Acute Toxicity Test

XCellR8’s GLP-accredited laboratory provides the first truly animal-free preliminary screen for human acute oral toxicity.

OVERVIEW

The AcutoX test is ideal as a rapid, cost-effective early screen in product development and to generate supporting information in a weight-of-evidence approach to safety assessment.

Acute toxicity describes the adverse effects of a substance that result either from a single exposure or from multiple exposures in a short period of time (usually less than 24 hours).

Our test is a non-regulatory human cell-based method, using human dermal fibroblasts in animal product-free culture conditions, and assesses cell membrane damage (Neutral Red Uptake method) as an endpoint. The output of the test is an IC50 (the dose of cytotoxic compound at which you achieve 50% viability) as an in vitro alternative to the traditional animal-based in vivo LD50. A prediction model is used to place test items into predicted GHS classifications for human acute oral toxicity (category 3, 4 or 5).

It has been internally validated using a panel of 20 well-defined cosmetic ingredients.

TEST SYSTEM: HUMAN DERMAL FIBROBLASTS

The test system comprises of human dermal fibroblasts that are incubated for 24hrs with test items and controls prior to endpoint measurement with Neutral Red. The human dermal fibroblast cultures used in this test are obtained commercially as cryopreserved primary cells. They are originally derived from donor tissue (for example, following plastic surgery) with informed consent for the tissue to be used for research purposes, in adherence with the Human Tissue Act (UK) 2004. The cells have been extensively QC tested for a range of parameters including viability upon thawing from cryopreservation, proliferation rate, morphology and sterility (absence of bacteria, fungal growth and mycoplasma). They have also tested negative for HIV-1, HIV-2, HBV and HCV. They are maintained in the exponential growth phase in routine culture at 37°C / 5% CO2 prior to seeding into test plates. The culture medium and all culture reagents are free of animal-derived components, providing a fully human cell culture system.

SUMMARY OF THE TEST METHOD

Range finding experiment

• Human dermal fibroblasts are seeded into 96 well plates and cultured for 24hrs under standard culture conditions (37°C, 5% CO₂ and ≥95% relative humidity)
• Following this, the medium is removed and the cells are dosed with test items and controls
• The positive control used is Sodium Dodecyl Sulphate (SDS)
• The test items are dosed using a wide dilution series (2 fold dilutions) based upon the highest soluble concentration
• After a 24hr incubation with the test item, the cells are washed and then incubated with Neutral Red stain, which is incorporated into live cells
• The IC50 value is calculated for the test item and is used to set the dosing range for the main experiment
• One replicate is used to determine the appropriate dosing range
• A range of acceptance criteria must be met in order to confirm validity of the test

Main Experiment

• Setup and acceptance criteria are identical to the range finding experiment, with the exception of the test item dosing range
• This is based around the range finding experiment IC50 value and a narrower series of concentrations is used to further hone-in on the IC50 value
• Multiple replicates are used to demonstrate consistency of results
• The IC50 values derived are used to identify the GHS category of the test item using the prediction model developed for the assay
• The prediction model can sub-categorise into GHS Category 3 (Toxic if swallowed), 4 (Harmful if swallowed) or 5 (Potentially harmful if swallowed)

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