Acute Toxicity Test

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

 (OVERVIEW)

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 integrity (Neutral Red Uptake method) and cell metabolism (MTT method) as an endpoint. The assay includes a human liver extract (S9) to examine the toxifying or detoxifying effect of metabolism on a test item. 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 EPA and GHS classifications for human acute oral toxicity.

It has been internally validated using a panel of nearly 70 test items with a range of chemistries and from a variety of use cases.

TEST SYSTEM: HUMAN DERMAL FIBROBLASTS

The test system comprises of human dermal fibroblasts that are incubated with test items and controls prior to endpoint measurement with Neutral Red and MTT. 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 (with metabolism)

  • Human dermal fibroblasts are seeded into 96 well plates and cultured for 24hrs in a humidified tissue culture incubator set to 37°C and 5% CO2.
  • Media is removed and the cells are dosed with test items and controls.
  • The positive control used is 2-Butyne-1,4-diol
  • The test items are dosed using a wide dilution series (10-fold dilutions) in the presence of metabolically active liver extract (S9) and the relevant co-factors for 3 hours. based upon the highest soluble concentration.
  • Cells are washed and incubated for a further 21 hours.
  • After a 24hr (total) incubation, the cells are washed and then incubated with Neutral Red stain, which is incorporated into live cells.
  • The EC50 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 dosing range and several acceptance criteria must be met in order to confirm validity of the test.

Range finding experiment (without metabolism)

  • The Range finding experiment without metabolism follows the same protocol as above, save the following deviations.
  • Positive control Chlorpromazine Hydrochloride is used.
  • The cells are incubated with the test items for 24 hours.

 Main Experiment (with metabolism)

  • Human dermal fibroblasts are seeded into 96 well plates and cultured for 24hrs in a humidified tissue culture incubator set to 37°C and 5% CO2.
  • Media is removed and the cells are dosed with test items and controls.
  • The positive control used is 2-Butyne-1,4-diol
  • The test items are dosed using a 2-fold dilution based around the EC50 calculated in the range finding experiment, in the presence of metabolically active liver extract (S9) and the relevant co-factors for 3 hours
  • Cells are washed and incubated for a further 21 hours.
  • After a 24hr (total) incubation, the cells are washed and then incubated with Neutral Red stain, which is incorporated into live cells and MTT, which provides a readout of cellular metabolic health.
  • The cell viability and an EC 50 are calculated
  • Multiple replicates are used to demonstrate consistency.
  • EC 50values derived from this assay are used to identify EPA classification and chemicals that produce a similar EC 50

 Main Experiment (without metabolism)

  • The main experiment without metabolism follows the same protocol as above, save the following deviations.
  • Positive control Chlorpromazine Hydrochloride is used.
  • The cells are incubated with the test items for 24 hours.

    AcutoX Acute Toxicity Test

    At a glance

    What it measuresViability of fibroblasts
    ComplianceNon-regulatory
    Turnaround time6-8 weeks
    MethodNeutral Red uptake and MTT test with and without metabolism
    Sample required10ml (liquids) / 10g (solids)
    Test codeCT-044

    Always animal and animal product-free

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