Inhalation Toxicity Test

WHY CONDUCT AN INHALATION TOXICITY TEST?

This non-regulatory test, also known as a lung irritation test, provides a profile of damage to the lining of the human airway at a range of concentrations, resulting in a classification of inhalation toxicity potential.  Using the IC75 value, you can also place ingredients or formulations into rank order of irritation potential or compare them against industry benchmarks.

OVERVIEW

Inhalation toxicity is defined as the degree to which a test item causes damage
(toxicity) to cells of the human airway following exposure to a single substance or
mixture for up to 4 hours. It forms a critical part of product safety assessment for
consumers as well as occupational safety for manufacturing workers, and is relevant
for a wide variety of volatile chemical ingredients including cosmetic fragrances,
adhesives, fuels, environmental pollutants and nanoparticles.

The test described here is a non-regulatory method for the assessment of chemicals in
terms of their potential to cause irritation to the human airway epithelium. The method can
be used to differentiate between products that have toxic potential to airway tissue or not,
and to place chemicals or products into a rank order of irritation potential.

The test method utilises a 3D human airway tissue model (EpiAirway™), which mimics the
biochemical and physiological properties of the epithelial tissue of the human respiratory
tract. The test item is directly applied to the tissue surface in an aqueous or corn oil vehicle,
depending upon solubility. Cell viability is measured by enzymatic conversion of the vital dye
MTT into a blue formazan salt that is quantitatively measured after extraction from the airway
tissues. Toxicity is determined by calculation of the IC75 value: the concentration of the test
item required to reduce the viability of the airway model to 75%. IC75 values are then used
to classify a test item as toxic should it fall below a threshold value.

TEST SYSTEM: RECONSTRUCTED HUMAN BRONCHIAL EPITHELIUM

EpiAirway™ is a respiratory tract model composed of normal human
tracheal/bronchial epithelial cells which have been cultured to form a multi-layered,
highly differentiated model. It closely resembles the epithelial tissue of the respiratory
tract, consisting of a pseudostratified epithelial structure complete with tight junctions
as well as microvilli, cilia and mucin secretion at the apical surface, analogous to the
normal human in vivo tracheal/bronchial epithelium. Transepithelial electrical
resistance of the tissues also mirrors that seen in vivo. The cells are both
metabolically and mitotically active and release many of the pro-inflammatory agents
(cytokines) known to be important in inhalation irritation and inflammation.
EpiAirway™ is grown on special platforms at the air-liquid interface, allowing for
direct application of test items in a way that accurately models “real life” airway
exposure. Where appropriate, caps can be used to prevent excessive evaporation of
volatile substances.

SUMMARY OF THE TEST METHOD

• EpiAirway™ models are pre-warmed in a cell culture incubator (37°C / 5% CO2)
  overnight. The culture medium is replaced prior to treatment and any
  accumulated mucus removed by gentle washing.
• Initially, the EpiAirway™ models are subjected to a Range Finding Experiment
  where the test item is applied to the apical surface of the tissues over a broad
  range of concentrations for three hours, to determine an approximate IC75
  (duplicate models per condition).
• The negative control consists of ultrapure water.
• Test items and controls are removed by gentle washing and the viability of the
  EpiAirway™ models is assessed by MTT conversion. MTT solution is applied to
  the surface of the models and placed into a cell culture incubator for 90 minutes.
  The blue formazan metabolite produced by viable cells is then extracted
  following the addition of extractant solution and gentle shaking for two hours at
  room temperature.
• Duplicate samples of the extracted formazan solution are transferred to a
  microplate and the formazan product is quantified by absorbance spectroscopy
  (wavelength 570 nm).
• Absorbance readings of the formazan product from EpiAirway™ models
  incubated with test items are compared with those of negative controls to
  calculate percentage viability and an IC75 value.
• The IC75 values obtained from this initial Range Finding Experiment determines
  the concentrations used in the Main Experiment, which facilitates definitive IC75
  value determination. The Main Experiment is performed as described above for
  the Range Finding Experiment, with the only deviations being the concentration
  range used for each test item, performing each condition in at least duplicate, and
  including a positive control (14.7 mg/ml formaldehyde).
• A range of acceptance criteria must be satisfied in order for the experimental run
  to be valid.
• Test items with IC75 values below specific thresholds are classified as toxic. The
  threshold is 200mg/ml for the GHS system (distinguishing categories 1-3 from 4-
  5); or 150mg/ml for the EPA system (distinguishing categories I-III from IV).
• The IC75 values can also be used to place a range of items into a rank order of
  irritation / toxicity potential, and compared with industry benchmarks.
• A detailed study report is provided, along with additional support for the
  interpretation of data where required.

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