Métodos alternativos para determinação da potência de veneno e antiveneno botrópico: aplicações e perspectivas para o controle da qualidade / Alternative methods for potency evaluation for Bothrops venom and antivenom: applications and perspectives for quality control

Introdução: Acidentes com animais peçonhentos são classificados como doenças tropicais negligenciadas e são atualmente a mais frequente causa de intoxicação em humanos no Brasil. O único tratamento disponível é a rápida administração de antivenenos específicos e de qualidade garantida. Para assegurar a eficácia e a segurança desses produtos, são realizados ensaios de determinação da potência in vivo para veneno e antiveneno, desde as etapas de produção até sua liberação final. Apesar dos diversos estudos sobre métodos   alternativos ao ensaio murino, nenhum método foi efetivamente validado. Objetivo: Compilar os métodos alternativos desenvolvidos para os antivenenos botrópicos, avaliando sua disponibilidade, perspectivas e aplicações em laboratórios de produção e controle da qualidade. Método: Foi realizada uma busca nas bases PubMed, BVS e Scopus entre novembro de 2021 e junho de 2022. Foram identificados 89 trabalhos, dos quais 31 foram selecionados de acordo com os critérios de elegibilidade. Resultados: Nos métodos alternativos identificados, observamos a preferência de 42,80% dos estudos por metodologias que utilizem linhagens celulares como método alternativo aos ensaios murinos, sendo que a maioria destes trabalhos 58,30% optou pela linhagem celular Vero. Conclusões: Pela diversidade das toxinas encontradas em cada gênero de serpentes, entende-se que é de extrema importância que o ensaio de potência dos antivenenos tenha como base a avaliação e a quantificação precisa da inibição da atividade biológica dos venenos. Ensaios de citotoxicidade são amplamente utilizados e têm acumulado evidências de sua adequação como importante ferramenta alternativa ao ensaio murino para o controle da qualidade de veneno e antiveneno antibotrópico.

Introduction: Accidents with venomous animals are classified as neglected tropical diseases and are currently the most frequent cause of intoxication in humans in Brazil. The only available treatment is the rapid administration of specific, quality-assured antivenoms. To ensure the efficacy and safety of these products, in vivo potency determination tests for venom and antivenom are performed during the production stages, until final release. Despite several studies on alternative methods to the murine assay, no method has been effectively validated. Objective: To compile alternative methods developed for Bothrops antivenoms, assessing the availability of the methods and the prospects and applications in Bothrops venom and antivenom production and quality control laboratories. Method: A search was conducted in PubMed, BVS, and Scopus databases between November 2021 and June 2022. 89 articles were identified, of which 31 were selected according to the eligibility criteria. Results: We observed in the alternative methods identified a preference of 42.80% of the studies for methodologies that use cell lines as an alternative method to the murine assays, and most of these works (58.30%) opted for a VERO cell line. Conclusions: Due to the diversity of toxins found in each genus of snakes, it is understood that the potency assay for antivenoms should be based on the evaluation and precise quantification of the inhibition of biological activity of venoms. Cytotoxicity assays are widely used and have been accumulating evidence of their suitability as an important alternative tool to the murine assay for quality control for Bothrops venom and antivenom.

Comparison of the different protocols of the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) by evaluating the eye irritation potential of surfactants.

The Hen's Egg Test - Chorioallantoic Membrane (HET-CAM) is a valid alternative method used to assess the potential for eye irritation from chemicals. This method is the only one that mimics the conjunctivae of the eye and aims to semi-quantitatively evaluate the irritant potential of a chemical on the chorioallantoic membrane surrounding the chicken embryo in egg by observing the irritation effects on the membrane immediately after the pure or diluted chemical is applied. The purpose of this study was to compare the different protocols of the HET-CAM, the French and German protocols, by evaluating the eye irritation potential of surfactants. The comparison led to the optimization of the French protocol, generating an adapted one, to reduce subjectivity in the test evaluation, ensuring more accurate results and greater quality control. The comparison showed that there are no significant differences between the results obtained in the French and German protocols. HET-CAM is known to overestimate the results and to be able to accurately identify non-irritant products and it is a great candidate to be part of a Bottom-up test strategy. It also can be used in a battery of tests to completely replace rabbits.

Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology.

Read-across (RAx) translates available information from well-characterized chemicals to a substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g., EU-REACH) already allow this procedure to be used to waive new in vivo tests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimization to allow a higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target in the RAx procedure, and (iii) how to deal with issues of ADME that may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluate the RAx process were identified. Here, in particular, the RAx guidance, being worked out by the European Commission's H2020 project EU-ToxRisk together with many external partners with regulatory experience, is given.

A comparison of pyrogen detection tests in the quality control of meningococcal conjugate vaccines: The applicability of the Monocyte Activation Test.

The meningococcal C conjugate vaccine (MenCC) is an interesting model with which to test the efficacy of the Monocyte Activation Test (MAT) as an alternative method of pyrogen testing in the quality control of vaccines. The MenCC that has been produced by Bio-Manguinhos in Brazil is in the final development stage, and, as recommended in the guidelines for MenCC production, its pyrogen content must be determined by using the Limulus Amoebocyte Lysate (LAL) assay and the Rabbit Pyrogen Test (RPT). This represents an ideal opportunity to compare LAL and RPT data with data obtained by using a MAT system with cryopreserved whole blood and IL-6/IL-1ß as marker readouts. In order to assess the compatibility of the MAT with MenCC, endotoxin and non-endotoxin pyrogen content was quantified by using MenCC samples spiked with lipopolysaccharide (LPS), lipoteichoic acid or zymosan standards. The presence of the aluminium-based adjuvant interfered with the MAT, increasing the readout of IL-1ß in LPS-spiked MenCC batches. This infringed the product-specific validation criteria of the test, and led to IL-6 being chosen as the more suitable marker readout. No pyrogenic contaminants were identified in the MenCC batches tested, demonstrating consistency among the different systems (MAT, RPT and the LAL assay). In conclusion, the introduction of the MAT during MenCC development could contribute to the elimination of animal tests post-licensing, ensuring human protection based on an effective non-animal based method of quality control.

Applicability of the Monocyte Activation Test (MAT) in the quality control of the 17DD yellow fever vaccine.

The need for alternatives to animal use in pyrogen testing has been driven by the Three Rs concept. This has resulted in the inclusion of the monocyte activation test (MAT) in the European Pharmacopoeia, 2010. However, some technical and regulatory obstacles must be overcome to ensure the effective implementation of the MAT by the industry, especially for the testing of biological products. The yellow fever (YF) vaccine (17DD-YFV) was chosen for evaluation in this study, in view of: a) the 2016-2018 outbreak of YF in Brazil; b) the increase in demand for 17DD-YFV doses; c) the complex production process with live attenuated virus; d) the presence of possible test interference factors, such as residual process components (e.g. ovalbumin); and e) the need for the investigation of other pyrogens that are not detectable by the methods prescribed in the YF vaccine monograph. The product-specific testing was carried out by using cryopreserved and fresh whole blood, and IL-6 and IL-1ß levels were used as the marker readouts. After assessing the applicability of the MAT on a 1:10 dilution of 17DD-YFV, endotoxin and non-endotoxin pyrogens were quantified in spiked batches, by using the lipopolysaccharide and lipoteichoic acid standards, respectively. The quantitative analysis demonstrated the correlation between the MAT and the Limulus amoebocyte lysate (LAL) assays, with respect to the limits of endotoxin recovery in spiked batches and the detection of no pyrogenic contamination in commercial batches of 17DD-YFV. The data demonstrated the applicability of the MAT for 17DD-YFV pyrogen testing, and as an alternative method that can contribute to biological quality control studies.

Métodos alternativos para a detecção de pirogênios em produtos e ambientes sujeitos a Vigilância Sanitária: avanços e perspectivas no Brasil a partir do reconhecimento internacional do Teste de Ativação de Monócitos / Alternative methods for the detection of pyrogens in products and environment subject to public health surveillance: advances and perspectives in Brazil based on the international recognition of the Monocyte Activation Test

Introduction: The detection of pyrogens is essential for the quality control of injectable products. The Rabbit Pyrogen Test remains widely used, despite the existence of alternative methods such as the Monocyte Activation Test (MAT). Objective: To review the use of alternative methods for pyrogen testing, pointing out advances and perspectives from the recognition of MAT by the European pharmacopoeia and its acceptance for regulatory purposes in Brazil. Method: A search was performed on the PubMed and BVS databases, with further classification, categorization by topic and critical analysis of the results. Results: Twenty-four papers were identified, addressing topics such as applications of MAT, its validation and comparisons with in vivo tests. MAT presented better results when compared to other tests, both in the evaluation of biological products and in the detection of non-endotoxin pyrogens. Limitations to diffusion include difficulties in obtaining whole human blood as a source of monocytes, for which several alternatives have been proposed. Conclusions: MAT is a promising method, with application in safety evaluation of new technologies. Its application in Brazil depends on a national implementation policy, which might include greater integration between BraCVAM, Concea and RENAMA in search for its recognition for regulatory purposes.

Introdução: A detecção de pirogênios é imprescindível no controle da qualidade de produtos injetáveis. O Teste de Pirogênio em coelhos ainda tem larga aplicação, apesar da existência de métodos alternativos como o Teste de Ativação de Monócitos (MAT). Objetivo: Revisar o uso dos métodos alternativos no teste de pirogênio, apontando avanços e perspectivas a partir do reconhecimento do MAT pela Farmacopeia Europeia e sua aceitação para fins regulatórios no Brasil. Método: Uma busca foi realizada nas bases PubMed e BVS, com posterior classificação, categorização por assuntos e análise crítica dos resultados. Resultados: Foram identificados 24 trabalhos, abordando temas como as aplicações do MAT, sua validação e comparação com testes in vivo. O MAT apresentou melhores resultados quando comparado a outros testes, tanto na avaliação de produtos biológicos como na detecção de pirogênios não-endotoxinas. Limitações para sua difusão incluem a dificuldade de obtenção de sangue total humano como fonte de monócitos, para o qual diversas alternativas têm sido propostas. Conclusões: O MAT se mostra um método promissor, com aplicação na avaliação da segurança de novas tecnologias. Sua aplicação no Brasil depende de uma política nacional de implantação, que inclua maior Integração entre BraCVAM, Concea e RENAMA na busca por seu reconhecimento para fins regulatórios.

Biopharmaceutical Assessment and Irritation Potential of Microemulsions and Conventional Systems Containing Oil from Syagrus cearensis for Topical Delivery of Amphotericin B Using Alternative Methods.

The aim of this study was to compare the biopharmaceutical characteristics and irritation potentials of microemulsions (MEs) and conventional systems (CSs) containing oil from Syagrus cearensis for topical delivery of Amphotericin B (AmB). Pseudo-ternary phase diagrams were constructed using a water titration method to develop the MEs, and the CSs were prepared according to the classical technique of phase inversion. In the skin permeation and retention study, dermatomed pig skin without stratum corneum was used as an alternative disturbed skin model. The irritation potential was evaluated using three different methods, chorioallantoic membrane assays (HET-CAM and CAM-TBS), and bovine corneal opacity and permeability (BCOP) test. The optimized formulation (ME1) consisting of 0.1% (w/w) Amphotericin B, 9.1% (w/w) catolé oil, 81% (w/w) Smix (1:1, Tween 20 and Kolliphor EL) possessed droplet size of 31.02 ± 0.9 nm, zeta potential of -23.4 mV, and viscosity 0.63 ± 0.1 Pa.s. ME1 exhibited greater retention of AmB in to skin layers (84.79 ± 2.08 µg cm-2) than all the others formulations. In general, MEs showed higher drug release and retention than CSs and all of the formulations showed greater retentivity than permeability. Only MEs developed using Labrasol/Plurol Oleique (L/PO) as the surfactant and co-surfactant exhibited a moderate irritation potential; all other MEs and CSs were classified as non-irritants or slight irritants. The results indicate that formulations containing oil from S. cearensis are promising alternatives for the delivery of AmB targeting the treatment of cutaneous leishmaniasis.

International Harmonization and Cooperation in the Validation of Alternative Methods.

The development and validation of scientific alternatives to animal testing is important not only from an ethical perspective (implementation of 3Rs), but also to improve safety assessment decision making with the use of mechanistic information of higher relevance to humans. To be effective in these efforts, it is however imperative that validation centres, industry, regulatory bodies, academia and other interested parties ensure a strong international cooperation, cross-sector collaboration and intense communication in the design, execution, and peer review of validation studies. Such an approach is critical to achieve harmonized and more transparent approaches to method validation, peer-review and recommendation, which will ultimately expedite the international acceptance of valid alternative methods or strategies by regulatory authorities and their implementation and use by stakeholders. It also allows achieving greater efficiency and effectiveness by avoiding duplication of effort and leveraging limited resources. In view of achieving these goals, the International Cooperation on Alternative Test Methods (ICATM) was established in 2009 by validation centres from Europe, USA, Canada and Japan. ICATM was later joined by Korea in 2011 and currently also counts with Brazil and China as observers. This chapter describes the existing differences across world regions and major efforts carried out for achieving consistent international cooperation and harmonization in the validation and adoption of alternative approaches to animal testing.

Brazilian Center for the Validation of Alternative Methods (BraCVAM) and the process of validation in Brazil.

The need for the creation of a Brazilian centre for the validation of alternative methods was recognised in 2008, and members of academia, industry and existing international validation centres immediately engaged with the idea. In 2012, co-operation between the Oswaldo Cruz Foundation (FIOCRUZ) and the Brazilian Health Surveillance Agency (ANVISA) instigated the establishment of the Brazilian Center for the Validation of Alternative Methods (BraCVAM), which was officially launched in 2013. The Brazilian validation process follows OECD Guidance Document No. 34, where BraCVAM functions as the focal point to identify and/or receive requests from parties interested in submitting tests for validation. BraCVAM then informs the Brazilian National Network on Alternative Methods (RENaMA) of promising assays, which helps with prioritisation and contributes to the validation studies of selected assays. A Validation Management Group supervises the validation study, and the results obtained are peer-reviewed by an ad hoc Scientific Review Committee, organised under the auspices of BraCVAM. Based on the peer-review outcome, BraCVAM will prepare recommendations on the validated test method, which will be sent to the National Council for the Control of Animal Experimentation (CONCEA). CONCEA is in charge of the regulatory adoption of all validated test methods in Brazil, following an open public consultation.

In vitro and in vivo evaluation of efficacy and safety of photoprotective formulations containing antioxidant extracts

ABSTRACT Chronic exposure to solar radiation could contribute to premature skin aging and skin cancer. Skin presents its own antioxidant defense, however when defenses are out of balance, reactive oxygen species could damage biological structures. In the present work, an oil-in-water photoprotective emulsion was developed and Bauhinia microstachya var. massambabensis Vaz, Fabaceae, extracts at 1% (obtained by extraction with different solvents) were added to this emulsion. In vitro and in vivo efficacy and safety of the formulations were evaluated. Spectrophotometric methods and in vivo Colipa test were performed to evaluated efficacy of the formulations, through sun protection factor (SPF) determination and UVA protection factor assessment. To the in vitro safety assessment HET-CAM, CAM-TBS and Red Blood Cell tests were performed. Results showed that both extracts contributed to a higher in vivo photoprotection (SPF 18) when compared to the formulation without extract (SPF 13), this result could be attributed to the antioxidant activity of the plant extracts that act by capturing reactive oxygen species. Concerning safety, all formulations were considered non-irritant according to in vitro tests. Formulations containing extracts could be considered efficient and safe for cosmetic use since they presented higher sun protection factor and passed the toxicity tests.

Applicability of the Monocyte Activation Test (MAT) for hyperimmune sera in the routine of the quality control laboratory: Comparison with the Rabbit Pyrogen Test (RPT).

Pyrogen tests are safety assays performed during the routine quality control of injectable products required by regulatory agencies. Currently, there are three available testing possibilities: 1) the Rabbit Pyrogen Test (RPT); 2) the Bacterial Endotoxin Test (BET); and 3) test systems using human whole-blood or monocytes, termed Monocyte Activation Test (MAT). Although BET is often considered as a replacement for the animal test, it is unable to detect pyrogens other than endotoxin. MAT is based on the human fever reaction and thus, most closely reflects the human response. The aim of this study was to conduct a parallel comparison of the RPT and MAT for hyperimmune sera (HS) batches analyzed during the routine of a quality control laboratory. MAT was performed in the same 43 batches of HS previously tested using RPT. The results showed that MAT presented 100% sensitivity and approximately 85% specificity as compared to RPT, i.e., no false-negative results were obtained. Few suspicious samples, which were negative in the RPT after retesting, provided divergent positive results suggesting a lower limit of detection of MAT. MAT is thus able to detect contaminants in biological products such as HS batches.

Preparation and Evaluation of Chitosan Submicroparticles Containing Pilocarpine for Glaucoma Therapy.

The objective of the present study was to optimize the preparation of chitosan submicroparticles and to assess whether they enhanced ocular permeation of pilocarpine. Submicroparticles were produced by spray drying and characterized to determine process yield, encapsulation efficiency, morphology, size distribution, drug-polymeric matrix interaction, porcine sclera permeation as well as ocular irritancy and drug retention. Quantification of pilocarpine using High Performance Liquid Chromatography was found to be selective, linear, precise, accurate and robust. The spray drying method proved to be simple and reproducibly produced particles with satisfactory yields, thus showing potential for industrial scale applications. The pilocarpine-loaded chitosan particles exhibited adequate morphological characteristics as well as high encapsulation efficiency. The particles produced were on a submicrometric scale and compatible with intraocular administration. In pilocarpine-loaded particles, the interaction between pilocarpine and chitosan polymeric matrix resulted in delayed release of the drug, attributed to formation of a reservoir system. The best fit for drug release was obtained using the Higuchi equation. The chitosan submicroparticles enhanced the permeation effect and increased the passage of pilocarpine through porcine sclera and also demonstrated low irritancy potential. Therefore, the particles produced can be considered a promising system for the ocular delivery of pilocarpine.

Assessment of pyrogenic response of lipoteichoic acid by the monocyte activation test and the rabbit pyrogen test.

Lipoteichoic acid (LTA) is a non-endotoxin pyrogen of a great importance in the pathogenesis of sepsis. The Rabbit Pyrogen Test (RPT) is able to detect all types of pyrogens but involves the use of animals. The Bacterial Endotoxin Test (BET) cannot fully replace the RPT because it only detects endotoxins. The Monocyte Activation Test (MAT) is sensitive to all types of pyrogens and it is based on the same biological mechanism that is responsible for the fever reaction in humans. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has recommended its use for other pyrogens than endotoxin because its equivalence to RPT can be demonstrated. The aim of this study was to evaluate the pyrogenic responses of the RPT and MAT that was induced by LTA. Different LTA concentrations were assayed by the MAT in parallel to the RPT. The results showed that the MAT was more sensitive than the RPT, demonstrating that the MAT detected LTA. This result may contribute to the acceptance of this test by the Brazilian regulatory agencies as a replacement for the animals used in the RPT.

Participation of Brazil in the World Congresses on Alternatives and Animal Use in the Life Sciences: an increase in commitment to the Three Rs.

Many Brazilian researchers have long been interested in the development and use of alternative methods. Most of their research groups work in isolation, due to the lack of funding for collaborative studies. Despite these problems, since the Third World Congress on Alternatives and Animal Use in the Life Sciences, Brazilian researchers have strongly participated, not only by presenting posters and oral presentations, but also by being involved in the World Congress Committees. The Brazilian Center for the Validation of Alternative Methods (BraCVAM) must play an important role in the development and validation of alternative methods, through the active participation of the National Network of Alternative Methods (ReNaMA). In Brazil, Law 11,794/2008 regulates the use of animals in experimentation and education, and Law 9,605/1998 clearly states that use of the original animal test is not permitted, if an alternative method is available. Therefore, given the current legal framework, it is very important that all the Ministries involved with animal use, and the organisations responsible for funding researchers, strive to increase the financial support of those groups that are involved in the development and use of alternative methods in Brazil.

A Reassessment of the in vitro total protein content determination (TPC) with SIRC and 3T3 cells for the evaluation of the ocular irritation potential of shampoos: comparison with the in vivo Draize rabbit test

The aim of this work was to determine the correlation between results obtained from the Draize test and from the Total Protein Content Determination (TPC) to assess the ocular irritancy potential of 20 shampoos. For TPC, two established cell lines (SIRC and 3T3) were used. The concentration that induced 50 percent inhibition relative to controls (IC50) was calculated for each product. Among shampoos tested with SIRC, only one had a false positive result. However, for the 3T3, three false-negative results were found. Pearson coefficient related to the in vivo value of maximum average score (MAS) was -0.58 (p=0.007) with SIRC and -0.73 (p=0.007) with 3T3. These results showed that the TPC assay was capable to predict the ocular irritant potential of shampoos, and therefore was a promissory tool to be used as a preliminary assay for the detection of irritant products and to be part of a battery of screening tests to minimize the animal use in the Draize Test.

Influência do erro de micropipetas na dosagem espectrofotométrica do ensaio de endotoxina bacteriana (LAL cromogênico) aplicável a produtos sujeitos à Vigilância Sanitária / Effect of micropipette inaccuracy on performance of spectrophotometric assay for bacteria endotoxin dosage (chromogenic LAL) employed in products subjected to sanitary surveillance

Segundo a norma ISO 17.025, os instrumentos usados em experimento devem ser calibrados. No caso de micropipetas não há limite máximo de erro definido, sendo difícil estipular até que ponto uma micropipeta pode continuar a ser usada. O objetivo do trabalho foi o de determinar a porcentagem de erro no volume da micropipeta que não interfira nos resultados das diferentes etapas do ensaio toxicológico Lisado de Amebócitos de Limulus (LAL). Os percentuais de erro foram obtidos do levantamento de 44 certificados de micropipetas. Foram utilizados uma pipeta monocanal calibrada, de volume variável e kit LAL Cromogênio QCL-1000 (Lonza). A curva de endotoxina foi construída com as concentrações de 0,1; 0,25; 0,5 e 1 UE/mL. Os percentuais de erro de ±2%, ±4% e±10% foram comparados com a concentração de referência (0,5 UE/mL), e analisados pelo Student t-test (p<0,05). A análise estatística mostrou que, para a endotoxina padrão e para o substrato do LAL, erro acima de 2% interferenos resultados, cuja correção deve ser feita pelo ajuste do volume na pipeta. Nenhum erro testado causou interferência estatisticamente significativa na reação da cor do substrato. Micropipetas com erro superior a ±2% devem ser corrigidas.

Proposal for a Brazilian centre on alternative test methods.

Several initiatives have recently taken place in Brazil in order to foster the creation of centers dedicated to alternatives to animal testing. In 2008, Vanessa Sá-Rocha organized a meeting with Brazilian regulatory authorities and the major stakeholders in the field of testing to foster discussions on the process of funding, development, and validation of alternative methods in Brazil. Octavio Presgrave published a scientific article on "The Need for the Establishment of a Brazilian Centre for the Validation of Alternative Methods." Also in 2008, Jadir Nunes, together with Dermeval de Carvalho, prepared and presented a proposal to the Brazilian National Agency of Health Surveillance (ANVISA) for the creation of a Centre for the Validation of Alternative Methods. ECVAM and other European stakeholders have been involved in the initiatives. Furthermore, also in 2008, a new legislation has been adopted in Brazil regarding the use of animals for scientific purposes ("lei Arouca"). The legislation establishes, among other provisions, the task of monitoring and evaluating the introduction of alternative methods. However, the legislation does not provide for promotion of or information about, existing alternative methods to the larger Brazilian scientific community. In order to streamline the different activities, Chantra Eskes acted as a facilitator by establishing a new joint proposal with the current Brazilian stakeholders, aimed at setting up a Brazilian Center on Alternative Test Methods.

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.

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.

The need for the establishment of a Brazilian Centre for the Validation of Alternative Methods (BraCVAM).

Brazil has recently approved legislation for regulating experimental animal use. On the other hand, it is a requirement that the safety of many products on the market is controlled on the basis of animal testing. Some groups at official laboratories, universities and industries are studying alternative methods, but there is no approved mechanism for funding collaborative studies, nor is there an institution responsible for coordinating these studies. These shortcomings obstruct the development of these assays in Brazil. The creation of a Brazilian Centre for the Validation of Alternative Methods (BraCVAM) would facilitate the development and validation of tests in all the institutions working on alternative methods in Brazil, and could also offer support to other Latin American countries.