## 4.3.9. Human toxicity testing - II. In vitro tests

(Draft)

Author: Nelly Saenen

Reviewers: Karen Smeets, Frank Van Belleghem

Learning objectives:

You should be able to

- argue the need for alternative test methods for toxicity
- list commonly used in vitro cytotoxicity assays and explain how they work
- describe different types of toxicity to skin and in vitro test methods to assess this type of toxicity

Keywords: In vitro, toxicity, cytotoxicity, skin

Introduction

Toxicity tests are required to assess potential hazards of new compounds to humans. These tests reveal species-, organ- and dose- specific toxic effects of the compound under investigation. Toxicity can be observed by either in vitro studies using cells/cell lines (see section on in vitro bioassay) or by in vivo exposure on laboratory animals; and involves different durations of exposure (acute, subchronic, and chronic). In line with Directive 2010/63/EC on the protection of animals used for scientific purposes, it is encouraged to use alternatives to animal testing (OECD: alternative methods for toxicity testing). The first step towards replacing animals is to use in vitro methods which can be used to predict acute toxicity. In this chapter, we present acute in vitro cytotoxicity tests (= quality of being toxic to cells) and skin corrosive, irritant, phototoxic, and sensitivity tests as skin is the largest organ of the body.

1. Cytotoxicity tests

The cytotoxicity test is one of the biological evaluation and screening tests in vitro to observe cell viability. Viability levels of cells are good indicators of cell health. Conventionally used tests for cytotoxicity include dye exclusion or uptake assays such as Trypan Blue Exclusion (TBE) and Neutral Red Uptake (NRU).

The TBE test is used to determine the number of viable cells present in a cell suspension. Live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, whereas dead cells do not. In this assay, a cell suspension incubated with serial dilutions of a test compound under study is mixed with the dye and then visually examined. A viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm. The number of viable and/or dead cells per unit volume is determined by light microscopy using a hemacytometer counting chamber (Figure 1). This method is simple, inexpensive and a good indicator of membrane integrity but counting errors (~10%) can occur due to poor dispersion of cells, or improper filling of counting chamber.

Figure 1. Graphical view hemacytometer counting chamber and illustration of viable and non-viable cells.

The NRU assay is an approach that assesses the cellular uptake of a dye (Neutral Red) in the presence of a particular substance under study (see e.g.Figure 1 in Repetto et al., 2008). This test is based on the ability of viable cells to incorporate and bind neutral red in the lysosomes, a process based on universal structures and functions of cells (e.g. cell membrane integrity, energy production and metabolism, transportation of molecules, secretion of molecules). Viable cells can take up neutral red via active transport and incorporate the dye into their lysosomes while non-viable cells cannot. After washing, viable cells can release the incorporated dye under acidified-extracted conditions. The amount of released dye can be measured by the use of spectrophotometry.

Nowadays, colorimetric assays to assess cell viability have become popular. For example, the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tests cell viability by assessing activity of mitochondrial enzymes. NAD(P)H-dependent oxidoreductase enzymes, which under defined conditions reflect the number of viable cells, are capable of reducing the yellow formazan salt dye into an insoluble purple crystalline product. After solubilizing the end product using dimethyl sulfoxide (DMSO), the product can be quantitatively measured by light absorbance at a specific wavelength. This method is easy to use, safe and highly reproducible. One disadvantage is that MTT formazan is insoluble so DMSO is required to solubilize the crystals.

2. Skin corrosion and irritation

Skin corrosion refers to the production of irreversible damage to the skin; namely, visible necrosis (= localized death of living cells, see section on cell death) through the epidermis and into the dermis occurring after exposure to a substance or mixture. Skin irritation is a less severe effect in which a local inflammatory reaction is observed onto the skin after exposure to a substance or mixture. Examples of these substances are detergents and alkalis which commonly affect hands.

The identification and classification of irritant substances has conventionally been achieved by means of skin or eye observation in vivo. Traditional animal testing used rabbits because of its thin skin. In the Draize test, for example, the test substance is applied to the eye or shaved skin of a rabbit, and covered for 24h. After 24 and 72h, the eye or skin is visually examined and graded subjectively based on appearance of erythema and edema. As these in vivo tests have been heavily criticized, they are now being phased out in favor of in vitro alternatives.

The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT) are in vitro assays that can be used to identify whether a chemical has the potential to corrode or irritate skin. The method uses a three-dimensional (3D) human skin model (Episkin model) which comprises of a main basal, supra basal, spinous and granular layers and a functional stratum corneum (the outer barrier layer of skin). It involves topical application of a test substance and subsequent assessment of cell viability (MTT assay). Test compounds considered corrosive or irritant are identified by their ability to decrease cell viability below the defined threshold level (lethal dose by which 50% of cells are still viable - LD50).

3. Skin phototoxicity

Phototoxicity (photoirritation) is defined as a toxic response that is elicited after the initial exposure of skin to certain chemicals and subsequent exposure to light (e.g. chemicals that absorb visible or ultraviolet (UV) light energy that induces toxic molecular changes).

The 3T3 NRU PT assay is based on an immortalised mouse fibroblast cell line called Balb/c 3T3. It compares cytotoxicity of a chemical in the presence or absence of a non-cytotoxic dose of simulated solar light. The test expresses the concentration-dependent reduction of the uptake of the vital dye neutral red when measured 24 hours after treatment with the chemical and light irradiation. The exposure to irradiation may alter cell surface and thus may result in decreased uptake and binding of neutral red. These differences can be measured with a spectrophotometer.

4. Skin sensitisation

Skin sensitisation is the regulatory endpoint aiming at the identification of chemicals able to elicit an allergic response in susceptible individuals. In the past, skin sensitisation has been detected by means of guinea pigs (e.g. guinea pig maximisation test and the Buehler occluded patch tests) or murine (e.g. murine local lymph node assay). The latter is based upon quantification of T-cell proliferation in the draining lymph nodes behind the ear (auricular) of mice after repeated topical application of the test compound.

The key biological events (Figure 2) underpinning the skin sensitisation process are well established and include:

1. haptenation, the covalent binding of the chemical compounds (haptens) to skin proteins (key event 1);
2. signaling, the release of pro-inflammatory cytokines and the induction of cyto-protective pathways in keratinocytes (key event 2);
3. the maturation, and mobilisation of dendritic cells, immuno-competent cells in the skin (key event 3);
4. migration of dendritic cells, movement of dendritic cells bearing hapten-protein comples from skin to draining local lymph node;
5. the antigen presentation to naïve T-cells and proliferation (clonal expansion) of hapten-peptide specific T-cells (key event 4)

Figure 2. Key biological events in skin sensitisation. Figure adapted from D. Sailstad by Evelin Karsten-Meessen.

Today a number of non-animal methods addressing each a specific key mechanism of the induction phase of skin sensitisation can be employed. These include the Direct Peptide Reactivity Assay (DPRA), ARE-Nrf2 Luciferase Test Method: KeratinoSens, Human Cell Line Activation Test (h-CLAT), U937 cell line activation test (U-SENS), and Interleukin-8 Reporter Gene assay (IL-8 Luc assay). Detailed information of these methods can be found on OECD site: skin sensitization.

References

EUR-lex site. https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32010L0063.

OECD site. Alternative methods for toxicity testing. https://ec.europa.eu/jrc/en/eurl/ecvam/alternative-methods-toxicity-testing.

Episkin model. http://www.episkin.com/Episkin

OECD site. Skin sensitization. https://ec.europa.eu/jrc/en/eurl/ecvam/alternative-methods-toxicity-testing/validated-test-methods/skin-sensitisation.

Repetto, G., Del Peso, A., Zurita, J.L. (2008). Neutral red uptake assay for the estimation of cell viability/cytotoxicity.Nature protocols 3(7), 1125.