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On-line version ISSN 1678-4324
Braz. arch. biol. technol. vol.55 no.3 Curitiba May/June 2012
HUMAN AND ANIMAL HEALTH
Andréa Martins da NóbregaI; Eloisa Nunes AlvesI; Rosaura de Farias PresgraveI; Rodrigo Netto CostaII; Isabella Fernandes DelgadoII, *
IDepartamento de Farmacologia e Toxicologia
IIDeparatmento de Imunologia; Instituto Nacional de Controle de Qualidade em Saúde; Fundação Oswaldo Cruz; Rio de Janeiro - RJ - Brasil
In an attempt to build the evaluation strategies to assess the human eye irritation, a reassessment of some in vitro tests is necessary, particularly concerning the non-irritants, mild and moderate irritants. Thus, the correlations between results obtained from the Draize test with the in vitro methods HET-CAM (Hens Egg Test-Chorion Allantonic Membrane) and RBC haemolysis assay to assess the ocular irritancy potential of 20 eye drops were examined. Parameters such as accuracy (%) and specificity (%) were determined. All results were correlated with RBC correctly with the results obtained in the Draize test. The HET-CAM presented four false-positive results, showing a tendency of data overestimation. Despite the high specificity provided mainly by the RBC, it would be necessary to test a wider range of products representing all the scales of irritation to confirm its ability to be used as a first alternative to test products that could be presumptive non-irritants.
Key words: Alternative methods, eye irritation, Draize Test, low-irritant, HET-CAM, RBC
As a result of a number of injuries and deaths from the consumer products in the 1930s, the United States Congress approved the Federal Food, Drug and Cosmetic Act (1938), which obligated premarket safety testing for a variety of products. Animal-based toxicological test methods were developed at that time in response to a recognized need by the industry and government regulatory agencies for supplying reliable products to ensure the consumer safety (Wax 1995).
One component in the risk assessment of the cosmetics - and other products that may come into contact with the human ocular surface - is the determination of its potential to induce eye irritation. The Draize eye irritation test (Draize et al. 1944) has been used for about 60 years in attempt to predict human eye injury and it is based on topical instillation of the potential irritant and scoring of ocular adverse effects by examination of the rabbit eye (Wilhelmus 2001). This test has been a subject of controversy among the animal rights groups and even in the scientific community. It has been criticized on the basis of dose volume, methods of exposure, use of animals as models, number of animals, observation and scoring, including the laboratory procedure variability, interpretation of results and the over prediction of human response (Princen 2006). However, the Draize eye irritation test continues to be the primary method accepted by the regulatory agencies worldwide (Vinardell and Mitjans 2008; Scott et al. 2010).
Humane and scientific concerns regarding the use of animals in toxicology have prompted development of many in vitro methods as potential alternatives for animal tests. Despite some progress in the areas of reduction and refinement, no single test has emerged as being acceptable as a complete replacement for the conventional Draize test, so that development of a battery of non-animal eye irritation alternative tests is actively being proposed (McNamme et al. 2009; Barile 2010).
On the development of in vitro eye irritation tests, a variety of methods such as cell-based cytotoxicity, reconstituted tissue models, chorioallantoic membrane (CAM) methods, isolated organ assays and other systems have been developed since the 1980s (North-Rooth et al. 1985; Luepke and Kemper 1986; Pape et al. 1987; Roguet et al. 1992; Balls et al. 1995; Harbell et al. 1997; Spielmann et al. 1997; Jones et al. 1999; Pape et al. 1999; Steiling et al. 1999; Cooper et al. 2001; Hutak et al. 2003; Eskes et al. 2005; Doucet et al. 2006; Vinardell and Mitjans 2006; Alves et al. 2008; Cater and Harbell 2008; Schutte et al. 2009; Takahashi et al. 2009). On the other hand, the relevance of some currently used in vitro tests for a reliable prediction of human eye irritation, particularly concerning mild and moderate irritants, is still uncertain. Recently, two alternative methods, i.e., the Bovine Corneal Opacity and Permeability (BCOP) and the Isolated Chicken Eye (ICE), have been scientifically validated (OECD 2009). However, those assays are solely employed to corrosive and severe ocular irritant ingredients, being not applicable to a great number of ingredients with low-irritant potential or products that produce human eye distress in the absence of macroscopic adverse clinical evidence. The present challenge is to develop a non-animal test method - or an assessment strategy - suitable to evaluate mild and moderate irritant materials (Debbasch et al. 2005; McNamme et al. 2009; Barile 2010).
In attempt to build up such an assessment strategy to evaluate the human eye irritation, a re-evaluation of some currently used in vitro tests is needed, particularly concerning the non-irritants, mild and moderate irritants. Therefore, in the present study, we investigated the irritation potential of products previously tested in vivo: (i) in the Red Blood Cell (RBC) haemolysis assay, and (ii) in the Hen's Egg Test-Chorioallantoic membrane (HET-CAM). According to a recently proposed in vitro testing strategy approach for eye irritation called "Botton-Up Approach" (Scott et al. 2010), these two assays are part of a group of in vitro tests that have the greatest potential to distinguish the non-classified substances from irritants. Thus, the aim of the present study was to investigate the relevance of HET-CAM and RBC test methods in the assessment of irritant potential of low-irritant products and to correlate their results with those of in vivo Draize eye irritation test.
MATERIAL AND METHODS
The products used in the present study comprised 20 eye drops acquired from the commercial establishments in Rio de Janeiro, Brazil. A description of the product formulations used in this study can be found in Table 1.
The in vivo test
The Draize test was previously performed in the laboratory routinely and the results described belonged to the database bank. Thus, the in vivo test was performed as follows. Five male or female New Zeland albino rabbits, weighing 2-3kg, were used in the in vivo test. The animals were maintained in individual cages, with water and food ad libitum, at 20+2°C and humidity of 70%. The protocol of the ocular irritation test was approved by the Animal Use Ethics Commission (CEUA/FIOCRUZ). One hundred microlitres of each product were instilled into one of the eyes, following by massaging for 30 seconds, while the other eye was used as control. The reading were performed at 24, 48, 72 h and seven days after the application, and the corneal, iris and conjunctival alterations were graded according to the Draize scale (Draize et al. 1944). To classify the eye irritation potentials of the products, the Kay and Calandra methodology was used (Kay and Calandra 1962), which took into account the persistence and severity of the irritation response. After the last reading, the animals were euthanasiated by intravenously injection of 100mg thiopental/Kg.
Preparation of the erythrocyte suspension
Defibrinated sheep blood was obtained from the Laboratory Animals Breeding Center (CECAL) at the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. Erythrocytes were separated by centrifugation at 1302g for 15 minutes at room temperature, washed three times with phosphate-buffered saline solution (PBS, pH 7.4), and re-suspended at a concentration of 8x109 cells/ml in PBS supplemented with 10nmol/l glucose. This suspension was maintained at 4°C for up to four weeks. The cells were returned to room temperature prior to the use.
The assay procedure
The assay was performed according to the method of Pape et al., (1987). Eight concentration of each product diluted in PBS were incubated with erythrocytes (8x109 cells/ml) for ten minutes, with constant shaking at room temperature. The incubation was terminated by a high-speed (7520g) centrifugation. The extent of haemolysis was determined spectrophotometrically (UV-160A UV/VIS dual-beam spectrophotometer; Shimadzu Corporation, Kyoto, Japan) at 540nm against a blank, which contained only the sample diluted in PBS. The hemoglobin denaturation was assessed by monitoring the absorbance at 575 and 540 nm (UV-160A; Shimadzu Corporation), against a blank containing only the sample diluted in PBS. The denaturation index was calculated as DI [(R1-Ri)/(R1-R2)]x100, where R1, R2 and Ri were the ratios between the absorbance readings of the hemoglobin released from the erythrocytes when in contact with: (i) distilled water; (ii) sodium lauryl sulphate at 1mg/ml and (iii) the product under analysis, respectively.
The extent of haemolysis, expressed as a percentage, was calculated as the absorbance of an erythrocyte suspension incubated with each product, relative to that of a completely hemolysed control (100%) at 540nm. The H50 was determined from the concentration-response curves. The irritation potential (IP), defined as the H50/DI ratio, was used for comparison with the data obtained in vivo.
The HET-CAM assay was carried out in accordance with the official method (Journal Officiel de la Republique Française 1996). Ten-day-old fertilized eggs from White Leghorn chicken were incubated on an automatic rotating device (Premium Ecológica, Minas Gerais, Brazil) at 38.0 ± 0.5°C and 70% relative humidity. The eggs were obtained from the Immunobiological Technology Institute (Bio-Manguinhos) at the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. On day 10 of incubation, the egg shell was opened at the side of the air chamber and the inner egg membrane was carefully removed avoiding any damage to the fine blood vessels of CAM. Three hundred microliters undiluted test products were applied to the chorioallantoic membrane. Four eggs were used for each product. After 20 seconds of contact, the membrane was rinsed with 37°C -isotonic NaCl solution. The time up to appearance of the reactions (hyperaemia, haemorrhage, and coagulation) was assessed and the overall irritation score (0 to 21) was calculated as the mean sum of individual scores of all the endpoints from three replicate assays.
The results obtained in this study were analyzed by using the performance comparisons between the in vitro assays and the Draize eye irritation test, derived from the contingency tables. The following values were calculated: specificity (the ratio of in vivo non-irritants classified in vitro as non-irritants); accuracy (the ratio of product classes [irritants and non-irritants] correctly classified in vitro) and false positives. Sensitivity (the ratio of in vivo irritants classified in vitro as irritants) was not determined, since not a single positive result was obtained in the in vivo test, and therefore also there were no false negatives.
The in vitro and in vivo irritation potential of the products analyzed in this study are described in Table 2. All the eye drops tested in vivo (N = 20) were classified as non-irritants. This same classification was obtained in the RBC assay. However, in the HET-CAM test, the products CO03, CO05, CO07 and CO17 were classified as slight irritants.
The predictive abilities of the RBC and HET-CAM assays in relation to the Draize Test were evaluated. For such, a contingency table comparing both in vitro assays with the in vivo test was set up (Table 3). The comparison of the RBC assay with the Draize Test showed 100% specificity and accuracy. When the HET-CAM assay was compared with the Draize Test, specificity and accuracy were 80%. Four products showed false positive responses (CO03, CO05, CO07 and CO17), as demonstrated in Table 4.
For validation, new toxicological test methods must demonstrate their reliability, which means repeatable and reproducible, and that they are relevant, which means the method is predictive and has a biological basis for the stated purpose (Salem and Katz 2003). For the assessment of eye irritation, the Draize test - despite its limitations -continues to be the only test accepted by the international regulatory agencies and no in vitro test has successfully been validated to fully replace it for regulatory purposes (Barile 2010).
Although the in vitro HET-CAM test has been described as a suitable alternative method for the assessment of eye irritation of water-soluble, especially surfactant-containing materials (Luepke and Kemper 1986; Steiling et al. 1999), some authors have reported a poor correlation of HET-CAM results with those of in vivo Draize test (Doucet et al. 1999; Debbasch et al. 2005). Despite the fact that HET-CAM had been accepted by the European Union to identify the ocular corrosives and severe irritants in July 2004 (McNamme et al. 2009), after about two years, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) did not recommended the HET-CAM as a screening test for the detection of ocular corrosives and severe irritants by not considering in this test a sufficient performance or an adequate amount of robust data to substantiate its use for the regulatory hazard (ICCVAM 2006). Consequently, the HET-CAM was not recommended as a screening test for the specific detection of that kind of irritants, but was deemed useful for other objectives such as for example, to identify the non-irritants (Scott et al. 2010; Scheel et al. 2011). In this specific goal, the RBC - an assay that is based on scientific and industry experience - is accepted as being "suitable for purpose" although not officially accepted by the regulatory agencies (McNamme et al. 2009), is quite similar to the HET-CAM in the recent proposed eye irritation testing strategy called "Bottom-Up approach" to test the materials estimated to have a no to low eye irritancy potential (Scott et al. 2010), what would really be interested in the area of quality control of products, subject to the action of Sanitary Surveillance (e.g. cosmetics such as shampoos, conditioners, makeup, medicinal products for ophthalmic use).
The HET-CAM provides information on the effects that may occur in the conjunctiva following exposure to a test substance. In the case of RBC, the effects are more related to the corneal damage (McNamme et al. 2009). The extent of involvement of the different ocular structures in the irritation is usually a reflection of the severity of the response. All categories of irritants cause some degree of alteration in the conjunctiva. The corneal injury ranges according to the potential irritant: (i) Slight irritants tend to affect the superficial corneal epithelium, (ii) Moderate irritants affect beside epithelium, the superficial stroma, and (iii) Severe irritants affect deeper layers of the stroma. In other words, depending on the mechanism of injury caused by a test substance (membrane lysis, protein coagulation, saponification or action on macromolecules), its effect will be better correlated with a specific ocular structure and which in turn will be better related to some specific in vitro methods. The HET-CAM and RBC, both have in common the feature to assess the potential of a test substance to disrupt the cellular membranes (Scott et al. 2010). Thus, in order to identify the irritants over the entire potency range for all kind of chemical classes, it is generally accepted that a battery of alternative methods will be required, since no single assay will fully replace the Draize test (Barile 2010).
In the present study, 20 eye drops presumably non-irritants, having been confirmed as such in the Draize test were tested in the in vitro HET-CAM and RBC assays to correlate their results with those of Draize Test with the main purpose of getting a preliminary evaluation of their real applicability on the first step of the "Botton-Up Approach". In this strategy, if test results indicated that the product or material was non-irritant, no additional tests would be needed. Otherwise, if a test substance was classified with any degree of irritation, an additional appropriate validated and highly reliable in vitro test to identify the real classification of the substance would be needed in the next step of the proposed battery. However, to definitely implement this type of approach, it would be necessary that international regulatory authorities accept the scientific validity of assays capable of accurately identifying negative results, without enforcing the need for confirmatory animal results (Scott et al. 2010).
With the data found in the present evaluation, The RBC was better than the HET-CAM in terms of predictability, because all the 20 results obtained in the RBC assay were correctly correlated with the Draize Test (Table 2). These results were consistent with recently published studies that showed good applicability of RBC in the evaluation of ocular irritation induced by the surfactants (Mehling et al. 2007; Mitjans et al. 2008) and finished products (Alves et al. 2008). The HET-CAM assay gave both specificity and accuracy of 80%, overestimating the in vivo results. The fact that HET-CAM method overestimated the results obtained in vivo had been described previously and was probably due to the conditions of exposure to the product which was applied directly into the chorio-allantoic membrane, taking into account the fragility of blood capillaries of the chorioallantoic membrane and the high, non- physiological osmolality of some test substances (Hagino et al. 1999; Debbasch et al. 2005).
Finally, it is noteworthy that the prospect of definitive replacing the Draize eye irritation test by an in vitro strategy requires a wide knowledge of the models involved, including a better understanding of the mechanisms of eye irritation, and, therefore, further studies in this area are still required. The key challenge for the regulatory agencies and industries that have an interest in this theme is to develop an evaluation strategy, which will be built on scientific knowledge.
The results suggested that both the RBC and the HET-CAM test were feasible and easy to perform, when compared to the Draize test, but the HET-CAM tended to overestimate the results in vivo. The RBC assay showed to be more susceptible to evaluate the low-irritant products, as it correlated exactly with the in vivo test. However, it would be necessary to conduct a more comprehensive evaluation with a larger number of products representing various categories of irritation in an inter-laboratory study to confirm these results.
Andréa Martins da Nóbrega has a fellowship of PIBIC (CNPq/Fiocruz). This study was supported by PDTSP-SUS/Fiocruz and MCT/CNPq 14/2008. Isabella Fernandes Delgado is recipient of fellowship from CNPq.
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Received: January 13, 2011
Revised: July 15, 2011
Accepted: February 16, 2012