predictor of genotoxicity.

The principle of the test is fairly simple. A bacterial strain, with a genetic defect, is placed on minimal medium containing the chemical in question. If mutations are induced, the genetic defect in some cells will be restored, thus rendering the cells able to synthesize the deficient amino acid caused by the defect in the original cells.

- Escherichia coli reverse mutation assay

The Ames assay is basically a bacterial reverse mutation assay. In this case different strains of E. coli, which are deficient in both DNA-repair and an amino acid, are used to identify genotoxic chemicals. Often a combination of different bacterial strains is used to increase the sensitivity as much as possible.

- In vitro mammalian chromosome aberration assay

Chromosomal mutations can occur in both somatic cells and in germ cells, leading to neoplasia or birth and developmental abnormalities respectively. There are two types of chromosomal mutations:

Structural changes: stable aberrations such as translocations and inversions, and unstable aberrations such as gaps and breaks.

Numerical changes: aneuploidy (loss or gain of chromosomes) and polyploidy (multiples of the diploid chromosome complement).

To perform the assay, mammalian cells are exposed in vitro to the potential carcinogen and then harvested. Through microscopy the frequency of aberrations is determined. The chromosome aberration can be and is performed with both rodent and human cells which is an interesting feat regarding the translational power of the assay.

- In vitro mammalian cell gene mutation test

This mammalian genotoxicity assay utilizes the HPRT gene, a X-chromosome located reporter gene. The test relies on the fact that cells with an intact HPRT gene are susceptible to the toxic effects of 6-thioguanine, while mutants are resistant to this purine analogue. Wild-type cells are sensitive to the cytostatic effect of the compound while mutants will be able to proliferate in the presence of 6-thioguanine.

- Micronucleus test

Next to the four mentioned assays, there is a more recent developed test that already has proven to be a valuable resource for genotoxicity testing. The test provides an alternative to the chromosome aberration assay but can be evaluated faster. The test allows for automated measurement as the analysis of the damage proves to be less subjective. Micronuclei are “secondary” nuclei formed as a result of aneugenic or clastogenic damage.

It is important to note that these assays are all described from an in vivo perspective. However, an in vivo approach can always be used, see two-year rodent assay. In that case, live animals are exposed to the compound after which specific cells are harvested. The advantage of this approach is the presence of the natural niche in which susceptible cells normally grow, resulting in a more relevant range of effects. The downside of in vivo assays is the current ethical pressure on these kind of methods. Several instances actively promote the development and usage of in vitro or simply non-animal alternative methods.

- Two-year rodent carcinogenicity assay

For over 50 years, 2-year rodent carcinogenicity assay has been the golden standard for carcinogenicity testing. The assay relies on the exposure to a compound during a major part of an organism's lifespan. During the further development of the assay, a 2-species/2-gender setup became the preferred method, as some compounds showed different results in e.g. rats and mice and even between male and female individuals.

For this approach model organisms are exposed for two years to a compound. Depending on the possible mode of exposure (i.e. inhalation, ingestion, skin/eye/... contact) when the compounds enters the relevant industry, a specific mode of exposure towards the model is chosen. During this time period the health of the model organism is documented through different parameters. Based on this a conclusion regarding the compound is drawn.

Non-genotoxic carcinogens

Carcinogens not causing direct DNA-damage are classified as NGTX compounds. Due to the fact that there are a large number of potential malign pathways or effects that could be induced, the identification of NGTX carcinogens is significantly more difficult compared to GTX compounds.

The two-year rodent carcinogenicity assay is one of the assays capable of accurately identify NGTX compounds. The use of transgenic models has greatly increased the sensitivity and specificity of this assay towards both groups of carcinogens while also improving the refinement of the assay by shortening the required time to formulate a conclusion regarding the compound.

An in vitro method to identify NGTX compounds is rare. Not many alternative assays are able to cope with the vast variety of possible effects caused by the compounds resulting in many false negatives. However, cell morphology based methods such as the cell transformation assay, can be a good start in developing methods for this type of carcinogens.

References

Stanley, L. (2014). Molecular and Cellular Toxicology. p. 434.