Testing vaginal irritation with the Hen’s Egg Test-Chorioallantoic Membrane assay

Rita Palmeira-de-Oliveira

doi:10.14573/altex.1710091

Abstract

The HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) assay is an in vitro alternative to the in vivo Draize rabbit eye test. This qualitative method assesses the irritancy potential of chemicals. The chorioallantoic membrane responds to injury with an inflammatory process similar to that in the rabbit eye’s conjunctival tissue. Regarding topical toxicity assessment of medical devices, ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irritation potentials for the eye and the vaginal epithelia are similar. The aim of this work was to apply the HET-CAM assay to test the irritancy potential of vaginal formulations. Vaginal semisolid medicines and lubricants currently marketed were tested along with the Universal Placebo formulation that has been shown to be clinically safe. Nonoxynol-9 (N-9), a known vaginal irritant, was enrolled as positive control (concentrations ranging from 0.001 to 100% (v/v)). The assay was conducted according to the ICCVAM – Recommended Test Method (NIH Publication No. 10-7553 – 2010). Formulations were then classified according to irritation score (IS), using the analysis methods (A) and (B). The studied vaginal formulations showed low potential for irritation. N-9 was classified as a severe irritant at concentrations above 2%, which is in line with clinical data, envisaging a possible in vitro/in vivo correlation. IS (B) was considered a more detailed classification output. Although still requiring further validation, the HET-CAM assay seems an ideal prospect for in vitro vaginal irritancy testing.

Highlights

Topical toxicity has been a main topic of work within the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM)
Regarding topical toxicity assessment of medical devices, the ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irritation po
At the concentration of 0.5% (v/v) the difference between the two testing criteria results was statistically different, it must be noted that standard deviations were relatively high

Summary

Introduction

Topical toxicity has been a main topic of work within the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM). Having a regulatory responsibility, the EU and associated laboratories have been working on developing and validating toxicity test methods for eye irritation, phototoxicity, skin corrosion, irritation and sensitization. The European Commission has undertaken several regulatory decisions in pursuance of the 3Rs policy (Replacement, Reduction, Refinement) such as the approval of the REACH (Registration, Evaluation and Authorization of Chemicals) legislation (applicable to the fields of raw materials, drug products and medical devices, for example) and the prohibition of the marketing of cosmetic products tested in animals (EC, 2003). The assessment of the irritancy and toxicity of products for vaginal administration does not yet include validated in vitro methods. Regarding topical toxicity assessment of medical devices, the ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irritation po-

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