''In vitro'' toxicity testing is the

scientific Science is a systematic endeavor that builds and organizes knowledge in the form of testable explanations and predictions about the universe. Science may be as old as the human species, and some of the earliest archeological evidence for ...

analysis Analysis ( : analyses) is the process of breaking a complex topic or substance into smaller parts in order to gain a better understanding of it. The technique has been applied in the study of mathematics and logic since before Aristotle (3 ...

of the

toxic Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subs ...

effects of

chemical substance A chemical substance is a form of matter having constant chemical composition and characteristic properties. Some references add that chemical substance cannot be separated into its constituent elements by physical separation methods, i.e., w ...

s on cultured

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...

mammalian Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur o ...

cells. ''

In vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology ...

'' (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and food additives. ''In vitro'' assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g., EPA; NIEHS/NTP; FDA), to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects.

Improvement over animal testing

Most toxicologists believe that ''in vitro'' toxicity testing methods can be more useful, more time and cost-effective than toxicology studies in living animals (which are termed ''in vivo'' or "in life" methods). However, the extrapolation from ''in vitro'' to ''in vivo'' requires some careful consideration and is an active research area. Due to regulatory constraints and ethical considerations, the quest for alternatives to animal testing has gained a new momentum. In many cases the in vitro tests are better than animal tests because they can be used to develop safer products. The

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studied 1,065 chemical and drug substances in their ToxCast program (part of the

CompTox Chemicals Dashboard The CompTox Chemicals Dashboard is a freely accessible online database created and maintained by the U.S. Environmental Protection Agency (EPA). The database provides access to multiple types of data including physicochemical properties, environm ...

) using ''in silica'' modelling and a human

pluripotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta. According to Latin terms, Pluripotentia means the ability for many thin ...

stem cell-based assay to predict ''

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

'' developmental intoxicants based on changes in cellular

metabolism Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run c ...

following chemical exposure. Major findings from the analysis of this ToxCast_STM dataset published in 2020 include: (1) 19% of 1065 chemicals yielded a prediction of

developmental toxicity Development of the human body is the process of growth to maturity. The process begins with fertilization, where an egg released from the ovary of a female is penetrated by a sperm cell from a male. The resulting zygote develops through m ...

, (2) assay performance reached 79%–82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with ''in vivo'' animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain.

Examples of cell viability and other cytotoxicity assays used for ''in-vitro'' toxicology

Many methods of analysis exist for assaying test substances for cytotoxicity and other cellular responses.

Hemolysis assay

The hemolysis assay examines the propensity of chemicals, drugs or

medication A medication (also called medicament, medicine, pharmaceutical drug, medicinal drug or simply drug) is a drug used to diagnose, cure, treat, or prevent disease. Drug therapy ( pharmacotherapy) is an important part of the medical field and ...

, or any blood-contacting medical device or material to lyse

red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

s (erythrocytes). The lysis is easily detected due to the release of

hemoglobin Hemoglobin (haemoglobin BrE) (from the Greek word αἷμα, ''haîma'' 'blood' + Latin ''globus'' 'ball, sphere' + ''-in'') (), abbreviated Hb or Hgb, is the iron-containing oxygen-transport metalloprotein present in red blood cells (erythrocyt ...

MTT

MTT assay is used often in determining cell viability and has been validated for use by international organisations. MTT assay involves two steps of introducing the assay to the chemicals and then a solubilisation step.

MTS

The

colorimetric Colorimetry is "the science and technology used to quantify and describe physically the human color perception". It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color ...

MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) in vitro assay is an updated version of the validated MTT method, MTS assay has the advantage of being soluble. Hence, no solubilisation step is required.

ATP

ATP assay has the main advantage of providing results quickly (within 15 minutes) and only requires fewer sample cells. The assay performs lysis on the cells and the following chemical reaction between the assay and ATP content of cells produces luminescence. The amount of luminescence is then measured by a photometer and can be translated into number cells alive since *ATP assay assumes alive cells still have ATP inside them, and *Luminescence level recorded is proportional to the ATP content in the sample cells.

Neutral Red

Another cell viability endpoint can be

Neutral Red Neutral red (toluylene red, Basic Red 5, or C.I. 50040) is a eurhodin dye used for staining in histology. It stains lysosomes red. It is used as a general stain in histology, as a counterstain in combination with other dyes, and for many stai ...

(NR) uptake. Neutral Red, a weak cationic dye penetrates cellular membranes by non-diffusion and accumulates intercellularly in lysosomes. Viable cells take up the NR dye, damaged or dead cells do not.

Enzyme-linked immunosorbent assay (ELISA)

ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presen ...

kits can be used to examine up and down regulation of proinflammatory mediators such as cytokines (IL-1, TNF alpha, PGE2).... Measurement of these types of cellular responses can be windows into the interaction of the test article on the test models (monolayer cell cultures, 3D tissue models, tissue explants).

Types of ''in vitro'' studies

Broadly speaking, there are two different types of ''in vitro'' studies depending on the type system developed to perform the experiment. The two types of systems generally used are : a) Static well plate system and b) the multi-compartmental perfused systems.

Static well plate system

The static well plate or layer systems are the most traditional and simplest form of assays widely used for in vitro study. These assays are quite beneficial as they are quite simple and provide a very accessible testing environment for monitoring chemicals in the culture medium as well as in the cell. However the disadvantage of using these simple static well plate assays is that, they cannot represent the cellular interactions and physiologic fluid flow conditions taking place inside the body.

Multi-compartmental perfused systems

New testing platforms are now developed to solve problems related to cellular interactions. These new platforms are much more complex based on multi-compartmental perfused systems. The main objective of these systems is to reproduce in vivo mechanisms more reliably by providing cell culture environment close to the in vivo situation. Each compartment in the system represent a specific organ of the living organism and thus each compartment has a specific characteristics and criteria. Each compartment in these systems are connected by tubes and pumps through which the fluid flows thus mimicking the blood flow in the in vivo situation. The draw back behind the use of these perfused systems is that, the adverse effects ( influence of both the biological and non-biological components of the system on the fate of the chemical under study) are more compared to the static systems. In order to reduce the effect of non-biological components of the system, all the compartments are made of glass and the connecting tubes are made up of teflon. A number of kinetic models have been proposed to take care of these non-specific bindings taking place in these in vitro systems. To improve the biological difficulties arising from the use of different culture in vitro conditions, the traditional models used in flasks or micro-well plates has to be modified. With parallel development in micro-technologies and tissue engineering, these problems are solved using new pertinent tools called "micro-fluidic biochips".

References

{{DEFAULTSORT:In Vitro Toxicology Toxicology Alternatives to animal testing