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The ultimate goal of toxicological studies is to ensure that newly developed substances, such as therapeutic drugs, agricultural or household chemicals, food additives, and cosmetics are safe for human, animal and environmental health. Traditionally, most of the available toxicity data comes from animal toxicity testing, but these tend to have significant limitations when translating them to human risk assessment. Despite their utility, commonly employed animal-to-human extrapolation of experimental results are still challenging, since animal models do not ideally reflect the fate and effect of a chemical in humans due to interspecies differences, different dosing schedules, inadequate statistical power, selection of outcome measures or follow-up duration etc. Thus, drugs showing safety and efficacy in preclinical animal models may show very different pharmacological properties when administered to humans. For example, corticosteroids are widely teratogenic in animals but not in humans, while thalidomide is not a teratogen in many animal species but it is in humans. Recent experience in a phase 1 study of the monoclonal antibody TGN 1412 resulted in life-threatening morbidity in all six healthy volunteers, reflecting inadequate prediction even in non-human primates of the human response.

The toxicity testing paradigm was introduced in 2005 by the U.S. EPA and the U.S. National Toxicology Program (NTP), the goal of which was to develop the area using advances in toxicogenomics, in vitro biology, computational sciences, and information technology, to rely increasingly on human data as opposed to animal data, and to offer increased efficiency in design and costs. In 2007, the US National Research Council released its expert panel report appealing for moving away from whole-animal testing to one founded primarily on in vitro methods that evaluate changes in biological processes using cells, cell lines, or cellular components, preferably of human origin with the emphasize on understanding of toxicity pathways (NRC-NAS, 2007).


Current advances in in vitro methodologies facilitate rapid hazard identification using high-throughput screening approach in mammalian cells cultured in monolayer 2D or biomimetic 3D structures. In vitro assays bring a number of technical advantages to the conventional method of testing substances on animal models, such as the ability to elucidate cellular-response networks and toxicity pathways; the use of concentrations relative to human exposure etc. In vitro 3D cultures that produce biomimetic models have been widely hailed as an advanced screening method by showing how substances influence in vivo cellular interactions. Data obtained using initial in vitro toxicity testing methods help to improve subsequent study design, which may significantly reduce the use of animals. 

Overall, in vitro model systems ensure tight control of chemical and physical environment during the experiment, enable to address the toxicity pathways issue, provide human relevant and high throughput data, and are relatively cheap and simple to procure. However, the application of in vitro methods as standalone approaches in risk assessment is still a problem regarding extrapolation the intact organism. A disadvantage is the absence of the in vivo biokinetics that may lead to a misinterpretation of in vitro data. It is possible that in the in vivo systems, the cells expose to the lower concentrations, since the chemical compound cannot simply reach the target cells. On the other side, drugs may accumulate in certain organs, tissues or cells, resulting in a prolonged or enhanced exposure of the target tissue. For this reason, using an in vitro system may result in an underestimation of the biological activity of the compound under study.


Some basic toxicity testing using human cells in vitro is already a conventional method and often conducted to evaluate endpoints like cytotoxicity, protein binding, enzyme induction/inhibition, membrane permeability etc. In the field of toxicology of cosmetic ingredients, regulatory agencies accept in vitro tests for dermal absorption, irritation and corrosivity, as well as ocular irritation and corrosivity. Below is a list of common validated in vitro toxicology methods.


The following in vitro studies are usually conducted first. If there is any positive response in the in vitro study, a follow-up in vivo study for the same endpoint is usually required.

  • OECD TG 471 Bacterial reverse mutation test (Ames test)

  • OECD TG 473 In vitro mammalian chromosome aberration test 

  • OECD TG 476 In vitro mammalian cell gene mutation test

  • OECD TG 482 DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro


The following methods may be used to replace the OECD TG 404 Acute Dermal Irritation / Corrosion traditional in vivo test.

  • OECD TG 430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test

  • OECD TG 431 In Vitro Skin Corrosion: Human Skin Model Test

  • OECD TG 432 In Vitro 3T3 NRU Phototoxicity

  • OECD TG 435 In Vitro Membrane Barrier Test Method for Skin Corrosion

  • OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method


The following methods may be used to replace the OECD TG 405 Acute Eye Irritation / Corrosion traditional in vivo test.

  • OECD TG 437 In Vitro Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants

  • OECD TG 438 In Vitro Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants

  • OECD TG 460 Fluorescein Leakage Test Method (FL)

  • OECD TG 491 Short Time Exposure Test Method (STE)

  • OECD TG 492 Reconstructed human Cornea-like Epithelium Test Method (RhCE)


The following methods alone cannot replace the  traditional OECD TG 429 and OECD TG 406 in vivo test.​

  • OECD TG 442D In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method

  • OECD TG xxxx In Vitro Skin Sensitisation: Human Cell Line Activation Test (h-CLAT)

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