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\def\citeauthor{~Imtiyaz~Ansari,
              ~Nitesh~Gupta,
              ~Abhaykumar~Yadav}
\def\citetitle{A review on: Alternatives to animal experimental models}
\def\copyrightstmt{This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial- ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.}
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\begin{document}


\def\RunningAuthor{Imtiyaz Ansari et al.}
\firstPage{1}
\articleType{Review Article}
\receivedDate{12 Jul 2021}
\acceptedDate{17 Aug 2021}
\revisedDate{15 Aug 2021}
\journalVolume{11}
\journalIssue{2}
\journalDoi{ijrpp.v11i2.1416}
\copyrightYear{2021}
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\def\journalTitle{International Journal of Research in Phytochemistry and Pharmacology}

\title{A review on: Alternatives to animal experimental models}
\author{Imtiyaz~Ansari\textsuperscript{*},  
            Nitesh~Gupta, 
            Abhaykumar~Yadav~\\[5pt]{Department of Pharmacology, Oriental College of Pharmacy, Sector 2, Behind Sanpada Railway Station, Sanpada West, Navi Mumbai-400705, Maharashtra, India}}

\begin{abstract}
An English solicitor, Jeremy Bentham, disdained the unregulated use of livestock, in the literature of 1780, which cast doubt on the inability to pay morale to animals. Animal experiment was fundamental to scientific science during his life, but it has also been a question of heated popular and intellectual discourse from decades. Since then, general understanding and attitudes have strengthened against the unethical or cruel application of animals for research purposes. Russell \& Burch provided in 1959 the principles set out during UFAW of the clinical lab method. The alternative measures, replacements, or non-animal steps shall be used as alternatives for live animal methods, or methods of measurement without live animal use. The term 'alternative' refers to approaches or strategies that substitute the overall use of laboratory animals, minimize the number of animals used, or enhance a current system or technology to reduce animal stress. In recent years, the creation of alternatives to animals has been accelerated by designing projects with the intention of developing and introducing alternatives. This report summarizes the key potential approaches to animal science. An integrated use of these techniques will offer an insight into the minimal use of animals for experimental research.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Alternatives,\newline Ethics,\newline In-silico,\newline Microdosing,\newline 3 Rs,\newline Danio Rerio,\newline Drosophila Melanogaster
\end{keywords}
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\makeatletter{\Large$^\ast$}Corresponding Author\par Name:\ Imtiyaz~Ansari~\\ Phone:\ 7977221955~\\ Email:\ imtiyaz.ansari@ocp.edu.in
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\section{Introduction}
Animals are widely used for the growth of medicinal therapy, toxicity assessment of prescription, human use food safety assessment and other uses of science, industry and health. Since at least 500 BC, livestock have been researched. By using animal experiments, researchers will explore various diseases in drug production. Animals are affected by pathogenic agents and studies are carried out. These diseases are created artificially by laboratories in an effort to reproduce human diseases. This allows scientists to design a new treatment by studying animals. In try to determine out how the disease progresses in the body; researchers will use animal experiments. Through examining animal models, scientists learn what causes and produces disease, and which genetic, climate or dietary factors contribute to disease development. A wide variety of various species, such as cats, rabbits, goats, dogs, albino-rats, monks, monkeys, frogs, etc. are used for this research, although the usual models for animals are mice. The least regularly used birds and fish\unskip~\cite{1203411:23114143}. The core principles in research on animal rights and ethics were the three Rs principles established in the 1959 book "The Principles of Humane Experimental Technique." (WMS Russel and RL Burch). Replacement, reduction and refinement of the three Rs specifically justify its use of alternative methods, reduction of laboratory models and rehabilitation the three Rs values were universally and uniformly adopted internationally and were incorporated in regulatory policies and form part of animal quality care. CPCSEA in India adopted another R for rehabilitation in contrast to the present R. The fourth R is explicitly the legal duty of researchers to animals after experiments. When budgets are made for a research project, the costs of laboratory animal recovery and post care should be weighed. Nearly three decades ago, science addressed the need to curtail unnecessary animal slaughter, and significant progress has since been made in dealing with the issue. The European Center of Alternative Methods (ECVAM) was founded in Italy in 1991 and became fully operational in 1993. One of the first to propose a complete ban on animal use in cosmetic science has been the European Union and since then numerous centers worldwide have been set up in order to establish alternatives to experimental animals. In India, CPCSEA is the authorized organization in responsible for overseeing and authorizing working protocols and animal research methods, under the Government of India Ministry of Forests and Environment. The approval and evaluation of alternate solutions was conducted out from the three big bodies known as validation agencies ICCVAM the ECVAM and the OECD \unskip~\cite{1203411:23114131}.

\textbf{3 Basic RS and the } \textbf{Addition of 4th R}

In 1959,' The Principles of Humane Experimental Technique' was released by British scientist William Russell and British microbiologist Rex Burch \unskip~\cite{1203411:23114134}. This research dealt with the aims of replacement, reduction and refinement (3Rs):


  
  \begin{enumerate}
  \item \relax Replacement in animal research with other approaches. 
  \item \relax Reduction in the number of tested livestock. 
  \item \relax Refining animal research to minimize discomfort. 
  \item \relax Rehabilitation has been applied by animal advocates today.
  \end{enumerate}
  This groundbreaking work sets the foundations for ethical treatment for animals used in science and the need to consider suitable alternatives. And today there is considerable importance to the basic ideas set forward over fifteen decades ago. Replacing 'greater' animals for 'lower animals requires substituting them. Some studies can replace warm-blooded animals including micro-organisms, plants, larvae, reptiles, amphibians and mammals. In order to teach animal or human body structure, mechanical or electronic models, audiovisual equipment or in vitro modelling, live animals can alternatively be supplemented by non-animal model models like dummies for initiation to the analysis. Reduction helps minimize the number of animals necessary for the experiment or instruction of the principle. Strategies for doing so include: pilot studies for detecting such possible complications before using several animals; planning a sample that utilizes animals as its own controls; gathering maximal data from each animal; and even concurrently collecting data on more than one experiment. Refining requires improvement of experimental procedures to reduce pain and/or anxiety wherever possible. Rehabilitation in compliance with the Animal Cruelty Control Act (PCA) of 1960 \unskip~\cite{1203411:23114144}.

\textbf{Alternatives to Animal Models} \textbf{\space }

Because of scientific breakthroughs and biomedical sciences, a range of alternatives for fair medicinal goods have been created. These alternatives have advantages in minimizing jobs, economic conditions and the use of time. The most common solutions are detailed in the following:

\textbf{In Vitro Testing (Cell and Tissue Culture)} \textbf{\space }

Through use of in vitro cell/organ cultures that need cells to develop from outside organism in the research setting can be an excellent option for animal studies. Liver, kidney, liver, skin, etc., cells and tissues are separated from the animal and can be retained within an appropriate medium of development for a couple of days to several months or for a few years. Culturing the in vitro of animal/human cells requires separating them and forming them as a monolayer over the surface of plates and flasks. Often used as cellular enzymes can contain cellular components, such as membrane fragments. Different kinds of cultures, including cell culture, callus culture, tissue culture and organ culture, are used for different purposes. Technical gain is easier to follow, time-consuming and cost-effective. These methodologies periodically help to check their toxicity and usefulness for the preliminary screening of potential drug molecules/chemicals. Nearly all cosmetics, medications and chemicals are toxicity and effectiveness tested using these techniques. For e.g., the eye irritancy test. In order to check the irritability of substances, Draize test, which includes animals (mainly rabbits). It's really painful because any time a new animal is used as an alternative that uses bovine corneal organ culture. In the laboratory the bovine cornea is grown for up to 3 weeks and the in vitro chemical irritation test uses various analytical approaches to assess the toxicological impact \unskip~\cite{1203411:23114138}.

\textbf{Micro-Organism Tests}

In metabolism, genetics, and biochemistry, microorganisms are appropriate as models, and may even act as models of more complex systems. For example, for the study of the normal and abnormal development of human embryos, insights into the basic mechanisms of gene expression are applicable. Investigators have also shown that yeast has estrogen receptors that are close in affinity to rat uterus receptors \unskip~\cite{1203411:23114132}.

The Ames test for the reverse mutation in \textit{Salmonella typhimurium} is the most widely used mutagenicity test. By exposing an already mutated strain to potential mutagens, mutagenicity is known. The bacteria recover their ability to generate the amino acid histidine if the mutation is reversed and will proliferate in a culture medium deficient in histidine. In order to decide if the metabolic products of a substance can be mutagenic, even if the substance itself is not, liver preparations from rats or other rodents are used to generate at least some of the possible metabolic products, the Ames test, as well as most other mutagenicity tests involving micro-organisms, does not completely avoid animal use. Microorganism systems may fail to detect mutagenic changes that could occur in whole animals or humans may be overpredicted by the detector. For example, the metabolism mechanism may not be capable of reproducing conditions in vivo, or mutation may not be the initiating event in the case of carcinogenicity screening. On the other hand, such systems may suggest mutagenicity when the mutation is reversed by the DNA repair system of mammals. 

Other bacterial tests have been developed using \textit{S. typhimurium, Escherichia coli,} and \textit{Bacillus subtilis}. These systems do not seem to offer any particular advantage over the Ames test, although thorough the evaluation is hindered by the absence of a comparable outcomes database. Tests have also been developed for molds, fungi, and yeasts \unskip~\cite{1203411:23114142}.

\textbf{In Silico Techniques: (Computer Models and Simulations)}

"In silico approaches" (in silico = on a computer) are increasingly pertinent as alternatives to animal testing. In determining the dangers of researching drug tolerances or likely modelling life cycles, these computer-controlled research and simulation approaches are often used for other applications. Consequently, computer simulations increasingly use neurobiological scientific data to reflect and predict central nervous system functions. Computer simulation is also used in higher education to simulate dynamic biological interactions\unskip~\cite{1203411:23114136}.

Like the key and key hole, biologically active substances are either ideal and have an effect on cell surfaces, which come in contact with receptors. In this area, one of its pioneers has created a model that allows results to be calculated even though the receiver is not among the reacting partners. This technique is called pseudo-receptor modelling and it was a very original concept in the area of computer-assisted drug design. It helps the reconstruction of a 3-dimensional configuration of an unknown bioregulator based on the ligand structures (the recognized bioactive compounds). It incorporates existing methods, but it significantly extends the possibilities by generating an explicit receptor model. This model will subsequently be used to estimate the qualitative binding strength of the new drug molecules. The relevance of pseudo-receptor models for minimizing and replacing animal models derives from the fact that the technique would be used in circumstances where only in vivo methods could be used, where little to no information of the true biological receiver is available. Pseudo-receiver modelling makes it possible to screen possible drug modules that are first functionally connected to an unknown bioregulator. QSAR approaches have been very useful in basic pharmacological research. In comparison, the figures for animals in the pharmaceutical sector have been greatly decreased. These are simpler, easier and more effective methods. In future the QSAR approaches should also include potential metabolic products of the test compounds. At the process, the side effects of materials are to be hoped for. Millions of livestock per year have the ability to use QSAR approaches to limit the scientific use of animals. The introduction of the QSAR methods could replace, alone, the EUR 950 million expense of animal studies programmed under the EU REACH press release (16 October 2003). According to a German chemicals firm, the introduction of QSAR methods could lead within a few years to an additional 30\% decrease in the amount of laboratory animals. In the past, the number of animals has reduced by 30\% when utilizing isolated organs \unskip~\cite{1203411:23114128}. Although in-silico is somewhat advanced as a substitute for animal, but sometimes the results of computer models or simulations require confirmation of the whole animal, its 100\% substitution for animals is still not necessary. In one research used in existing pharmacological schedules, computer modelling models have been found satisfactory for their user-friendly and adaptable accuracy.

\textbf{Computer Assisted Learning}

Computer-aided pharmacology learning can substitute, minimize and perfect by minimizing experimental replication. In addition, the apprehension of unambiguous and imperfect data processing attributable to differences in the animal's brief survival time is safeguarded.

In view of the real-life scenario of new medication criteria, it wasn't just campaigners condemning animal experimentation that can remove the need animal research but none of them will discuss the name of medications in the absence of an expert research scientist working on animal testing. 

In normal life, animals can be substantially decreased by simulated tests to generate and test medicines. Finally, both technical and non-training bodies created applications for virtual animal studies that can minimize and replace animal trials and simplify presentations as well as teaching by software that displays complete video demonstrations of various procedures including the insulation and mounting of animal tissue. In the end many animal experiments are now designed for virtual animal testing. Ex Pharm Pro programmer is available on the Internet which has similar related work and can be applied to instruct and learn undergraduate and postgraduate studies. This software will easily understand the defined saving time experiment without losing any animal \unskip~\cite{1203411:23114141}.

\textbf{Microbiological Systems}

In toxicology and carcinogenesis (cancer producing) research, these are widely used. These are focused on chemicals' capacity to cause mutating changes in the DNA of a cell, which is the cell's genetic information core, e.g., Ames Test that can recognize 80-90\% of all researched carcinogenic chemicals. It is mostly used as a screening method and must be confirmed by means of animal studies.

Another example is the use of fungi in drug metabolism studies. Fungi may minimize the general need for laboratory animals. Selected fungal classes were demonstrated to be capable of metabolizing a wide range of drugs. The most promising one is Cunningham Ella elegans. A series of anti-coagulants, diuretics, anticonvulsants and hemorheological agents have been investigated using these fungi (which make red blood cell membranes more elastic for oxygen delivery to tissues). This approach is being improved by scientists and not intended to replace drug tests entirely with rodents, including such rats and hamsters. The harmful RtxA1 modulation inducing acute cytotoxicity was investigated in recent studies using Vibrato vulnificus bacteria. There has become an opportunity for the control of chronic diseases. The ultimate benefit is that the development facilitation mechanism is easily regulated, non-mammalian and highly predictable. This would lead to a decline in livestock demand, but definitely would not eliminate all animal science. Researchers have worked with lots of healthcare companies for the best possible way of implementing the modern approach to drugs manufacturing \unskip~\cite{1203411:23114139}.

\textbf{Microfluidic Chip Testing}

Micro fluidic chip testing is a more nuanced and improved form of testing in contrast to in-vitro testing; biologic pain testing and drug effectiveness and safety must be examined for microfluidic chip testing. Just 2 cm large structurally microfluidic chips are available, each with a tissue sample from different parts of the body, and contain an organized collection of tiny chambers. These chambers are also compared to the microchannels from which replacement blood flows on the physical law that defines microscale fluid flow behavior. Finally, the checked material is applied to the fluid that will circulate in microchannels and nourish the chip's compartmental tissues and sensors to gather data and send it for analysis to a connected device. It can therefore give us a better understanding of a specific tissue being extracted from microscale details. In addition, this method often has disadvantages as the procedures are screened and examined at the levels of cell or tissue, but there can be multiple involvement of organs that cannot replace the whole body. It is still necessary to perform definitive trials on animals \unskip~\cite{1203411:23114137}.

\textbf{Microdosing}

Throughout the initial stages of economic research, this microdosing procedure has been performed to test the effectiveness and safety of medicines. Data on physiology of the body used for evaluating the substance being studied or tested are produced by this process. The micro specification test relies on the very specific accelerated spectroscopy device's ultra-sensitivity. 40\% of medications fail in Stage I~clinical trials, according to reports. This step analysis takes a period of 18 months and costs about \textsterling 3-5 million. Thus, microdosing will scan for sooner, easier and cheaper effectiveness medicines. It takes just 4-6 months and costs about \textsterling 0.25 a million per medicinal product. The estimation of human metabolism is outstanding in its precision \unskip~\cite{1203411:23114135}.

The convergence of many market factors has resulted to modifications of pharmaceuticals. Among the few important ones are public scrutiny, the high rates of failure of research agents and concerns regarding the need for animals in testing. Specific variations throughout this method are being taken in order to tackle these problems. Microdosing is one change which can save money and resources by giving humans extremely small, radio-labeled doses of research agents. The pharmacodynamic pattern of substances shall be determined. Although not all agencies agree that their promise will be honored, the potential is enormous. Thus, human microdoses seem to hold a significant promise as an evaluation method. Individual microdosing can be effective in many medicines that can also be used consecutively as science and experience in the Preclinical trials are more refined. When a step 0 research will begin in human trials, microdosing could be a proven pharmaceutical production strategy. The true result of Preclinical micro dosage experiments, is that the technique can provide details within the provided and appropriate range in relation to therapeutic dose evidence under which circumstances it can estimate. Keeping track of ongoing quick advancement in drug manufacturing innovation and without a doubt, reducing the period of drug discovery, the price falls dramatically. Unless further detail is given, it is thought that a microdosing technique will contribute to normal animal-to-human allometric scaling; redefining existing Preliminary research designs. This technique will help minimize animal experimentation by discovering potential drug candidates \unskip~\cite{1203411:23114140}.

\textbf{Vertebrates Animals}

Because of the genetic relationship to higher vertebrates, including mammals, lower vertebrates are an attractive option. Moreover, the experimental use of lower vertebrates involves fewer ethical issues.

\textbf{Example {\textendash} Danio rerio. (zebra fish)}

Danio rerio is a small, roughly 1.5-inch-long freshwater fish, commonly known as a zebra fish. It has a virtually translucent body during early development, making it easier to visually view internal anatomy. Optical clarification allows precise observation of developmental stages, detection of mutagenesis phenotypes, simple sampling, determination of the toxicity outcome test and direct observation by means of light microscopy on the expression of the genes. The use of the laboratory is assisted by limited volumes, brief life cycles and high fertility. By selecting D, there are reductions in the operating room, the expense of the lab solutions, chemical experiments and workers. Rerio is an option to animals. Its eggs and offspring may be made and used for processing in plates of cell culture and Petri dishes. Zebras are an appealing choice for molecular and genetic analysis because whole genome sequences are available. This is specifically used for detecting of numerous chemical and pharmaceuticals toxicological investigations in such a plethora of requests from early-to-early years. This is also commonly used during the observation and research of defects and organ growth disorders related to exposure to research molecules in cancer, cardiac disorders, neurological malfunctions, behavioral dysfunction. Modeling of certain human diseases in zebra fish could be used to improve the phenotype of the disease and malfunctions in the development of organs \unskip~\cite{1203411:23114130}.

In both animal and human health research and much more recent times, aquaculture, the Danio rerio model was widely utilized. Throughout the recent century employment of Danio rerio method in the science establishment increased significantly, even though its rodents are by far the most often used model to follow. A multitude of international regulatory bodies as required. In reference to the models of these more documented animals, zebra fish model results to lessen any utilization of the effort and cost. Which has a recent update and predictive capacity compares favorably with the in vitro results. By use of mammals in research could then be substituted and minimized using this model and the matters due to just the wellbeing of those animals can be greatly reduced \unskip~\cite{1203411:23114129}. 

\textbf{Invertebrates Animals}

As a general option for experimental use, invertebrate organisms are widely regarded. Various conditions, including parkinsonism, endocrine and cognitive malfunction, muscle hyperplasia, wound healing, cell maturity, retroviral biology with conditions, cell death and diabetes and toxicology, have been investigated. Invertebrate organisms are underdeveloped and do not have innate immune responses that induce certain limitations on their utilization in human diseases. It has many advantages, however, such as short durations, small sizes and easy anatomy, which means that one test allows a large number of invertebrates that suffer from less ethical problems to be investigated in a limited time. In comparison with poultry, their living prices are lower. For example, millions of flies can accommodate a habitat, only some mouse would be preserved.

\textbf{Example {\textendash} Drosophila melanogaster}

Drosophila melanogaster is an arthropod in the Drosophila family. Approximately 100 years ago this insect was adopted as a biological model and was significant for the development of genetics and related fields. Traditionally used as a model for genotoxicity, the above fly has only been incorporated recently as a model for systemic toxicology study or an alternative model for the toxicology trial. In 1894, for their work with embryogenesis, Ed Lewis received a Nobel prize for physiology and medicine for her groundbreaking research into the concept of the hereditary construction of fly. In scientific studies on its use of animal models, Melanogaster reached ECVAM \unskip~\cite{1203411:23114133}.\textbf{\space }It is a genuinely appealing model for studying many of the key features of the fly. As mentioned above, for just over 14,000 genomes, four genes, three of them carry most of the genome, have been completely sequenced and recorded and coded. About 75\% of human disease-related genes have been measured by successful ethologists. However, the gross fly- and mammal resemblance of the nucleotides or protein sequences in the preserved functional regions usually amounted to around 40\% among the homologues. The fly's life span is very short. Within 10 to 12 days at 25\text{\textordmasculine}c, a single breeding pair produces 100 genetically identical descendants. Only a handful of descendants are produced every 3-4 months in accordance with the normal rodent models. As various examples, the fly, the fetus, the caterpillars, the pupa and adults may be included, with their own unique advantages that define the developmental period. The embryo is also used in fundamental current psychology on habitat, cell destiny forecasting, organogenesis and neuronal and axon trajectory growth. During development and physiological analysis, the larva is also used as well as forging simple actions, usually the walking third-star larva. Innermost indissoluble epithelia are considered to be the possible structures of the fly in the larva. Since the end of the third stage, the structures undergo major morphological changes which led to the final structure of the adult. Duration of pupal. The analysis of molecular and genetic processes in pupa behind hypothesized disc structures provided an important understanding of human biology in relation to fly biology. The pupa therefore represents an efficient model for analyzing those cell phases. The adult fly is not a higher species, but a very smart and complicated being. The adult fly has mechanisms which carry out reproductive functions of the heart, liver, kidney, bowels and mammals. It's extraordinary to remember the adult fly. Approximately 1 million neurons - circadian rhythms, sleeping, cognitive, courtesy, sleep, aggression, flight, shape of discrete and complicated behavior. Flies are substantially the same as the noticeable way on mammalian systems in reactions to certain medicines within the CNS \unskip~\cite{1203411:23114127}.
    
\section{CONCLUSION}
The ethics of animals is a question as important as the wellbeing of humans. To incorporate 3 Rs effectively during the experimental use of livestock, further effort must be made. Different methods were proposed to animal use, which have to be successfully implemented. Bioinformatics software, in vitro cell cultures, enzymatic screens, and model organisms are useful to integrate varied computer models. Modern computer methods, data analysis and computational procedures may produce correct information in attempt to comprehend the results of alternative protocols. Such integrated approaches will lead to the limited involvement of animal model proceedings. 

\textbf{Funding Support}

The authors declare that they have no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that there is no conflict of interest for this study.
    

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