In vitro Methods for the Assessment of Primary Local Effects of Topically Applied Preparations

Abstract

Three ‘alternative’ methods for the screening of the primary irritation potentials of topically applied preparations and raw materials are presented as a test battery and their in vitro endpoints are compared with in vivo data. The first method is the intermediate test on the chorioallantoic membrane of hens’ fertilized eggs, which is generally proposed for the prediction of the irritation potential of chemicals. Relevant test parameters, such as the time-dependent appearance of hemorrhages, vascular lysis and coagulation, are used to classify the test substances. Secondly, cytotoxicity tests with Balb/c 3T3 fibroblasts are used to characterize the influence of test materials on cellular homeostasis and viability, which is quantified by the dose-dependent uptake of Neutral red. As a second parameter changes in the cellular protein level can be easily measured by subsequent staining with Kenacid blue. Finally, the third approach to in vitro evaluation is presented in the form of the red blood cell assay, a rapid photometric assay which permits distinguishing basically between damage to the membrane and to proteins as endpoints which correlate with lesions observed on the conjunctiva, iris and cornea in the eye and the acute inflammatory responses evoked during epicutaneous irritancy testing. Changes in the optical behavior of oxyhemoglobin are used as the inherent indicator for monitoring both endpoints and their interrelations. These methods are proposed and can be used advantageously for testing newly developed topical preparations and their ingredients in view of their local irritation potential without further use of animal testing. Moreover, they can also be used for screening chemicals.

A Reassessment of the In Vitro RBC Haemolysis Assay with Defibrinated Sheep Blood for the Determination of the Ocular Irritation Potential of Cosmetic Products: Comparison with the In Vivo Draize Rabbit Test

“…The principal challenge for cosmetic industries and regulatory agencies remains the development of alternative methods that permit the determination of eye irritation and, at the same time, generate results that are consistent with data obtained in rabbits. In this context, the RBC haemolytic assay has been proposed as an alternative method to the Draize eye test by many researchers (15)(16)(17)(18)(19)(20)(21)(22). Initially described by Pape et al (20) to illustrate the phenomenon of haemolysis induced by irritating substances or products, the RBC assay was later standardised by Pape et al (21) to permit the estimation of the irritation potential of tensoactive agents and of cosmetic products that contain these agents, such as shampoos and soaps.…”

Alternative methods for the replacement of eye irritation testing

“…The amount of hemoglobin can then be measured photometrically. The assay is used for surfactants and is applicable for water soluble and water dispersible substances (Pape and Hoppe, 1991;Lewis et al, 1993;Pape et al, 1987). extent than two-dimensional cell layers (Sun et al, 2006).…”

Alternative Methods for Eye and Skin Irritation Tests: An Overview

“…Red blood cells (RBCs), which are readily available, have a long scientific history of being used in the study of the lysis of plasma membranes. The RBC test was developed to assess initial cellular reactions to the irritation caused by certain chemicals 27–29. Certain classes of chemical irritants damage cell plasma membranes and denature several types of proteins.…”