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WET LAB GENETOX SERVICES

GenTox studies are conducted to assess the mutagenic potential of chemicals. As a part of safety evaluation process, regulatory authorities all over the globe require information on the genotoxic potential of the new drugs.

STANDARD METHODS

Toxometris.ai provides the full range of genetic toxicology studies, conducted in accordance with GLP standards and regulatory guidelines. A standard battery for genotoxicity testing is performed based on the guidance S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, which combines and replaces two ICH guidances ICH S2A and ICH S2B. Additionally a DNA strand break single cell gel electrophoresis (“Comet”) assay (OECD 475) is offered.

Gene Mutation

  • Ames Mutagenicity Assay (OECD 471)

  • Mouse Lymphoma Assay (OECD 476)

Chromosome Damage In Vitro

  • Micronucleus Assay (OECD 487)

  • Chromosome Aberration Test (OECD 473)

Chromosome Damage In Vivo

  • Micronucleus Assay (OECD 474)

  • Chromosome Aberration Test (OECD 475)

 

HIGH-THROUGHPUT METHODS

The high-throughput approach is important during early drug discovery process, when the screening of multiple compounds need to be performed at the same time to speed up the lead selection process and reduce unfavorable failures on late stages of drug development process.

Toxometris.ai provides a high-throughput gene mutation assay performed in 384-well plates using two TA98/TA100 or four TA98/TA100/TA1535/TA1537 Salmonella strains. Rat liver S9 fraction is used as a component of the assay to determine if generated metabolites are potential mutagens. The bacterial cytotoxicity assay is performed in parallel.

 

MODE OF ACTION ANALYSIS

Genotoxins induce damage to genetic material either directly or indirectly by various mechanisms. Additional insight to understand mode of action along with standard assays are often needed to move the drug candidate efficiently forward to market. Toxometris.ai offers a wide range of studies associated with the elucidation of the mechanism of DNA damage response machinery activation, oxidative DNA damage, and epigenotoxicity. 

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INTRACELLULAR PROTEIN RELEASE-BASED ASSAYS

One of the most reliable methods for studying cell death measuring the leakage of cellular components from compromised cultured cells when membrane integrity is altered.

  • Lactate dehydrogenase (LDH) release colorimetric or fluorescence assay

  • Glucose 6-phosphate dehydrogenase (G6PD) release colorimetric or fluorescence assay

 

METABOLISM-BASED ASSAYS

The measurement of metabolic activity can be achieved using tetrazolium salt as dead cells cannot metabolize the substance. The incubation of viable cells with tetrazolium salt leads to the production of colored formazan product, associated with metabolic activity.

  • MTT colorimetric assay

  • XTT colorimetric assay

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CELL MEMBRANE PERMEABILITY ASSAYS

Reliable and rapid tests to detect cytotoxic chemicals that affect cell membranes.

  • 7-Amino Actinomycin D (7-AAD) cell viability flow cytometry assay

  • Propidium Iodide (PI)  cell viability flow cytometry assay

  • Trypan blue exclusion test

  • Neutral red uptake assay 

 

APOPTOSIS ASSAYS

A broad range of chemotherapies work by interfering with the mechanisms of cell death, and inducing apoptosis remains a promising strategy for cancer drug discovery.

  • Caspase activation assay

  • FITC-conjugated Annexin V assay

  • DNA fragmentation assays

 

AUTOPHAGY ASSAYS

Autophagy inhibitors are considered as potent chemosensitizers and might therefore be introduced into combination regimens for optimal anticancer therapies.

  • LC3B immunofluorescence assay

 

CELL PROLIFERATION ASSAY AND CELL CYCLE ASSAY METHODS

Besides cell viability, proliferation assays are a fundamental tool in the drug development process and are used to evaluate antiproliferative potency of compounds.

  • Clonogenicity assay

  • Bromodeoxyuridine (BrdU) incorporation assay

  • Cell cycle analysis by DNA content (PI staining)

  • Cell proliferation Ki67 immunostaining assay

  • Endogenous lysosomal beta-galactosidase (SA-beta-gal) colorimetric senescence assays

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VALIDATED AND ACCEPTED IN VITRO ALTERNATIVE METHOD

Advanced alternative methods and their combinations are commonly used for safety assessment of drug candidates. Several alternative methods, which were scientifically validated and accepted by competent regulatory bodies, can be used for regulatory toxicology purposes, thus reducing or fully replacing living animals in toxicology experimentation.

CYTOTOXICITY SERVICES

Cytotoxicity is an ability of certain chemical compounds to destroy the living cells. In vitro cytotoxicity assays performed during early screenings identify and define safety thresholds for subsequent use. In vitro toxicity tests are found to be advantageous in preclinical studies because of their eligibility, cost effectiveness, and reproducibility. Toxometris.ai offers cytotoxicity assays in numerous tissue-specific cell types (purchased from cell banks such as ECACC and ATCC), providing greater predictably of target organ toxicities and identifying multiple toxicity endpoints.

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CARCINOGENICITY

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Carcinogenicity studies are generally required for pharmaceuticals that are expected to be used continuously for at least six months or intermittently for the treatment of chronic or recurrent conditions. The objectives of carcinogenicity studies are to identify a tumorigenic potential in animals and to assess the relevant risk in humans. Standard carcinogenicity evaluations period using animals is 18–24 months.

Toxometris.ai offers a Cell Transformation Assays (CTAs) in Syrian Hamster Embryo cells (SHE) performed at pH 6.7 and at pH 7.0. This method will give a reliable result in up to the 4 weeks.

Endorsement of Scientific Validity: EURL ECVAM (2012)

International Regulatory Acceptance: OECD GD No. 214 (2015)

Most drugs undergo chemical alteration, known as biotransformation or metabolism, by various bodily systems to create compounds that are more easily excreted from the body. This process has a huge impact on the efficacy and safety of drugs and considers as one of the main lynchpins in the discovery and development cycle of a new pharmaceutical.

Toxometris.ai offers a human cytochrome P450 (CYP) n-fold induction in vitro test method, which is a reliable and relevant in vitro tool for the assessment of human CYP induction for routine testing of drug candidates.

 International Regulatory Acceptance: Draft OECD TG xxx (2014)

Many types of chemicals have been reported to induce phototoxic effects defined as a toxic response elicited by topically or systemically administered photoreactive chemicals after the exposure of the body to environmental light.

Toxometris.ai offers the in vitro 3T3 NRU phototoxicity test was shown to be predictive of acute phototoxicity effects in animals and humans in vivo.

International Regulatory Acceptance: OECD TG 432 (2004)

Pyrogenicity refers to the induction of fever and is identified as a subset of acute systemic toxicity. The pyrogenicity testing is a part of mandatory release tests for parenteral pharmaceutical products and medical devices. Life-threatening fever reactions induced by pyrogenic substances may lead to the fatal systemic response of the patient’s innate immune system.

Toxometris.ai offers five human cell assays for detecting pyrogenicity produced by gram-negative endotoxins: Human whole blood IL-1; Human whole blood IL-6; Human cryopreserved whole blood IL-1; PBMC IL-6; PBMC IL-6, as well as Monocyte-Activation Test, which detects both endotoxin and non-endotoxin pyrogens in one in vitro test.

Endorsement of Scientific Validity: ESAC (2006); ICCVAM (2008).

International Regulatory Acceptance: European Pharmacopeia, Monocyte activation test (2.6.30) (2010); revised (2.6.30) (July 2017); FDA Guidance: Pyrogen and Endotoxins Testing (2012)

Reproductive toxicity is defined as adverse effects of a chemical on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring. Developmental toxicity pertains to adverse toxic effects to the developing embryo or fetus.

Toximetris.ai offers in vitro embryonic stem cell test for embryotoxicity based on differentiation of mouse embryonic stem cells, where three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts.

 Endorsement of Scientific Validity: ESAC (2002)

Eye corrosion or irritation is the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible or fully reversible within 21 days of application on animal. Before there is any animal eye irritation/corrosion testing all existing information on a test material should be reviewed. If a test material can be classified, no testing is required. In vitro alternatives that have been validated and accepted may be used to make classification decisions.

Toxometris.ai offers Short Time Exposure (STE) in vitro test method for identifying chemicals inducing serious eye damage that can be used under certain circumstances and with specific limitations for hazard classification and labeling of chemicals (substances and mixtures) that induce serious eye damage as well as those that do not require classification for either serious eye damage or eye irritation, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labeling of Chemicals (GHS).

Endorsement of Scientific Validity: ICCVAM (2013)

International Regulatory Acceptance: OECD TG 491 (2020)

Dermal penetration testing, also known as percutaneous penetration, measures the absorption or penetration of a substance through the skin barrier and into the skin. Both in vivo and in vitro methods are used for determining the percutaneous penetration of a substance. Selection of the method used may result in obtaining different types of information and relevance of the test results to human dermal exposure.

Toxometris.ai offers in vitro methods to determine dermal absorption of chemicals applied to the surface of a skin sample separating the two chambers of a diffusion cell.

Endorsement of Scientific Validity: OECD Expert Group (2011)

International Regulatory Acceptance: OECD 428 (2004)

Skin corrosion refers to the production of irreversible damage to the skin manifested as visible necrosis through the epidermis and into the dermis, following the application of a test chemical [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)].

Toxometris.ai offers an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures, as well as partial sub-categorisation of corrosives, in accordance with UN GHS.

Endorsement of Scientific Validity: ESAC (1998; 2000; 2006; 2009); ICCVAM 2002

International Regulatory Acceptance: OECD 431 (2004; updated 2019); OECD Summary Document 190 (2013)

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METABOLISM

PHOTOTOXICITY

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PYROGENICITY

REPRODUCTIVE & DEVELOPMENTAL TOXICITY

EYE CORROSION/EYE IRRITATION

DERMAN ABSORPTION/PENETRATION

SKIN CORROSION

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SKIN IRRITATION

Skin irritation refers to the production of reversible damage to the skin occurring after exposure to a substance or mixture [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)].

Toxometris.ai offers an in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Endorsement of Scientific Validity: ESAC (2007; 2008; 2009); JaCVAM (2013)

International Regulatory Acceptance: OECD TG 439 (2010; updated 2021)

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SKIN SENSITIZATION

A skin sensitizer refers to a substance that leads to an allergic response following skin contact as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The assessment of skin sensitisation typically involves the use of laboratory animals (e.g. guinea-pigs). Today a combination of non-animal methods (in silico, in chemico, in vitro) within Integrated Approaches to Testing and Assessment (IATA) is able to fully substitute for the animal tests currently in use given the restricted mechanistic coverage of each of the currently available non-animal test methods.

Toxometris.ai offers in vitro test methods, which address mechanisms of key events of the skin sensitization based on the analysis of keratinocytes and/or dendritic cells activation. These test systems are able to discriminate between skin sensitisers and non-sensitisers in accordance with the UN GHS.

Endorsement of Scientific Validity: EURL ECVAM (2014); EURL ECVAM (2015)

International Regulatory Acceptance:

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