Acute Toxicity Test

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.