Vaginal irritation test

EPIVAGINAL™ ET-50 METHOD

WHY CONDUCT A VAGINAL IRRITATION TEST?

The EpiVaginal™ ET-50 test provides a scientifically advanced approach for feminine product safety testing, using a human reconstructed vaginal epithelium. It enables the rapid generation of reliable, human-relevant data on the vaginal irritation potential of a product or ingredient, without the use of animals.

OVERVIEW

EpiVaginal™ utilises tissue engineering technology to create a human vaginal epithelium,
which closely parallels both the structure and cellular physiology of the in vivo tissue. It can
be used as a model for short or long-term exposure to diverse substances such as
spermicides, hormones, anti-microbial agents, products of intimate hygiene, contraceptives
and lubricants, as well as any personal care products applied to the skin in the intimate area.

The test item is applied topically to the surface of the model. 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 vaginal tissues.

The exposure time causing a 50% reduction in tissue viability (ET-50) is determined and test
items are assessed for vaginal irritation potential against well-defined, published
benchmarks. The ET-50 value can also be used to place a series of items into a rank order
of vaginal irritation potential.

A special extended protocol is also available for mild and ultra-mild test items. It is possible,
upon special request, to save the culture medium from the basal side of the tissues for the
analysis of inflammatory mediators such as LDH, PGE-2 or IL-1α.

TEST SYSTEM: RECONSTRUCTED HUMAN VAGINAL EPITHELIUM

EpiVaginal™ is a reconstructed human vaginal epithelium composed of normal human
ectocervico-vaginal (ECV) epithelial cells. The in vitro model closely resembles the
histological, ultrastructural, and protein expression properties of native tissue, including a
multilayered three-dimensional morphology, inter-digitation of cells, glycogen production,
and cytokeratin expression. The cells are metabolically active and can release inflammatory
agents.

EpiVaginal™ is grown on a microporous membrane at the air-liquid interface, allowing for
direct application of test items onto the tissue surface. It is therefore suitable for testing a
wide variety of ingredients and formulations, irrespective of their solubility in aqueous cell
culture medium.

SUMMARY OF THE TEST METHOD

  • EpiVaginal™ models are pre-incubated for 60 minutes or overnight at 37°C/5% CO 2 .
  • The test item is applied to the tissue surface (100µl or 100mg)
  • Controls consist of ultrapure water (negative control) and 1.0% Triton X-100 (positive control).
  • The dosed vaginal models are placed in a cell culture incubator for 1 hour, 4 hours and 18 hours, using triplicate models for each time point. For mild and ultra-mild test items, a 48 hour exposure time is included.
  • After appropriate incubation period, tissues are gently rinsed to remove residual material.
  • If required, culture medium is collected for subsequent analysis of LDH, PGE-2, IL-1α or other inflammatory markers.
  • The viability of the vaginal models is assessed by enzymatic conversion of the vital dye MTT. A solution of MTT is applied to the basal side of the tissues, which are placed into a cell incubator for 3 hours. The blue formazan metabolite produced by viable cells is then extracted into isopropanol by incubation at room temperature for 2 hours or overnight.
  • Duplicate samples of the extracted formazan solution are transferred to a microplate and the formazan product is quantified by absorbance spectrophotometry (wavelength 570nm).
  • Absorbance readings quantifying the amount of formazan product are used to calculate the ET-50 value for test items and controls. (ET-50 is the time taken to reduce the viability of the EpiVaginal™ model by 50% compared to the negative control). In some cases, based on the initial range finding experiment,
    additional time points may be necessary (between 4-8 hours or 8-12 hours) to define an accurate ET-50 value.
  • The value obtained from this initial 16 minute incubation with testsubstance determines the time points for 2 further incubations, which are then carried out using a repeat of the above process. Time points are determined as follows. If the viability is >90% after the initial 16 minute exposure, subsequent time points are 64 and 256 minutes. If the viability is <90% but >30% after the initial 16 minute exposure, subsequent time points are 4 and 64 minutes. If the viability is <30% after the initial 16 minute exposure, the subsequent time points are 1 and 4 minutes. Triplicate positive controls (0.3% Triton X-100) are incubated for 4, 15 and 45 minutes.
  • Acceptance criteria must be satisfied for the experimental run to be valid. The ET-50 value for the positive control should be between 0.75-1.75 hours.
  • ET-50 values are compared to a range of published benchmark values, to assess the irritancy level of the test item.

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Vaginal irritation test

At a glance

What it measuresViability of vaginal epithelium tissue
ComplianceNon-regulatory
Turnaround time6-8 weeks
MethodEpiVaginal™
Min. sample required10ml (liquids) / 10g (solids)
Test codeCT-046

Always animal and animal product-free

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