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In vitro Toxicity Evaluation
Toxicology Seminar
Autumn 2024
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Phases And Stages of Drug Development
Adapted from: PANDEY, Abhay. Phases of Drug Development Process, Drug Discovery Process. NorthEast BioLab [online]. 5. červen 2020 [vid. 2023-10-16].
Dostupné z: https://www.nebiolab.com/drug-discovery-and-development-process/
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Position of in vitro methods among the
processes of toxicity testing
in silico in vitro in vivo
Example of testing strategy – e.g. skin sensitization test
a. in silico (structure characteristics, pKa, log P etc.)
b. in vitro – human skin models
c. in vivo – animal model – usually albino rabitt
Prediction of the effect –
based on known
characteristics, similarities
etc.
Simplified live models –
Effect assesment, dose
determination etc.
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In vitro models
= living system simplified as compared with the in vivo model
Types of in vitro models
• Subcelular models (e.g. isolated mitochondria)
• Cell cultures
• Tissue cultures
• Isolated organs
• 2D vs. 3D modely
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Cell line
̶ Permanent cell lines
(immortalized – they will proliferate “indefinitely”)
̶ Single cell type
̶ Fully adapted to in vitro conditions
̶ They are derived from tumor cells or transformed from normal cells by
physical or chemical mutagens
̶ Sources cell lines:
̶ ATCC (American Tissue and Cultures Collection)
̶ ECACC (European Collection of Cell Cultures)
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In suspension x Adherent
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Culture conditions
• !! Sterile conditions !! Risk of contamination
• Laboratory equipment
– incubator, flowbox etc.
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Culture conditions
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In vivo x in vitro models
++ --
In vivo
Possible toxicokinetic testing
Monitoring of the effect of systemic
regulation
In vivo models can not be fully eliminated !!
Financial aspects, time-consuming
Ethical aspects
Interindividual differences
In vitro
Testing a larger number of compounds in a
short time-period
Plenty of biological material as a model
Reproducibility
Possibility of using human cell cultures
Determination of organ-specific toxicity (eg.
hepatotoxicity, nephrotoxicity etc.)
No information about systemic regulation
For the replacement of in vivo model
validation techniques are required
The problem with the extrapolation of
data
Not all cell types can be cultured in vitro
Culturing under non-physiological
conditions (culture media, cell lines in the
absence of tissue context)
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In vitro cell culture model for the evaluation of toxicity
̶ One cell type – homogenous properties e.g
expression of specific receptors, overexpression of cell
cycle regulators etc.
̶ Enable us to study the molecular basis of the toxic
effect – how the potential toxic substances affect their
biological targets
̶ The results correspond to the effect of the substances
without any interactions with other cell types or tissues
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Cytotoxicity
• It means the response of cells to the
effect of toxic substance
• Cytotoxic effect:
– Changes of cell morphology (augmentation,
multinuclear cells, granularity of cell surface etc.)
– Changes in cell metabolism
– Inhibiton of proliferation (changes in cell cycle
progression etc.)
– Cell death (apoptosis, necrosis, autophagy etc.)
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Dye-exclusion test
• Evaluating of the cell viability using intracellular cell dyes e.g.
erythrosin B, trypan blue, neutral red
• Viable (live) cells stay unstained x dead cells are stained
because of the penetration of the dye through impaired cell
membrane
• Hemocytometer (e.g. Bürker chamber) + microscope
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Cell viability
• Another method:
– Lactate dehydrogenase (LDH) analysis – impaired membrane integrity, then we can
detect LDH extracellularly
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Cell viability and proliferation
̶ Tetrazolium salts
̶ Enzymatic reduction of TS (changes of the color) – changes of the color correlate with the
intensity of cell metabolic activity
̶ MTT; XTT; WST-1 analysis
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Resulting parameters
Inhibitory
concentration
Lethal
concentration
Effective
concentration
Toxic
concentration
IC10 LC10 EC10 TC10
IC50 LC50 EC50 TC50
IC90 LC90 EC90 TC90
• Enable us to compare the effect of different substances
• Depend on the time of exposition, selected model etc.
• Dose-response curve
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In vitro toxicity tests – validated methods
Validated by OECD
(Organisation for Economic
Co-operation and
Development)
̶ Cytotoxicity
̶ Genotoxicity
̶ Eye irritation
̶ Phototoxicity
̶ Cardiotoxicity
̶ Nephrotoxicity
̶ Hepatotoxicity
̶ Reproductive toxicity
̶ Ecotoxicity
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Cytotoxicity
̶ E.g. Biological evaluation of medical devices
= Tests for in vitro cytotoxicity
̶ Fibroblast cell line 3T3 - Neutral Red Uptake Test (NRU)
̶ Incorporation of NR into the lysosomes of live cells – spectrofotometrical determination of
changes in color intensity
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Genotoxicity
̶ Principle: detection of mutation – DNA damage (changes in genetic information)
Standard testing series:
̶ The bacterial reverse mutation test (Ames Test)
̶ In Vitro Mammalian Chromosome Aberration Test (changes in cell ploidy, detection of
polyploidy)
̶ In vitro mammalian cell gene mutation test
̶ Etc.
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Ames test
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Eye Irritation/Corrosion
̶ Methods for testing the effect of potential ocular corrosive or irritant substances
Examples of in vitro test methods:
̶ BCOP (Bovine Corneal Opacity and Permeability)
̶ ICE (isolated chicken eye) test
̶ HET-CAM (Hen ́s Egg Test - Chorioallantoic Membrane)
̶ Fertilized eggs – substances are applied on the membrane – changes are detected
̶ e.g. hemorrage, koagulation, lysis …
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In vitro
Skin Corrosion Test: human skin model
̶ Episkin, Epiderm, SkinEthic....
̶ (reconstructed human epidermis – human keratinocytes – with functional stratum corneum)
Principle:
̶ Application of tested substance on the surface of human skin model - MTT analysis –
detection of changes in cell viability
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Reproductive toxicity
Embryonic Stem Cell test (EST test)
= evaluation of embryotoxic potencial of tested substances
Principle of the test: determination of inhibited differentiation of embryonic stem cells (ESC)
and inhibition of cell proliferation (ESC and 3T3 fibroblasts)
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Thank you for your attention