Alternatives to Laboratory Animals

There is limited availability of reliable and valid information on alternative methods to the use of experimental animals from open access data banks. In 1989, the Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) at the German Federal Health Office started a data bank to meet requirements of the German Animal Protection Act. In comparison with other data banks in this field, ZEBET's data bank comprises two types of evaluation: firstly, whether a specific method contributes to the Three Rs concept of Russell & Burch, i.e. replacement, reduction and refinement, and secondly, whether the method meets the criteria of validation as outlined in the report of the Amden workshop on validation of toxicity test procedures. This information is primarily available to the scientific and ethical commissions which have been introduced by the Animal Protection Act in order to reduce the numbers of animals used in experiments in Germany. However, other users of the ZEBET data bank are also welcome, e.g. local animal protection officers, interested scientists and the public. Although the language of ZEBET's data bank is German, the documentation also contains English summaries, key words, and updated lists of references. Here, the concept of ZEBET's data bank and its scientific background are described and compared with other alternatives data banks.

Non-animal procedures, including in vitro test systems and test strategies, can already make a significant contribution to the background to risk assessment — in predicting both the toxic potential and toxic potency of chemicals, as well as, in some circumstances, the toxic hazard they may represent under specified conditions of exposure. They can be particularly useful for investigating molecular and cellular mechanisms of chemical-induced toxicity, and for identifying species-specific effects, which greatly limit the value of data from laboratory animal studies in the human risk assessment process. Attention is focused on the need for greater effort to be invested in the development of non-animal procedures for evaluating the biokinetic factors which will determine the ultimate form and concentration of a particular chemical at possible sites of toxic action. The relative merits of correlative and mechanistic approaches to test development and test validation are discussed. The need for realism is emphasised, not only in relation to our expectations of the validation process, but also in terms of the current and future status of regulatory toxicology, in vitro or in vivo, as a scientific discipline. Finally, it is concluded that the intelligent and strategic use of in vitro test systems, in conjunction with predictive computer modelling, could markedly improve the scientific basis of human risk assessment.

Twenty years ago Imai & Nose introduced a whole-blood clotting test for the estimation of haemocompatibility of biomaterials in vitro In our paper a modification of this assay is described and the mechanism of clot formation further elucidated. It was found that neither the inhibition of platelet function nor the removal of platelets from blood significantly changed the clot formation rate on glass and polyvinyl chloride in comparison to the rate tor whole blood. Scanning electron microscopy demonstrated that platelets were not involved in clot formation near the blood/biomaterial interface. Thus, it was concluded that the system of contact activation of the coagulation cascade dominates during clot formation under static conditions. The latter conclusion was supported by the fact that preadsorption of human serum albumin or human fibrinogen onto the glass plates used, decreased the clot formation rate in the same manner.

In the framework of the multicentre evaluation of in vitro cytotoxicity (MEIC) programme, the first ten chemicals of the prescribed list were tested for acute toxicity in four standardised cyst-based aquatic invertebrate tests, consisting of two rotifer species (the estuarine Brachionus plicatilis and the freshwater Brachionus calyciflorus) and two crustacean species (the halophilic anostracan Artemia salina and the freshwater anostracan Streptocephalus proboscideus). Mortality was the test criterion and toxic effects, expressed as 24-hour LC50 values, were correlated with rodent and human acute oral toxicity data. Generally, a good correlation was obtained between any of the invertebrate tests and the rodent data. Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in man was slightly better than the rat test for eight (excluding diazepam and digoxin) and nine (including diazepam, excluding digoxin) of the ten substances. The aquatic test systems, however, appear to be more suitable for compounds soluble in water.

As a result of a workshop held at Täljöviken, Åkersberga, Sweden, on 27–29 May 1991, a multicentre collaborative research project was established, with the purpose of developing the concept of integrated in vitro toxicity testing. The first priority was the selection of tests within eight appropriate areas: basal cytotoxicity, irritancy, developmental toxicity, hepatotoxicity, nephrotoxicity, immunotoxicity, neurotoxicity and biokinetics. An ideal battery of tests for each area was identified. Since it was realised that it would not be feasible to include the full ideal list of tests in the project, a minimum test list was also agreed. For each area, ten calibration chemicals were selected. From these 80 compounds, 30 were selected for inclusion, together with 20 of the OECD test programme chemicals, in a first test set of chemicals. The toxicity of these 50 test set chemicals will be investigated in the minimum integrated test scheme.

The aim of the project is to employ non-animal methods to assess the toxicological properties of chemicals, and to improve this assessment through the use of knowledge about mechanisms of toxic action. The information produced will contribute to the establishment of a more-scientific and more-efficient toxicological procedure for hazard assessment. Questions concerning which parameters need to be investigated and combined to make hazard assessments, and which parameters relevant to in vivo toxicity can be determined in non-whole animal test systems, will also be addressed.

The HET-CAM (hen's egg test-chorio-allantoic membrane), described by Luepke in 1985, permits the study of immediate effects following the administration of test substances to the chorioallantoic membrane of 10-day-incubated White Leghorn chicken eggs. The results of a study of 60 chemicals and 41 cosmetic formulations are presented here.

Intralaboratory reproducibility was established with a double-blind study of 20 surfactants at two concentrations. The results show a high rank correlation between the scores given by both experimenters: the Spearman's rho is greater than 0.9 (p < 10^-8).

The 60 chemicals were studied at a concentration equivalent to 10% of the concentration tested in vivo. They were classified according to the three EEC categories of ocular irritancy, and when a correlation between the HET-CAM scores and historical Draize in vivo data was determined, the corresponding Spearman's rho value was 0.72 (p < 10^-4).

The 41 formulations (make-up removers, shower gels, shampoos, creams and body milks) were tested by two protocols: rinsed and non-rinsed. The correlation between the HET-CAM scores and the historical Draize in vivo maximum average scores was studied, and the rhos obtained were 0.77 and 0.76 (p < 10 ^-8), respectively.

The advantages of the HET-CAM method lie in its sensitivity, rapidity and moderate cost. Both chemicals and cosmetics formulations can be tested, and specific families can be assessed using modified and more-appropriate protocols. However, to ensure that a good reproducibility and sensitivity is provided, the use of reference materials is strongly recommended.

The satisfactory performances of the HET-CAM test in many previous evaluation studies (e.g. those of the BGA, CTFA, EEC and OPAL) show that it can be a useful assay as part of a battery of in vitro tests for the screening of new ingredients and formulations.

The in vitro agarose overlay method was investigated as an alternative to the Draize eye irritation test for the evaluation of the ocular irritancy of cosmetic products.

Modifications to the original protocol include the definition of an area of lysis (expressed as a weighty)/toxicity relationship for each product by a planimetric method and the creation of an agarose classification.

56 different cosmetic formulations, including emulsions, gels, lotions and tonics, were evaluated using this modified test, and the results were correlated with in vivo Draize data. Four different agarose classes were delimited: non-irritant; minimally irritant; mildly irritant; and irritant; corresponding respectively to the following scoring scales (x 10 ⁴g): 0; 0 <x<200; 200≤ x <450 and ≥ 450.

A high correlation between the agarose overlay method and the Draize test was observed when taking into account two classes: non-irritant (non-irritant, minimally irritant) and irritant (mildly irritant, moderately irritant [Draize] or irritant [agarose]) as shown by an 86% concordance value. In this study, the sensitivity was 92% (11–12 irritants were predicted by the agarose diffusion method) and the specificity was 84%. The overestimation error of 16% suggests that the agarose overlay method might be slightly more sensitive than the Draize eye test.

When applying the kappa test, the agarose overlay method reached a 64% coefficient, attesting that the good correlation with the Draize test was not due to chance. In relation to the Landis & Koch classification, this percentage places the agarose overlay method in the “good concordance” class.

Based on these results, the use of the agarose overlay method for screening the ocular irritation potential for the cosmetics industry can be considered to be a very interesting alternative to be included in a battery of tests.

The use of a microporous membrane as an interface between the cell monolayer and a substance dissolved in a non-aqueous, non-cytotoxic medium has permitted the study of the cytotoxicity of non water-soluble substances without the addition of any adjuvant for dispersion or solubilisation. The LC50 values of raw materials widely used in cosmetic and pharmaceutical products were established on human skin fibroblasts. This method was also applied to the study of cosmetic products. A relationship between exposure time and cytotoxicity was established for 169 products and a cytotoxicity index (MCI) was calculated. When the results were compared to the Draize maximal ocular irritation index (MaOI), a very good correlation was shown between MCI and MaOI.

The SIRC cell line, derived from rabbit cornea was used to assess the cytotoxicity of a wide range of surfactants. The measurement of their effect on the cell membrane, the critical target of surfactants, has been performed using the neutral red uptake (NRU) bioassay. The results obtained for 30 surfactants have been correlated with in vivo data (Draize test) obtained from a data bank. The cytotoxicity of the tested surfactants ranks as follows: cationic > anionic > amphoteric > non-ionic. A good correlation (r = 0.80 and p < 10^-4) was obtained. In the case of non-ionic surfactants (n 11) a higher correlation with in vivo data was obtained (r = 0.92 p < 10^-4), showing that the SIRC-NRU test is particularly useful for the evaluation of non-irritants or weak irritants currently used in cosmetic formulations. The results demonstrated that this easily performed method leads to few false negatives (6%) and thus merits its inclusion in a battery of screening tests for predicting eye irritancy without the use of animals.

International guidelines for the evaluation of ocular irritation advise that all the available information, structural and physicochemical, should be taken into account in order to minimise the testing of substances under conditions that are likely to produce severe reactions in laboratory animals.

In order to determine whether the pH and the acidic/alkaline reserve would be suitable parameters for the prediction of ocular irritation, we have measured, under standard conditions, the pH and the acidic/alkaline reserve of 166 chemicals (90 non-irritant, 22 irritant, and 54 severely irritant for the eye, according to the EEC classification and labelling system).

The combination of these parameters permitted the correct identification of 74% of the severely irritant chemicals and 97% of the non-severely irritant (non-irritant or irritant) chemicals. The predictive value for a severely irritant potential was 93%, and 88% for non-severely irritant chemicals.

This physicochemical approach, used as a first line screen for ocular irritation, is very simple and inexpensive.

In vitro evaluation of ocular irritation requires the use of a battery of tests, for example cell culture assays and the HET-CAM test. In order to check the field applications of the different methods and their complementation, we have studied the EYTEX™ physicochemical model in an internal validation study.

In this study, we compared the EYTEX™ results with the in vitro ocular irritation potencies of 52 formulations and 49 chemicals. The in vivo/in vitro correlation was highly significant, p < 0.01%, K = 0.8 (Kappa method). On the other hand, the concordance, sensitivity and specificity of this method were, 85%, 89% and 83% respectively. The compatability of 98% obtained showed the kit to be applicable to cosmetic formulations.

The EYTEX™ system has also been evaluated during a comparative study of EYTEX™ human clinical tests, performed on 12 finished products; we have observed a concordance equal to 92%, indicating an excellent correlation between the two methodologies.

These results suggest that the EYTEX™ test could be a reliable in vitro prescreening system for determining the eye irritation potential of cosmetic formulations, and that it should be accepted as a component of our battery of alternative methods.

Various cell culture models have been proposed as partial or total replacements for in vitro ocular irritation tests. The cytotoxic potential of substances can be evaluated according to various parameters for measuring cellular viability and metabolic capacity. Cell cultures have their limitations, but they also have advantages which justify the inclusion of some such models in a battery of in vitro tests that could replace the Draize test.

The so-called Draize eye test, the offical method used worldwide for the evaluation of ocular irritancy, has been heavily criticised in recent years concerning its scientific credibility and its extensive use of animals.

Numerous methods have been, or are being, developed with the aim of complementing and/or replacing the Draize test. Regardless of their nature, these new techniques will have to be validated to guarantee their scientific credibility. Used singly or in batteries, the alternative tests will have to evaluate all the components of ocular toxicity, and in particular the reversible or non-reversible nature of the observed effects.