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Zebrafish () and its embryo has become a popular replacement alternative among the scientists because of many scientific attributes. As it is a model commonly used in ecotoxicology, our plan is to use this model to identify causative factors leading to chronic renal disease of unknown origin prevailing among poor, farming communities in Sri Lanka. This paper describes briefly the training underwent at University of Antwerp, Belgium and how zebrafish model could be used to address an important public health issue in Sri Lanka.

Current regulatory requirements focus on assessment of acute irritation potential of chemicals and cosmetics in order to support risk management. A trend has been changed from to testing due to 3R (replacement, reduction, refinement) requirements has resulted from a recent animal testing ban in Taiwan. RhE (Reconstructed human Epidermis) models use normal human keratinocytes to form a multi-layered epidermis including a stratum corneum at the top, which function as a barrier. Some commercialized RhEs passed validation of skin irritation test (SIT) examined by ECVAM, but none of them are originated from Chinese heredity. Therefore, ITRI started a RhE project some years ago based on our long-term developed cell culture experiences, such as isolation of cells from human donors, cell expansion technology, and our own GTP/GMP qualified facilities. So far, a well differentiated and with reproducible barrier function epidermis named EPiTRI has been reconstructed. We developed a protocol for EPiTRI SIT in accordance to OECD TG 439. The protocol displays a result of sensitivity of 100%, specificity of 70%, and accuracy of 85% in international validation study. Thus, the human epidermal skin equivalent EPiTRI can be provided as an model for evaluation of skin irritation and a reliable method has been developed accordingly.

Reconstructed Human Epidermis (RHE) is an artificial epidermis made in such a way that it resembles human skin, and can be used for the identification of irritant chemicals, especially for cosmetic and topical medicinal products. Currently the new RHE is produced by European and American countries, whose skin physiology is very different from Indonesia. Based on this, the Center for Research on Drugs and Food, NADFC of Republic of Indonesia took the initiative to develop the reconstruction of keratinocyte, melanocytes and fibroblasts cells into RHE adapted to the anatomical and physiological functions of the skin of Indonesians. RHE is made from an epidermal layer composed of keratinocyte and melanocytes cells that are reconstructed with a dermis layer composed of fibroblast and collagen cells. Keratinocyte, melanocytes and fibroblasts cells are cultured on suitable mediums by adding a suitable growth medium. To find out that RHE has been successfully reconstructed, measured percentage of cell life, made histology preparation to see the existence of cell nucleus, and conducted Immunohistochemical examination to see existence of integration (bond) between antigen. From the research results can be seen that keratinocyte cells grown on culture medium Keratinocyte SFM (IX) with rEGF supplements; melanocyte cells grown on Melanocyte 254 culture medium with HMGS supplementation; and fibroblast cells grown on Fibroblast M 106 culture medium with LSGS supplementation. The percentage of epidermal cell life grew well in the planting of 10 × 10 cells /mL keratinocytes and 0.25 × 10 cells /mL of melanocyte cells and survived until the 11 day with live cell percentage of 93.45%. In making preparation for histology with HE staining, there is a cell life in RHE tissue. Used Immunohistochemical (IHC) examination using cytokeratin 10 antibody marker to view physiological function of epidermal tissue.

Alternative research is currently conducted all over the world and its recent progress is remarkable as 3Rs research. This article describes a review of alternative research and 3Rs in the world, Asia and Japan.

In this study, we considered whether acute oral toxicity hazard classifications for pesticide formulations and active ingredients (AIs) could be used to assign acute dermal toxicity hazard classifications using U.S. Environmental Protection Agency (EPA) and the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS) hazard categories. This retrospective analysis used highly curated acute toxicity data for 503 formulations and 297 AIs. Hazard classifications based on rat oral LD values were compared to hazard classifications based on rat dermal LD values for the same substance. The concordance of oral and dermal hazard classification was 62% for formulations and 64% for AIs using the EPA system and 71% for formulations and 55% for AIs using the GHS. Overprediction of dermal hazard was 38% for formulations and 32% for AIs using the EPA system and 28% for formulations and 41% for AIs using the GHS. Underprediction of dermal hazard was 1% for formulations and 3% for AIs using the EPA system and 1% for formulations and 3% for AIs using the GHS. While concordance overall was modest, the very low underprediction rates show that acute oral hazard categories are sufficiently protective for acute dermal hazard classification. Use of oral hazard data to also classify dermal hazard would obviate the need to perform acute dermal toxicity tests for classification and labeling and thereby reduce the number of animals used for acute systemic toxicity testing of pesticides.

We reviewed six key peer-reviewed publications that assessed the need for one-year studies of pesticide toxicity in dogs. Each of the six papers took a different approach to comparing the value of one-year studies relative to three-month studies, and despite the adoption of different databases and approaches, each study reached the same conclusion: the recommended limit to the testing of pesticide toxicity in dogs should be three months.

Therefore, the present review supports the conclusion that the routine inclusion of a one-year dog study should not be a mandatory requirement for the safety assessment of pesticides, since it is scientifically no longer justifiable. We recommend that the OECD should adapt a harmonized approach that is in line with the legislation in Europe and the USA.

United Kingdom was the first to ban animal testing of cosmetics, and it happened in 1998. The European Union (EU) went through legislations and endeavors implementing animal testing ban in a phased manner, and the final step was done in 2013. Consequent to this, there was enormous canvassing for ban of cosmetic testing on animals in India, to harmonize the marketing as well as import ban of EU. The Indian Drug and Cosmetic Regulatory Authority conceded the demand resulting in the final ban in 2014. This review traces the evolution of thought sequence resulting in the ban, and approval of adopting OECD Guidelines for testing of skin and eye irritation and corrosion.

In accordance with a notification issued by Japan’s Ministry of Health, Labour and Welfare (MHLW) in 2011, the Japanese Center for the Validation of Alternative Methods (JaCVAM) has accelerated applications for new test methods developed as alternatives to animal testing for the safety assessment of the manufacture and sales of cosmetic products and quasi-drug products that contain new ingredients. Taking advantage of this opportunity to strongly impact testing throughout Japan, researchers have been coordinating guidance on the use of such alternative test methods since 2012. Based on test guidelines issued by the Organisation for Economic Co-operation and Development (OECD) and JaCVAM evaluation documents, dermatologists and representatives of cosmetic companies as well as specialists from both the Pharmaceuticals and Medical Devices Agency (PMDA) and the National Institute of Health Sciences (NIHS) have drafted guidance documents for a number of alternative test methods.

Globally cancer is a major public health issue and is a second biggest cause of deaths. Although animal models have limitations in terms of predictive and translational value to humans, they have played a major role in understanding this disease and anticancer drug discovery. In cancer research the most commonly used animal species are mice, rats, hamsters, rabbits, Guinea pigs, fish and amphibians. Use of different cell lines in tissue culture system offers a great relief from use of animals at the same time provide important clues before embarking the animal experiments. Use of spontaneously developing tumor models are encouraged rather producing diseases in the animals. Beside the alternatives, refinement also plays important role in reducing the animal usage in cancer research. However, cancer research use whole body system to evaluate the new strategies for diagnosis and treatment of cancer. This paper highlights the available alternatives and refinements for animal experimentation. A positive note is that if coupled with the refinements, even a minimised number of animal usage shall also yield the meaningful and acceptable results.

The 3Rs principles – Replacement, Reduction, Refinement – were established in 1959 and since then have been adopted widely and particularly in Europe with the European Directive 2010/63/EU. The vaccine industry in Europe has been committed to the 3Rs principles for several years, including animal welfare as well as for reducing and replacing animal use in research, non-clinical safety and analytical testing.

Whereas animal testing has been successfully removed from lot release testing of well-characterized human vaccines, large numbers of laboratory animals continue to be used for safety and potency quality control testing for established inactivated vaccines such as rabies, pertussis, diphtheria, and tetanus vaccines.

Moreover, specifications for human vaccine batch approval often differ between various parts of the world, resulting in either duplication of animal testing or partial implementation of 3Rs for some vaccines when distributed worldwide. This reinforces the need for enhancing international harmonization and cooperation efforts.

In this chapter, we review the use of laboratory animals in human vaccines research and quality control and describe the vaccine manufacturing industry commitments and its concrete programs for implementing 3Rs principles in R&D and industrial operations processes. We highlight the successes as well as the barriers that are encountered when implementing 3Rs principles, as well as the ongoing efforts that include international collaborations with other industries, public organizations and Health Authorities for the acceptance of alternative methods.