Toxicology

Preface

Toxicology was my bread and butter. I did my studies in Kerala and completed my Ph.D. in University of Kerala. My Degree was in Zoology, Post graduation in Ichthyology and Ph.D. in Immunology. I was able to get a Post Doctoral Fellowship of Indian Council of Medical Research to work on the Toxicity of Bio implants, under the Guide ship of Dr.MS Valiathan, the founder Director of Sree Chitra TirunalInstistute for Medical Sciences and Technology. I was posted in the Material Toxicology Division of SCTIMST. Dr P.V.Vedanarayananwas the Head of the Toxicology Division. My post doctoral work turned me into a student of Toxicology. During  my Post Doctoral work I assisted Dr.Vedam to draw up a Testing protocol of Biocompatibility/Safety testing of Biomaterials. Later tried to standardise the new testing procedures using various samples supplied by V.S.S.C, Trivandrum, HAL Bangalore. I continued to work in the SCTIMST and was able to lead the Toxicology Division and theDivision of Laboratory Animal Science to get Accredited by COFRAC of Europe in ISO 17025 in 2003. So I have a solid 33 years of Experience in practicing Toxicology and Laboratory Animal Science. This background and the truth that the common person is not aware of the basics of Toxicology,even though he is exposed to many hazards in his daily life, made me to write a small book on Toxicology to educate the public. My effort to write the book is initiated by putting it down here below.

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1 – Learning Toxicology

Learning Toxicology is simple if it can be introduced from the home. We are yet to understand that Mother is the first and best teacher in the world. Home is the School. Mother has spent more time with the child before birth and started talking, singing and petting the child while he or she is in the womb. Child is slowly taught the relationships, acts and words of respect, introduced to the rooms, materials in the house, the sky, wind, birds, flowers, places of worship and through all these activities mother is teaching and child is learning. The child’s word power will be enormous. A child brought up by such a Mother will see his first Teacher in School with respect and be calm in the class. Why I explain this much is to show that the safety could also be taught in child hood itself, if mothers could be introduced to Toxicology. So learning is made easy and the child as he grows will try to avoid use and contact of hazardous materials in his life.

2 – Teaching Toxicology

Toxicology in general is the hazard in using a material. Air and water are not Toxic but can be hazardous if air gets into the intestine or blood vessel. Likewise water become hazardous if it enters the lungs and blood vessels. Talcum powder or other cosmetic items could become irritants or suffocating in large doses or if used without care. Nail cleaners can become a poison if consumed or lose eyesight if it falls in the eye. Toxicology is the result of misuse or excessive use of any material that we come across in daily life. Talking about hazards from materials that we need to use every day, it becomes boring as we have been using them each day for so long a period, just like the Mosquito repellent or sugar or cigarettes.

Literature on Teaching of Toxicology is interesting to read through. Toxicology is an important subject in Medical practice but it is not taught as a specific or special subject in the Medical curriculum. We all know the importance of poisoning in cases of suicides or murder and in medico legal cases. But Toxicology is not taught as a rule. In modern times majority of hospital admissions are due to unknown causes and after a long time it may turn out to be a case of misuse or over use of medicines either prescribed by a Doctor once and thereafter by self medication. There are occasions where many universities tried to put Toxicology in Medical curriculum in vain. But there are takers for Toxicology of Industry, Environment Etc. and are available as special courses in Universities.

My experience in teaching Toxicology is very interesting. For my first class the takers were Post Graduate degree holders in Medicine and Surgery. The attendance was poor and 50% of the attendee’s were sleeping. It was found out that the Classes were part of their Curriculum to expose them to Medical related subjects like Engineering, Toxicology, Material Science, Microbiology, Biochemistry etc. Further, I tried to teach Toxicology through  Popular science articles but did not get an appreciation note from any readers. My second chance to teach Toxicology was in Cochin University to the students of Environmental Toxicology. The students represented almost all disciplines, like Fishery science, Engineering, Chemistry and Biochemistry, Zoology, Bacteriology etc. Here I found batches of students who were really interested in the subject  and made use of the libraries of various departments of the University. Students came to the Hostel room where I stayed with questions and  for guidance and got their essays corrected by me. Here,now I am trying to put Toxicology Classes through my website “www.thebs-acfernandez.Info”. Why I take effort to teach is that, we have reached a stage and time we use more than 200 chemicals and are exposed to innumerable number of materials daily through Air, Water, Food, Cosmetics and Personal care materials, Cleaners and Fresheners, Detergents, Lotions and Repellents, Plastics and Furniture cushions and covers, Car interiors, Air conditioners, Particulate matters and Cigarette smoke etc., Drugs for Asthma, Allergy, Cardiac problems, Hypertension, Diabetics, Natural products, Ayurveda, Unani, just with out much attention , care to continue to consult a doctor systematically and seriously. We have to read the labels of materials we purchase, look at the expiry dates and precaution if any suggested before using each item. This should be the first step. Take steps in use of the quantity recommended. If we are serious and cautious in our daily deeds we are sure to avoid taking a lot of poison in small proportions. We have to remember, nature has build man in a sturdy manner that he has the best biological detoxifying machine.

3 – History of  Toxicology

Tracing back Toxicology, is really a retreat through olden literature in Chinese, Greek, Vedas, Persian, French, British etc. Chinese Medicine records that ShenNung (2696 B C ) tasted 365 herbs and authored a Treaties “Herbal Medical Experiment on poisons.” Homer describes the use of Poisoned arrows in his epics “The Odyssey and Iliad” in 860 B C. Here he enlightens on Tokkicon or poisoned arrows used very commonly on wars conducted during his life period and the root of the word, Toxicology. Hippocrates (400 B C ) in his writings showed that toxins or poisons exerted their effects by getting absorbed into the living system and this could be treated by preventing absorption by various means. In the Treaties “ De Historia Plantarum, 370 B C”, Theophrastus describes numerous Poisonous plants. Nicander of Colophon 185 B. C., physician to King Attalus of Bythnia studied use of poisons, its effects, symptoms of poisoning and the effects of anti dots and nature of recovery or death on prisoners, with King’s permission. The use of linseed for inducing vomiting and sucking of poison from the wounds made by venomous animals are his suggestions as part of treatment. Lucius Cornelius Sulla of Roman empire enacted the first law against poisoning in 82 BC to protect against careless dispensing. The Greek physician Discorides( 40 -90 AD ) travelled a lot, mostly along with the Greek Army and wrote about plants that have medicinal value as well as that were poisonous. He classified poisons as originating from Plants, animals and minerals. He is the one to suggest emetics as a means of treating poisonous cases especially if the route of poisoning is oral. IbnWahshya  (860 -930 ), an Arab Botanist and thinker wrote a book on poisons. Moses Maimonides(1135- 1204 A. D. ) , a Jewish Philosopher and physician wrote about Poisons and their Treatments and criticized some of the methods of treatments of Poisoning, practiced earlier. His writings are considered as earlier notes on the Topic toxicology. Matheu Joseph BenavanthuraOrfila  (1787 – 1853) a Spanish Scientist, known as the founder of Modern toxicology, as well as that of forensic toxicology. His book “ Treaties on Poisons” was a breakthrough  Moses Maimonides(1135- 1204 A. D. ) , a Jewish Philosopher and physician wrote about Poisons and their Treatments and criticized some of the methods of treatments of Poisoning, practiced earlier. His writings are considered as earlier notes on the Modern toxicology. Use of poisons in eliminating enemies saw a growth during this period. The beginning of 1900 saw the development of Chemistry in relation to Biology and the idea of mechanisms of action came to the fore. Rudolph Peters of Netherland, studied Arsenic gases used in the wars and came out with Anti Lewisite as an antidote for the exposure. From there on the Development of Science was very fast and embraced almost every aspect of life on earth. Man faced a situation where he has to find out ways to detoxify the Air, Water and the Soil he has to survive. His activities has contaminated even the Space and the two Arctic regions. So the modern world has the most modern techniques to assess the toxicity of any materials and to design a way to use them safely, with out which Human race could not survive for long, to write and read about Toxicity and Toxicology.

 4 – An Introduction 

Toxicology is commonly known as the study of poisons. The subject has been understood and explained in common terms from olden times. Toxicology was noted and in practice from the B.C era. It was connected with plant foods and agriculture products of different time eras. Some plants were found to be toxic or lethal to animals as well as to humans. Humans soon learned that these toxic materials could be used to the advantage to dissuade or eliminate predators or even fellow humans for various reasons. As time passed man learned that poisons are lethal but it depended on the strength and dose of the poison in question. He also understood that some poisons have medicinal properties at lower dose levels and useful for humans and animals. Some poisons were found useful to keep the pests away from the valuable agriculture plants as well as for safe storage of the plant products. As time passed it was found that poisons were also seen in animals. Modern times and industrial developments and the great world wars exposed the poisonous nature of metals, non metals, minerals, many manmade materials, radiation etc. Presently, Toxicology has become an interdisciplinary subject practiced in participation by Biologists, Biochemists, Chemists and Physicists. Further many other specialists like, Ecologist, Environmentalists, Drug designers, Cosmetic designers, Biomedical Device designers, Heavy Industrialists, Various transport designers including Space farers and Designers of Closed environments come into the realm of Toxicology as Man is the consumer of their products or the persons who has to endure the Toxic effects of their end products or bye product.  We will look into different faces of Toxicology, so that we will be able to understand Toxicology.

5 – Toxicology of the confined space

Confined space could start from an Air-conditioned room to, multistoriedoffice buildings, Drugs and Medical device factories Submarines, Spacecrafts commuting to and fro to ISS and the International space station orbiting in outer space. The common thing is that in a confined space the air is re-circulated fully or partially. In an air conditioned room the purity of the air depends on the furniture and equipments like Computer, Printer etc. Within, the materials that are used in the equipments and cosmetics used by the occupying persons.Here if you feel uncomfortable you can open the door or window and permit fresh air.

 In the case of Submarines, situation changes altogether and fresh air is very precious and not easily available. Unlike an air conditioned room the submarine has to accommodate more number of people, be underwater for days and months and the number of equipments are more in number. There will be out gassing from the equipments as well as an emergency fire. Further, the chances of contaminants polluting the inner air is more. There is no chances of getting fresh air if it is submerged. Here steps are taken to take care all these points from the designing to finish of construction of a submarine, to the possible extend. This includes from selection of construction materials, equipments, purging of unwanted gases, particulate matter etc. Even greater care is maintained in the selection of personnel working in the submarines. Though there are many number of pollutant in a working submarine we may look at the major ones. Carbon dioxide is the major one and is related to the number of human occupants. This is minimized by limiting the total number of staff in the submarine. Pollutions from the equipments and working of the submarine are anticipated and well managed by operation control. For maintaining the air clean, submarines usually will have more than one options at hand working and many as reserve. Likewise, we come across the confined environment of International space station where person spend years together, unlike a Submarine. We know that the confined environments of manned space crafts as well as that of ISS is able to provide healthy and safe living and working quarters for the Astronauts. Astronauts relay on recycled air and water to stay and work in the confined environment. For this in-flight or in-capsule life, continuous monitoring and post analysis of grab samples that are brought back into the labs on the earth are made use of. Further, there are operating procedures that are in place and strictly followed by different Nations participating in the ISS programmes, which is the corner stone of success. The various space modules whether it is a module with Astronauts or with Cargo, all have to adhere to the operating procedure. Behind all these success there is the concerted effort of Environmental Chemistry and Toxicological Science work of the past several decades and that of present. Even then the Toxicological emergencies could happen at unexpected events as it has happened in Appolo and Mir flights, but without mortality. It is the humans that act as monitoring as well as primary detoxifying agents in the Outer space. On alert from the occupants only many of the toxicological situations have been noted and rectified. In the ISS the lucky option is to shift to the U.S. port or Soviet port as and when one shows signs of contamination. It is the constant vigilance of the occupant Astronauts and the backup teams and strict follow up of work procedures make the Travel and life in outer Space Safe and Healthy.

6 – Toxicology of  Politics, Flood and other Disasters

6.1-Politics:-   What Toxicology has to do with Politics? Is it an imaginative Title? Or it is given a part in the different classes of Toxicology? Let us give a look at the different ways Toxicology is related to Politics and then take our opinion. The medical device Industry is rather new when compared to Lead Industry. For the last 40 years the Medical device industry follows Testing methods established in the beginning for regulating the Medical Device Products. Many advanced techniques in physical, chemical and sensing have come up but has not found a place in regulation. But many manufacturers are using modern methods to prove that their products are more safer to use. In the case of Lead, from good olden times to that of Notre-Dame fire the presence of Lead and the safety of the public is questioned and debated. The safety of any material is well documented and there are Food and Safety Units, Industrial Regulatory bodies, Environmental pollution Control Units, Explosive Use and Control set ups and a host of rules and regulations, National and Inter-National are in place.  But we can see that they are controlled by Political will and Pleasure. Take the case of development of Atomic power and Bombs, Exploration of Space, burning of rocket fuels and littering debris in outer Space, Dumping of Nuclear waste in the sea of the close by Nations, nonstop production of Chemicals for daily use, pest control, drugs, cosmetics, luxury products, food additives, colors, non eco friendly power production by coal, atomic power, exporting and importing of plastic and other hazardous waste etc. are controlled by Political decisions and not by any regulation, without respect to Human beings and other living organisms inhabiting the earth. The main curse behind all political decisions are kick back money, technically termed Commission percentage and not safety of living beings. These circumstances explicitly explain the Toxicity of Political activity, Nationally or Internationally.

6.2 -Flood and other Disasters:- Toxicology is the science of Poisons. What Toxicology has to do with Floods and Disasters? Money which is attached to corruption is the most powerful poison in this world from time immemorial. We all know that land slides happen during the flood because of not following rules or natural principles of cultivation by our forefathers who went to the near forest areas for practicing agriculture. Authorities have reports and reports regarding the use and misuse of cultivation and running stone quarries in the sensitive areas of hills in the border areas of Kerala State. The Government has rules and have empowered the last in line authority for scrutiny and implementation of the same.

7 – Classification of Toxicology

Analytical Toxicology, Applied Toxicology, Clinical Toxicology, Veterinary Toxicology, Forensic Toxicology, Industrial Toxicology, Environmental Toxicology are considered to be major divisions of Toxicology, in general. Toxicology as we know encompass the study of poisons, in the early years. At present, Toxicology touches any materials and it’s safety and it’s nature of hazard or risk in use or exposure. Thus Toxicology has become a broader Topic applied to various areas and subjects. When you want to study the hazards from food, it becomes food toxicology. You will observe that, to study food toxicology, the toxicologist has to have a team of experts to analyze the chemical composition, contamination if any, in the food before and after preparation, safety of packing material, bacterial contamination and sterility of the packed or served food. Likewise, drugs, cosmetics, dress materials, cleaning materials, insect repellents, construction materials of the Residence etc. needs to be safe for prescribed use and toxicology plays an important role in the day today life. Therefore, the general classification put up here is not a rigid one and could be added or changed. Academic toxicology, Regulatory toxicology, Occupational toxicology, Immune toxicology, Genetic toxicology, Contract toxicology, Political Toxicology etc. are some examples that we will come across. Toxicologist needs to understand the cause of toxicity and the extend of the damage brought about by the causative agent. For that he has to analyze the tissue samples, organ samples, blood, urine etc. and the causative material if any left. Spectroscopy, Chromatography and Immunological assays are used to detect, identify and measure the toxic material in question during a Toxicological study. Toxicologist has to evaluate the nature and mechanism of action of a poison or a material in biological systems, he applies various techniques available in Biochemistry, Pharmaco dynamics, Pharmacokinetics, Instrumental chemistry, Medicinal Chemistry etc. This area is denoted as Applied toxicology. When cases come up with over dose of medication, substance of abuse or poisoning, exposure to chemical burns or radiation in an Emergency Medicine Centre, the Clinician with special training in the management of such cases, evaluates each case, asses the extend of hazardous exposure and prescribe medical care for the patients. This area of Medical practice, where science of toxicology is employed in the treatment of patients is known as Clinical Toxicology. Like wise, the science of Toxicology used in conjunction with Veterinary Medicine is known as Veterinary toxicology. Other than the routine diagnosis and  treatment animals, the study of contaminants entering human food through animals, ecological and environmental contamination of animal habitat and foods, accidental poisoning from plants and venomous animals etc. also are relevant under this topic. Use of science of Toxicology in investigating the cause of poisoning, death by poisoning, identifying the nature, quantity and time of poisoning, misuse of drugs and chemicals falls under Forensic toxicology. Industry is an important word in modern world. Whatever we use in our daily life is produced in an industry. Any material or any product comes out of chemicals, physical forces, temperature and through lot of mechanization. In the manufacturing process, in transportation of raw materials and finished products the environment of production, packing, transport are polluted by the raw materials, accessories used to aid production process, packing, labeling etc. The work force and the people staying near and around the industry are sure to get a share of contamination. It may be through Air, Water or the soil where the waste material from the industry is dumped. This result in the deterioration of health of the industry worker as well as the people, animals and plants in the locality where the industry is situated. Such situations has initiated the subject Industrial Toxicology. The occurrence of contaminated Air, Water and Soil is very common in any places on the Earth, even the Arctic regions and the Space. As we are aware, the environment is contaminated and the branch of toxicology concerned with the health of Human beings, animals, plants and all living beings is, Environmental toxicology. Science of toxicology, assures safety and lead us to capably use anything that is hazardous to our advantage by designing safe protocols for use.

7.1 – Aquatic Toxicology: Toxicology of our water bodies are very important because we all depend on water for our daily life. Water is a major constituent of any living organism ie., plants, animals and humans. Further, more than 70 % of the earth is covered by water. These water resources may be streams originating from mountains, flowing down to form lakes, rivers and ending up in the Sea. These aquatic forms are abundant with plant and animal life depending on the depth and volume of flowing water or water contained. We humans collect water from these sources for drinking, house hold purposes and Industrial use. House hold use and Industrial uses of water turns water to be a contaminated with a multitude of chemicals like PCBs, DDT, TBT, Pesticides, furans, dioxins, phenols as well as atomic nucleotides. This can happen as a result of direct discharge from production units, from storage and transport units, as a wash off from end use areas. In such a back ground Toxicology of water bodies- Aquatic toxicology become very much relevant. Contamination of water need not be of whole water body and it may depend on the nature, physical and chemical characteristic of the contaminant and depend on the water currents and weather conditions such as prevailing air velocity.

 Aquatic Toxicology comprises the identification and estimation of contaminants noted in various water bodies. This will help us predict the contamination levels in the living organisms found in that particular water body, which in turn alert human beings about the hazards in consuming the fishes and water sourced from such environment. The analysis of aquatic environment and plants and animals inhabiting that environment is a team work of interdisciplinary scientists using physical, chemical, and biological techniques. Most of the animals are “consumers” since they are unable to prepare their own food but depend on consuming other smaller organisms in general. Therefore, water pollution will reach the higher organisms through a food chain. Aquatic Toxicology could also explain whether an Industry is maintaining its regulatory norms as laid down by the Regulators. There is a plethora of testing methods available to find out the potential contaminants in plants and fishes in Aquatic environment.

7.2 – Cosmetic Toxicology: Cosmetics and Toiletries are products very much different from Drugs, Agrochemicals, Pesticides in that, they are applied to the human body very much knowingly and willingly by human beings. Majority of us never think that the cosmetics and toiletries are made up of chemicals and can be hazardous to use. For example to make  soap only minimum of three chemicals are required, but the soaps available in the market may contain more than three. They may contain additives to impart color, smell, lather, softness, transparency, liquid nature etc..Likewise each cosmetic product you use will be a combination of various chemicals to attract you to buy that. That means, the ingredients of these cosmetics and toiletries will enter our body through the nostrils, mouth and skin. Most of our organs, lungs, eyes, facial tissue, lips, surface of body, hair are exposed to cosmetics and toiletries as we prepare ourselves to go for daily work, make up during office intervals, travel back and forth from work, getting a bath to freshen up at home and waxing up the skin to fight aging and to feel young. During the daily routine we do not get time to read the list of ingredients or directions for proper use. We select a cosmetic from an advertisement or advice of a friend. Toiletries and cosmetics are not regulated items like Drugs and chemicals. Further, the recent trend for adding natural products to cosmetics for enhancing their acceptance and may be to increase profits. So we may suffer allergies, irritant eyes, asthmatic symptoms, immunological reactions depending on the contents of these products. We are familiar with hazardous responses from hair dyes and baby powders. Therefore, Toxicology has an Important role to play in the production and use of cosmetics and Toiletries . The display of detailed content list and a certificate of Safety should become mandatory and cross checked by Regulators. These products have to undergo Toxicological tests for acute and chronic nature depending on the site of use and absorption characteristic of the tissue which comes under direct exposure. Further, as customers we have to be choosy when we buy Cosmetics and toiletries, not to buy hazard causing items paying hard earned money.

7.3 – Environmental toxicology: Air, Water and Earth constitute the environment. Toxicology of the Environment means that our air, water and earth are not pure or they are contaminated and we have to make use the Science of Toxicology to make them safe to live with. Whenever we are at some activity we tend to add something to the natural air. Travelling raises dust, fumes that carry burned out fuels and also move the air from one place to the other. While cooking we add fumes and heat to the air, Industries, Electricity generating Plants whether it is coal fired, oil fired or nuclear they all contaminate the air, water and earth. Same way water too is contaminated by our daily activities. Transport, power generation, cooking, cleaning, cooling etc. contaminate water and water bodies. Earth is contaminated by more ways than air and water. Major contamination is by construction activities and the pouring of chemicals and other contaminants  carried by air and water. Further earth is the only place which will take all the waste we generate, use and dump. Agriculture is carried out on the earth and all artificial manure and pesticides, weedicides we use end up on the earth and reach our body through the food we eat or water we drink. Rain water takes these chemicals to water bodies that are nearby or filter into ground water to contaminate our drinking water sources. Our streams, rivers and seas get contaminated and the fishes we harvest from them will also be tainted by various contaminants. We have noted how our environment is contaminated. It is no exaggeration to say that Mother’s milk is contaminated by chemicals that are prevalent in the air and water the family live. The air, water and soil of the Arctic area are contaminated by  products of Atomic Bomb Testings that were conducted in the past years by the super powers. Therefore Environmental Toxicology has become a need of the hour to regulate the contamination of our environment so as to retain a livable earth for human beings as well as other living things in the air, water or on the earth. Environmental toxicology is extensive that there should be an interdisciplinary team of people from toxicology, engineering, chemistry, ichthyology, biology, materiology,  geology, hydrology, aerology(atmosphere), maths, computer Science etc. who will together assess the contamination rate and predict the future and lay down guidelines for slowing down of the pollution rate.

7.4 – Industrial Toxicology: We are living in a world where over one lack things/ materials are produced by various Industries to meet the requirements of our daily life. Each industry in production will need the raw materials for production, need power, machinery, transportation, packing, storing, distributing the end products and people to operate the factory. All the processes mentioned should not be hazardous to the people working in the factory, people using the end product, should not pollute the air in the environment, should not contaminate the nearby water bodies or Soil. These are the minimum requirements that Govt. Pollution Control Bodies put forth. It simply means that, any Industry should be environmental friendly and safe. There for it is a routine practice for the Industry to have Environmental Engineers or Toxicologist in their pay-Roll. Further, Industry will have to submit yearly or six monthly report on various safety audits to the Pollution Control Boards. It is each producer’s responsibility to make sure that their products are safe to use by the customers or consumers. Therefore Companies send their products to Toxicology laboratories for toxicity and safety testing and get their product certified. Petrochemicals, Drug companies, Pesticide manufacturers, Cosmetic producers, Food and Drink makers, Residential and household product manufacturers, Milk and milk product makers are some examples of Industries. On analysis one can find out that the Petrochemical companies and Milk product companies are places of potential Occupational  as well as Environmental pollution. The procedures are Instituted for preventing Occupational hazards in the Industry as well as the Local environmental contamination and to get safe products to the consumers. Basically, Toxicological Tests are common but there are other factors linked to the Safety studies. Presently every industry is under  daily recording of  worker environment, environment out side, safety audits and preparation for Emergencies and disasters. The safety of the Industry depends on Toxicologists, Environmental hygienist and to the regulatory agencies concerned.

7.5 – Food Toxicology: Food, water and air are considered safe routinely.  Before 30 years we got the ingredients for food and prepared ourselves what we wanted to eat. We drank water from our wells or ponds near our land and breathe the air in our surroundings. Presently we use prepacked materials which are ready to be cooked or eat out or order food through “Uber”, get potable water through water supply department, breathe air that is available around us.

Food available ie. Grains, cereals, lentils, vegetables milk, fish, meat are now procured from far away farms and water bodies. They are produced using modern agriculture methods, processed mechanically and we get them in plastic containers or sachets. Some products come ready to be cooked and some needs to be processed at home from cleaning to cooking. We are sure that modern food we get, whether it is processed or natural it will be contaminated.

We will try to sort out the real problems that affect the food we eat and how they are going to affect the consumer. Knowing these basics will l become a tool to get and use safer food. The contamination of food by Chemicals comes first. This can happen as chemicals that were used during the cultivation, growth, reaping, storage, packing and transport of the agricultural products, as seed protector, sprouting agent, pest repellent, artificial manure, rodent repellent at storage and transport times. Natural foods derived from animals and various plants add to the potential of different chemicals other than mentioned in earlier para. Fungal toxins are complex contributors to the food toxicity. We can assume the different ways that our food gets contaminated. Purposeful addition of chemicals for preservation of foods, to give food items color, smell, taste and crispy consistencies also bring possibilities of contamination and hazards to the consumer. The only option is the use of available analytical methods to separate and identify the chemical contamination of food and ascertain the safety of food. Like drugs and Medical devices it has become the duty and responsibility of the Producer of food items to prove that his product is safe to consume.

 In modern times good agricultural practice instructs that that producer give an information sheet beginning from the selection of seeds to the end process of packing and transporting of the product, so that the customer get an idea about the possible contamination of the product. Another way out practiced by manufacturers who follow good manufacturing practice is to attach a Food Analysis Certificate of their product as per National Norms to give the Nutritive and Contamination level of his product. Food poisoning is very hazardous as it spreads fast and easily as most of the classic foods are produced by Great Manufacturers and a single batch may contain Lacks units of food packets.

7.6 – Drug Toxicology: Drug toxicology is a very broad area which needs more specific address for clarity. The title can simply mean that it is about the toxicity of the drug which is under development, study of use of illicit and unknown quantity of drugs in suicide or poisoning cases. Here we will learn about the role of toxicology in drug development and separately we will take up the latter under the title Forensic toxicology.

We know that science is always moving forward with innovations in the area of search for new medicines to increase the efficiency of treatment and complete cure and with lesser side effects. Researchers work to improve the preparation of old medical compounds, for refining the core compound of the drug etc. From the determination of the primary ingredient selection, purification, synthesis of the core drug, efficacy testing, clinical trials etc. the toxicology will be involved so that a perfect, safe and effective drug is developed and delivered to the people. This process may take years and lot of money. We have learned about the nature and type of various Toxicological tests, earlier. A battery of tests will be selected and applied for the Screening of drugs. We see that drug comes in various forms ie. Tablets, capsules, powder, ointments, liquids, drops, injections, drips, patches etc. All these along with their containers, packing materials, labels, sterility status will also contribute to the safety of drugs. So, an extensive reading is needed to understand drug toxicology in detail.

We know that what ever may be the matter or material in question a living being has to get exposed to it. Then only we will understand the material is harmful or not. We may get exposed materials through the air we breath, water we drink, or food that are produced through agricultural practice. That means that we can get exposed through our lungs, orally, through our skin.

7.7 – Acute Exposure: We now know what is meant by Exposure in Toxicology . Daily we are exposed to hundred and one items through the air we breath, water we drink, food we eat, the drugs we consume, the cosmetics we use almost every day, the secondary smoke from smokers around and so on. These are all exposures in general, may be in small doses which may not bring about any noticeable responses. But when we get exposed to some chemical or a poison and experience some untoward response from the body, the exposure become acute exposure to the particular chemical or poison.  Acute exposure of a poison at a small dose may not result in any harm to the body but exposure to even a very small dose of hydrogen cyanide will bring death. Acute toxicity response can be a result of a single dose or multiple doses during a day or 24 hrs and may be without knowledge of what the Toxin to which the exposure is made. On exposure to small doses/ an acute exposure to a hazardous material may initiate symptoms like nausea, vomiting, stomach pain, head ache, irritation of skin and eye, cough, oedema, seizures, respiratory failure etc. Toxicity data on almost every existing chemicals and xenobiotics are available. These data is arrived at after carrying out experiments in Laboratory animals.

7.8 – Chronic Exposure: In our day today life we may be exposed to some material each day repeatedly for over a period of time for more than 90 days, it may be called a chronic exposure. This may be due to work conditions, environmental conditions, or may be due to modern life style or use of certain drugs eg. for Asthma. These materials may be hazardous or debilitating in the long run. Exposure to Lead or Mercury through food or water, pesticides during agriculture work  or the vehicle solvents in many inhalation drugs. These kinds of chronic exposures to low concentrations have deleterious effects of living systems, symptoms slowly developing and expressing. The symptoms expressed owing to a chronic exposure may not subside as soon as the exposure is with held or stopped. These symptoms may be the outcome of the toxic or hazardous nature of the material to which the exposure is made. Hazardous substance need not always be a toxic material or a poison. Fibrosis of liver due to consumption of alcohol, kidney problems due to slow lead poisoning, neurological problems due to mercury poisoning, chronic bronchitis of chain smokers, fibrotic lung disease of coal workers, cancer of asbestos miners are some examples of chronic toxicity outcomes. Drugs for asthma, blood thinners, pain killers, thyroid problems are some examples which people take without medical supervision and end up in chronic exposure resulting in untoward results.

7.9 – Dose:-         Dose” simply means to the common man, the quantity of a medicine or drug prescribed by the Physician to be taken at a time. When we have fever, the doctor prescribes fever medicines. It is 500 mg of antipyretic for an adult, 3 times in a day, usually at 8 hr intervals, may be for 3 days or as advised by the Physician. Like this we know about the prescribed medicines for treating various illness have doses to follow. But in the case of Xenobiotics, when we are exposed, that can be a single dose as in the case of radiation or corrosive gases like chlorine or carbon monoxide, cosmetic cream applied to the skin. Here the dose is single exposure, but quantity of the xenobiotic is unknown and the effect of that exposure depends on the nature of the xenobiotic, period of exposure, the health of the exposed etc. How much xenobiotic was present?, the toxicologist has to estimate after studying the source, nature and period of exposure and clinical lab data from the exposed.

In the case of a drug or a xenobiotic the intake at a time could be “a dose”. The frequency at which the dose is repeated and the duration for which the dose is repeated is important and gives an idea of “total dose”.But when we take into consideration the response of the living system to eliminate the drug or xenobiotic, the quantity that is left in the body after elimination becomes the “internal dose”. The biological process involved in the elimination of Dose that is administered imposes problems in estimating the internal dose. “External dose” is another term denoting the dose equivalent received from radiation sources outside the body or living organism. Another common term used is “Threshold dose” which means the lowest amount or exposure level of a toxic substance at which a specified and measurable effect manifests. I hope, the reading on “Dose” will give an idea about the different terms used in Toxicology. When we are dealing with response, mechanisms, biochemistry, testing etc. in Toxicology, we will come across more terms related to toxicology, pharmacology and medicine and will elaborate on them.

7.10 – Response: We respond to friendly calls and conversations. Our response to fire is to douse it and call for help if required. In toxicology, response is the action or reaction of the organism towards exposure to a xenobiotic. We have seen that exposure can be through inhalation, ingestion, through skin etc. This response depends on the dose to which the organism was exposed. This reminds use of the basic principle in toxicology that the dose decides the response, whether a material is Toxic or Nontoxic. The living organism could be exposed to various chemical, radiological, so called inert materials through the environment in which they exist. These exposures bring about responses according to the concentration of these xenobiotic, finding a way into the organism. Take the case of burning of waste in the open. There is heat and smoke. The nature of heat and smoke varies with the content of the waste. Our response is to avoid the smoke, as it gives a burning sensation to the eyes and suffocating to the lungs. These are immediate response. In reality the acidic nature of the smoke makes it dissolve in the tears and form acid which gives irritation to the eyes and make it appear red in color and swollen. The same effect is happening with in the lungs and the cells in the lung gets swollen and suffocation happens. Here the response is from the tissue and as time pass, the acidic portion is scavenged by the body and relief is obtained. So the initial response of the body gradually may fade if the nature of exposure is acute or temporary. If the exposure is going to be continuous or the concentration of the exposure is going up, then the response will become severe and the organism continues to be in the smoky environment, the response becomes more suffocation because of lung tissue, failure to supply oxygen to the whole body, resulting in a stand still of body functions leading to death. The response depends on the status of the environmental conditions and availability of emergency medical assistance. To understand and asses the response of living organism to toxic materials, various methods have been developed and in place

From the previous readings we understood the basics of  Response. Now we will try to understand how Dose and Response relate to each other in a toxicological study setting. Dose, response relationship is described based on studies conducted on experimental animals, clinical trials in human beings and cell culture studies. When a population is exposed to a toxicant or poison the response may vary from person to person or to say there may be a group that is affected severely and another group affected in general. That indicates there could be variation in response at a particular dose. But in general you can observe an increase in response as the dose increase and reaches a stage where all exposed are severely affected or dead. The final dose which brings in death is known as the lethal dose. The lowest dose at which an observed response is evident is known as threshold dose. If you are plotting the response to a xenobiotic or a poison in a standard graph, the X axis for dose and Y axis for response, the point at which the response is noted becomes the threshold dose level and as the dose increases the curve also increases and the point at which the response is 100% severely affected or mortality. The threshold level will vary for each substance and the potency increases as the slope becomes greater. The quantity of xenobiotic or to a poison to which an individual or organism is exposed or administered over a period of time or in several and individual doses may be considered as administered dose, the dose at which the individual or organism responds initially is called the threshold dose, the division of the total dose and its administration at different period of time decreases the toxic effect. All these factors are necessary for determining dose response relationship. As we have noted earlier, the common dosage unit is mg per Kilogram body weight per day. As we read on we will come back to the basics of dose and response and their relationship to acute and chronic exposures and responses.

7.11- Route of Exposure. Exposure to a xenobiotic present in Air, Water or soil can happen through skin by dermal absorption, by inhalation through the respiratory system or by ingestion through the digestive tract. The same is true for drugs. Further, xenobiotics can enter the body via food, drugs that are consumed as routine in daily life or administered intentionally by self or a third person. These are called route of exposure. Oral, inhalation and dermal are the common routes. Substances can enter the body through the Parenteral route too, ie. the entry of xenobiotics through intramuscular, intra dermal, subcutaneous, intravenous or artery injections. Food, medicines, water are the common materials we ingest and contamination through these vehicles is possible. Through inhalation we breath in any contaminants that is very common in the immediate environment air surrounding us. Dermal absorption is common from the water we use, cosmetics, soaps and detergents, skin drugs or work related materials. Parenteral route commonly involved with prescribed medicines drugs of abuse or given by illegal means / options. Patterns of Exposure are important to be kept in mind. It can be one time, acute exposure or continuous, chronic exposure. This may be also a high dose acute exposure or a low dose chronic exposure.

When we talk about exposure we have to consider dose, age, body size and time also. Already we have noted basics about dose. An adult may need 650 mg (thrice a day) of antipyretic drug to control fever but  for a child 650 mg is toxic. Child may need only may be less than 80 mg. Another parameter is that 100 mg of “X” substance will lead to death of an adult as a single dose, it need not lead to death if taken 10 mg per day over a period of 10 days. Commonly the dose is set by mg per kilogram body weight per day or, mg/liter for liquids and, mg/m3 for air and other units like parts per million, parts per billion and parts per trillion are  well in use.

But when doses are compared with different species of animals it may be by body surface area. The unit of time during which a dose is administered is accepted is “a or one day”. When xenobiotics in question are strong poisons the dosage will be lesser that mg/kg, namely microgram, nanogram, picogram etc.

  7.12- Xenobiotics.Toxicology is all about materials or chemicals that are not normally found in an organism, a Xenobiotic. Living organisms will make reactions or responses towards the xenobiotics that enter the living organism. What is the xenobiotic in Question, the quantity that entered the body, the number of times the xenobiotic entered, the route of entry, the residue that is left in the organism etc. are questions which a Toxicologist ask and try his best to find out. Without these information he will not be able to access the true nature of the hazard and help to ameliorate the condition of the living organism. The xenobiotics that are usually hazardous are commonly known as poisons. In case the xenobiotic in question is a known material or poison, the toxicologist may get readily available information on the chemical nature, safe dose, possible responses of the living body towards the substance, degeneration within the body etc. and may be able to suggest methods to neutralize or detoxify the effects. The xenobiotics can enter the body from the environment or ingested knowingly or purposefully. The Air, Water and the Soil in the environment contain lot of xenobiotics, that will enter the living organism through inhalation, ingestion, absorption by skin 

8 – Testing in Toxicology

Testing in Toxicology have many branches or separate and individual divisions, with reference to the purpose or objective of testing. It will be easy to understand this general classification.

8.1 – Toxicology Screening of a person: Here the tests are carried out to diagnose the nature of poisoning a person might have exposed to. It may be self poisoning (suicide attempt), accidental poisoning, poisoned by another individual, poisoned under the working environment, insect or reptile bites etc.The route of exposure if known, the conduct of test will become easy and quick to perform. The main aim will be to ascertain the quantity of the poison, if known, otherwise it also will include to identify the poison. Toxicological screening of a person is very common in Industrial settings, Sports and Games, suspected poisoning and cases of unknown poisoning.The blood, saliva, breath and urine samples are usually collected for analysis.

A toxicology test can screen for drugs of abuse in daily life or by a sports person. It may not specify whether you are an addict but may say that you have been using it recently.

• Amphetamines
• Barbiturates
• Cocaine
• Methamphetamine
• Marijuana
• Opiates
• Phencyclidine (PCP)
• Drugs banned from competitive sports

Toxicology screening of dead bodies are carried out by Forensic toxicologists and they may take blood,urine, body fluids and parts of body organs as samples for analysis.

8.2 – Toxicology Screening for Materials in general: Another requirement for toxicology tests are for certifying any materials for human use. Any drugs, chemicals, cosmetics, house hold utensils, food and additives,etc. have to get a License to sell, they have to get a toxicity and safety data and approved by the Drug controller or  Food and Drug Administration. These commercial materials are submitted to Accredited laboratories for safety testing and get the reports and submit them for clearance for sale.

Examples of Materials that has to get Certified before release into the market.

Agrochemicals

Petrochemicals

Paints and Varnishes, Polishes

Drugs allopathy- ayurveda-Unani

Pharmaceuticals

Talcum powder, Body lotions and sprays, creams, make up materials.

Packaging materials for drugs, food and farm products.

Medical devices

Genetically Modified crops and Genetically modified organisms

Dyes, softeners, mordants and dye removers

Toys and devices meant for children.

Herbal formulations

Battery of Tests for Toxicological screening of Materials.

Dose finding Studies.

In-Vitro toxicity studies

Acute Toxicity tests

Chronic toxicity Tests

Reproductive Toxicity tests

Carcinongenicity

Inhalation Toxicity tests

Dermal Toxicity tests

Systemic toxicity tests

Implantation studies

Sensitization tests

The above noted are common Toxicological studies that are listed in a Battery of Tests. The toxicologist will select appropriate tests for each material after studying the physic chemical nature of the material submitted for Toxicological evaluations.Special studies could be conducted as required by the Customer or Product manufacturer or as demanded by the Drug Controller or FDA.

8.3 – Toxicological  Tests General: In vitro toxicity studies are used to determine the potential of a new pharmaceutical, agrochemical, food additive, or other chemical product to be hazardous to humansn. Studies are carried out using Cell culture techniques to evaluate the toxic potential of the test material. Toxicologist after evaluating the results will decide whether the test material need be tested for use and should undergo further testing using in vivo (animal ) studies. Results will also could be used to as a guide for finding appropriate dose for further studies.

8.3.1 – Acute and Dose Range Finding Studies:- As part of a general toxicology program, acute single dose toxicity testing in animals is used to help define and characterize the intrinsic toxicity of new test items and provide data for establishing acute exposure to that substance. Information generated in acute studies along with dose range finding assessments is used to guide the design and selection of dose levels for subsequent subchronic and chronic toxicity studies.

8.3.2 – Chronic Toxicity Tests:- Chronic toxicity tests are defined as tests that characterize adverse effects following repeated administration of a test substance over a significant portion of the life span of the test species. Establishing the duration of a chronic study is based on the anticipated human or environmental species’ exposure.

8.3.3 -Reproductive Toxicity Tests:- Reproductive toxicology is the study of the effects of chemicals on the reproductive and neuroendocrine systems, and also the embryo, foetus, neonate and prepubertal mammal. It could be used either as a single test or as a two generation test as desired by the Toxicologist.

8.3.4 – Carcinogenicity Test:- The major conclusions are that chemicals evaluated for long-term toxicity and carcinogenicity in experimental animals can be divided into three categories: (1) those that cause organ toxicity without cancer, (2) those that cause site-specific cancer with no associated toxicity and (3) those that cause toxicity and cancer.

8.3.5 – Inhalation Test:- Inhalation is the primary method for delivering pharmaceuticals to patients with lung or airway diseases and is also a leading route of unintended exposure during manufacturing, handling, or use of chemicals and agrochemicals. Safety assessment for inhalation toxicology requires specialized capabilities to successfully test the effects of inhaled compounds.

8.3.6 – Dermal Toxicity Tests:-  This test method provides information on health hazard likely to arise from short-term exposure to a test chemical by dermal route. Test chemicals should not be administered at doses that are known to cause marked pain and distress due to potential corrosive or severely irritant actions.

8.3.7 -Systemic Toxicity Tests:-  Acute systemic toxicity testing involves an assessment of the general toxic effects of a single dose or multiple doses of a chemical or product, within 24 hours by a particular route (oral, dermal, inhalation), and that occur during a subsequent 21-day observation period

8.3.8 – Implantation Studies:-  Candidate materials are made into desired strips and implanted in the test animals to study the tissue response of the biological system to the test material to understand the biocompatibility of the material.

8.3.9 – Irritation Test:- The  irritation Test is used for the determine capacity of chemicals and medical devices to cause irritation while in use. These tests are assessing the potential of a material or product to initiate irritation of respiratory system, tissue or organs, dermal or ocular systems.It may be redness, inflammation or scar formation.

8.3.10 -Sensitization Test:- The Sensitization test is used for the determination of sensitizing activity of chemicals and medical devices. These tests are assessing the potential of a material or product to cause a delayed hyper-sensitivity reaction.

9 -Safety / Toxicity Testing of Biomedical Devices

Medical devices are life saving devices and it is mandatory that they should not impose any health hazard to the user whether it is a human or animal. As we know medical devices are classified as per their use and nature of contact with the body. The length of time the medical device is used, whether its contact with the body is superficial or invasive, whether it is active with in the body or contains medications etc. etc. are to be considered when the tests necessary for screening them for safety. Contact of devices with the body less than 24 hrs is Limited duration, more than 24 hrs and up to 30 days, Prolonged duration, more than 30 days, considered Permanent. So it is going to be different for different medical devices. Today we will look into the basic tests applied for Medical Device safety screening.

In the present scenario whenever a material comes for Toxicological testing it comes along with a comprehensive, Performance testing report,  Material characterization report, ASTM Certificates from material procurement agency etc. will give the Toxicologist ample information to decide upon the different tests warranted for Toxicological testing.

Cyotoxicity, Irritation, Sensitization, Intracutaneousreactivity, Systemic Toxicity, Genotoxicity, Implantation, Haemocompatibilty, test for Biological reactivity, Pyrogen are the basic toxicology tests from which Toxicologist will select  screening tests that a medical device under consideration has to undergo.

Each of the above tests will use the extracts of the Test sample, strips of the test samples for carrying out various Toxicological Tests.

9.1 – What is a Medical Device: A medical device is a man made material or objects to alleviate or replace the functions of any body organ/ systems, without deforming or chemically degenerating or acted upon by body tissues and fluids. In this way medical devices are differentiated from drugs other chemical formulations.

While carrying out the intended activities a medical device becomes a Xenobiotic or foreign body and naturally invokes body response. These responses can be in the form of irritation, inflammation, necrosis, sensitization, cancer etc. and end up in the removal of the medical devices.

Therefore the materials that go into the fabrication have to qualify that they do not invoke body responses. Above we have noted only the general responses. Body responses to various biomaterials differ depending upon the nature and intended activity of the device as well as the different materials go into the fabrication of each medical devices specifically. Devices that act only on the surface of the skin, devices that penetrate the skin, devices that are going to stay for hours or days such as catheters, dialysis tubes, saline and blood distributing tubes, stents, heart valves etc. gives an idea of different type of medical devices. Dental materials, artificial dentures, contact lens other correctional devices for eyes, artificial hip, bones and knuckles, man made vertebra etc. are in common use at present.

9.2 – Testing  an Introduction: In our class we learned that what is testing in toxicology meant. Today we will try to see what is Toxicological Testing. We use  food, water, air, drugs, cosmetics, cleaners, perfumes, dress materials, materials for residents and offices, materials for transport vehicles, materials for air conditioning and keep the air tight compartments livable. We use different kind of fuels for various purposes, we use atomic power for discrete uses and so on. We have come a long way from our ancestry of the jungle and may be getting ready for a future which we may not be able to dream today. Finally we use medical devices as life saving products which amounts to more than sixty thousand in numbers under Medical prescription. In all these , the materials have to be safe to be used

has to undergo to get certified for use in general. Depending on the nature and period of use, many tests may be carried out for longer period of observation and collect more data for support of safety. Tests like Carcinogenicity Testing, Reproductive and developmental toxicity testing, Immunological profiles and Pharmacokinetic studies of test samples and end products are some examples of the same.  The onus of selling safe products is on the manufacturers. The end user ie. the Medical persons and Surgeons who prescribe  medical Devices are responsible for choosing Biocompatible products and for reporting the occurrence of hazards in the use of Medical devices.

Biological reactivity Test    Medical devices made up of Elastomers, plastics, polymeric materials are tested for adverse effects before use. The main objective is to be safe from change in material composition during processing, cleaning procedures, inks, adhesives, permeation of preservatives, expose to conditions of storage and transport, methods of sterilization etc. could affect the end use of the materials. There for these end products are tested for adverse effects. In this test the extracts of the materials are prepared and injected into a rodent species. This test is a standard test for Biologicals and Biotechnologically produced samples.

The medical devices undergo intracutaneous irritation test, systemic toxicity test and Implantation test in order to evaluate the local and systemic toxicity of the biomaterials that go into the fabrication of the medical devices and also that of the end products. In these tests different combination of extracts and solid materials are used. The USP class VI test and the ISO 10993 guidelines include intracutaneous, systemic toxicity and implantation tests they vary in many details such as specific activities like sample quantity and preparation, different types of extraction media and duration recommended, preparation and observation periods of test animals, processing of post harvesting of test samples.  Meeting the requirements of USP Class VI does not meet the requirement of Biocompatibility of a Medical Device. ISO 10993 requirement has to be met positively.

9.3 – Genotoxicity Test: This test was introduced into the battery of tests for testing the toxicity and safety of materials going into the fabrication of medical devices or end product, medical devices. No materials leaching from the medical devices should induce genetic damage to the patient. Genetic damages are caused by mechanisms like gene mutations or point mutations, chromosomal damage or translocations, small or large deletions and numerous chromosomal aberrations. Organization for Economic Cooperation and Development has proposed a battery of tests for identifying the different types of chromosomal aberrations like Ames Test- a test for gene mutations in bacteria OECD 471, an in vitro test for chromosome damage, OECD 473 ,an in vitro mouse lymphoma assay OECD 476 and an in vitro mammalian cell micronucleus test for chromosomal damage OECD 478. ISO outlines various tests for Genotoxicity in its standard ISO 10993-3 Tests for genotoxicity, carcinogenicity and reproductive toxicity. OECD and ISO covers similar techniques for genotoxicity testing. We can see that there is common methods in sample preparation and extraction methods adapting various extraction mediums, may be drawing from USP methods. The main aims to assure that the Medical devices do not carry any genotoxic potential.

9.4 -Haemocompatibility Test: Blood is a complex mixture of Plasma with albumins, coagulation factors and immunoglobulins, erythrocytes, leukocytes and platelets held together in a harmonious way. Biomaterials and medical devices that are destined to be in contact with blood should be blood compatible. They should not activate plasma, erythrocytes, neutrophils or platelets in order to be blood compatible. All new biomaterials or new composition of the biomaterials should be tested for blood compatibility or hemocompatibility. Heart valves, stents, cardiac pace makers, ventricular assist devices, vascular grafts, patches for  ventricular septal defects, cathwires, many nannoparticles, shunts and dialysis tubes, I.V tubes are widely used medical devices. The test for haemocompatibilty has been evolved through a long period from the initial Thrombogenic potential test. Presently the ISO test for haemocompatability is the universally accepted test.

More emphasis on carrying out haemocompatibility test is given on the preparation, environment, care and speed involved in the preparation of equipments, drawing of blood and conduct of the test. The peripheral blood samples has to be collected from healthy human beings who meet the requirements of the Standard. Use of appropriate anticoagulants, un traumatic drawing of blood with outvenostasis by using 21 gauge needle are recommended. Biocompatible syringes and containers for blood storage and above all to draw the sample just before the test so that the tests could be completed at the earliest after collection. This is very important as blood is a complex “organ” and any variation can bring in anomalies in the results.  In the test the collected blood is diluted indifferent tubes and the test samples are exposed to the test solution for the time and temperature recommended. After the completion of the test the results are evaluated as per the standard.

9.5 – Pyrogen Test: Medical devices, especially implantable ones are tested for substances that  induce fever or that are pyrogenic. This test is called Pyrogen test. Further the common use of the parenteral route or intravenous route for drug administration also warrants the products to be free of pyrogen causing substances. Pyrogens reach hypothalamus of brain which control the body temperature and initiate high fever. Endotoxins  which are pyrogens, are polysaccharides present in the gram negative bacteria. Another common pyrogen is interlukin-1 which is produced by macrophages when in contact with certain bacteria or viruses.

9.6 -Rabbit Pyrogen Test: Rabbits have a similar response like human’s to pyrogens and hence rabbits are used to Test for pyrogenic nature of Test samples of drugs, saline and medical devices qualitatively. This method can detect nonbacterial endotoxins as well as bacterial endotoxins. Rabbit pyrogen test is carried out under typical conditions like atypical size of the test article, the neutralizing effect on endotoxins due to the presence of some chemicals in the test article, the presence of pyrogenic substance other than endotoxins, may be due to regulatory complaiance.  Presently the LAL test (limulus amoebocyte lysate test for bacterial edotoxin detection) is the mandatory test for detection of pyrogenic contamination. LAL test is an in vitro test for pyrogen. It is a kinetic Calorimetric assay which can detect bacterial endotoxin levels in solutions at concentration as low as 0 .005 EU/ml. Under certain circumstances as mentioned above, rabbit pyrogen test is carried out. Rabbits are injected with test solutions in doses not exceeding 10 ml per kg with in a period not exceeding 10 minutes. The number and nature of animals used, preparation of animals and test samples and recording of observation etc. follow directions of Standards that are followed by the investigator.  USP, Federal Register, ISO 10993 are some of the Standards available Internationally.

9.7 – Implantation Test: Implantation test is carried out by placing test samples of candidate Biomaterials or end product of Medical Devices into the test sites of living laboratory animals  in order to find out the response of the surrounding tissue to the test samples. Test samples are generally of 1x1x10mm size and placed into the tissue using a trocar and needle of 18 or 19 gauge size. The paravertebral muscle of the rabbits are the standard site of Implantation test. Healthy albino rabbits of body weight of less than 2.5 kg are chosen for test. The period of observation depends on the end use of the end product and decided by the investigator. The test samples for implantation test are implanted on one side of the paravertebral muscles and the negative samples or control samples on the contralateral side. The test samples and implantation needles are sterilized and the paravertebral muscle on the both sides of the vertebral column clipped and sterilized with 70% alcohol or beta iodine solution. The animals are anaesthetized for implantation and the procedure carried out by a Veterinarian. The animals are observed for 72 hours for health and normal behavior and sacrificed giving an overdose of an anaesthetic. The test samples are recovered along with surrounding tissue for immediate macroscopic evaluation and after scoring the results the samples are fixed in 10% formalin or recommended solutions for histopathological studies and scoring. Depending on the end use of the end product or Medical device the observation periods may be extended up to 12 weeks. In the cases of special devices the site of implantation could be chosen as subcutaneous, bone, ocular, dentine, brain, mucosal etc. and the observation periods also will vary as per the decision of the investigator and depending on which Standard is being followed for Testing. From mice and rat to any species could be used for testing. Full procedure kindly do refer USP or ISO10993 standards.

9.8 – Systemic Toxicity: Systemic toxicity is the adverse effect of a material or chemical in a living system caused by the material to various body organs and organ systems through the circulatory or lymphatic systems from the place of entrance ie skin, inhalation, oral, intravenous. Likewise a biomaterial or medical device placed in the human being may exert adverse effects at distant places with in the body or organ tissue. These effects are termed acute, short term, long term or chronic depending on the nature of the response of the medical device in question. Medical devices are generally not soluble or do not change structure, shape, size or degrade while serving the intended function. But if they are not biocompatible materials leaching from them may systemically reach distant body sites as eyes, lungs, liver, kidney, brain etc. to cause various adverse effects.

In order to produce safe Medical devices the candidate materials that go into the fabrication of these medical devices and the end products are subjected to systemic toxicity tests. Depending upon their intended use and site of use they are subjected to standard systemic test for acute (up to 24 hrs), sub acute(14 to 28 days), sub chronic (up to 90 days)and chronic( up to animals life span) periods of observation after the initiation of the test. Mice and rats are commonly used for systemic toxicity studies but rabbits, dog or an appropriate animal can be used for test after giving valid reasons for the same by the investigator and approval by the Institutional animal’s ethics committee.

For systemic toxicity studies the test samples are selected and extracts prepared as described earlier using appropriate extracting media at required temperature. The route of exposure is decided by the investigator considering the end use and period of use of the medical device. The observation parameters and other data like blood picture, serum chemistry, urine analysis, histopathology of major organ systems will be carried out and recorded as per the work procedure accepted. Long term or chronic tests are also carried out the same way and to prove that the use of the tested device is safe in every respect. Infact the Implantation test, and Pyrogen test that are coming next in line to be explained could be called systemic test because the response to these tests could be local in the test site as well as in distant body organs or behavior. The systemic test could be carried out as per ISO1993 to get worldwide recognition for the results.

9.9 – Test for Irritation:

9.9.1-Intracutaneous Irritation Test:- Irritation test is carried out to prove that the candidate material supplied for biocompatibility testing is a non irritant material in the in vivo test system. We know that this material has undergone the in vitro cytotoxic test and gave negative results. Test samples are extracted in fluids selected at a given ratio of surface of the candidate material at controlled conditions of time and temperature. Two albino rabbits are selected and their dorsal side is clipped free of hair and free of cuts or aberrations. Recommended volume of extract is intracutaneously injected on different sites on one side and equal number of sites areinjected with a control solution intracutaneously. Toxicologist selects the type of the extracts that should be used considering the nature and end use of the candidate material. At 24, 48 and 72 hrs after the injection the injected sites are observed and scored for skin response towards the test extracts. Parameters observed are redness, swelling and necrosis. In some cases heat and pain may result after the injection. Observed results are then compared with the skin response towards the control injections. The response is rated as none, mild, moderate and severe and indexed as per the standard that is used for Guidance in carrying out the test. This test is mandatory for Medical devices so as to avoid skin, mucosal and eye irritation. Though the candidate material may be non irritant, but irritation maybe due to unintentional contaminants from mould releasing agents, remnants traces of cleaning or chemical sterilents used or even from the packing materials.

            9.9.2- Primary Skin Irritation Test:-In the Intracutaneous irritation test,  test extracts prepared under selected conditions of temperature and time is used and is considered a comprehensive test to get any irritant leachables from the candidate test material. In the primary skin irritation test the candidate material itself is exposed directly to the skin for different periods of time. For this test two albino rabbits with their dorsal side shaved are used. The samples placed on the back skin are held in place with occlusive patches. In some instances the Toxicologist may go for applying extracts instead of patches after giving reason for changing the nature of test. The observation period stars from 4 hours to 72 hours. The sites are observed for erythema (redness), edema (swelling/inflammation) and rated as mild, moderate, severe etc. The preparation of samples, size or quantity of samples, observation periods, rating of results etc. will vary with the standard one chose to follow. Occular irritation test. This a variation of the Intracutaneous irritation and the Primary skin irritation tests. This test is specifically recommended for drugs and Medical devices that will be in contact with the eye. Rabbits are the test animals. In the initial test only one animal is used for test. In order to confirm the results two more animals can be used. The lower eye lid of one eye is drawn out a little to produce a small pocket like sac and the test sample is placed in this pocket. The other eye serves as control. Use of extract or powder or solid is determined by the toxicologist and the Standard he chose to follow. Eye is evaluated for the erythema, especially of the Conjunctiva, response of iris to light, corneal opacity, nature of discharge from the eye. As in the above cases the period of observation is 24 to 48 hours and rating is carried out as in the guideline that is followed.

9.9.3- Sensitization Test:-Some of as allergic to dust, pollen, house hold or Office Keys, milk, some food etc. once we are allergic to some material or chemical in a material we avoid them. But if we happened to be in contact with them unknowingly we get allergic symptoms and if the contact is repeated the symptoms will be moving up the scale like moderate to severe and science say that you are sensitized to that particular material. Sensitization episodes can become fatal too. The medical devices are made up of various materials and it is imperative that the candidate material as well as the end products should be negative in Sensitization Tests. Most of the sensitization reactions to biomaterials have been noted as the dermal cell mediated rather that humeral or antigen antibody responses. Dermal sensitization reactions in laboratory animals are marked by redness and swelling. Sensitization reactions in humans appear only after continued use of a biomedical device such as permanent implants or use of natural latex gloves. These sensitization responses may be the result of a chemical eluted from the device that is an allergen, over a period of time.

Summary of the tests are given below as quoted from ISO documents:

9.9.3.1- Methods of Test:-“Biomaterials and other device materials are tested for the presence of sensitizing chemicals using guinea pigs, a species known to be nearly as responsive to dermal sensitizers as human beings are. Guinea pig sensitization tests require six to eight weeks and thus take the longest time to complete of all the acute biocompatibility tests described in the 10993 standards.

The repeated-patch, or Buehler, test method involves exposing the shaved backs of guinea pigs directly to the test material under

occlusive dressings for a minimum of six hours. This procedure is repeated as many as three times a week for three weeks. This part of the test is often referred to as the induction phase. Following a two-week rest or recovery period to allow for the development of a delayed response, the animals are challenged by a final exposure to a patch of the biomaterial. The repeated-patch model is used primarily for topical devices such as dermal electrodes and surgical gowns and drapes, since in these cases the method of applying test materials to the animals simulates clinical use.

In the maximization, or Magnuson-Kligman, test method, fluid extracts of the test material are prepared in saline and vegetable oil, and separate groups of guinea pigs are exposed repeatedly to the two types of extracts. The guinea pigs are first injected with an extract along with an adjuvant intended to enhance an immune response, then receive a topical application. Following a two-week rest or recovery, the animals are covered with a topical patch containing the extract. Generally considered more sensitive than the repeated- patch model, the maximization test is used for device materials that will contact areas other than skin. The use of both a saline extract and an oil extract simulates extraction by bodily fluids and by intravenous liquids and other pharmaceutical products that first contact the device and then the patient.”

10 – Sample preparation

The materials that go into the fabrication of Medical devices as well as the completed Devices has to undergo Testing for toxicity and safety, before going into the market. Depending on the end use, medical devices are grouped into different classes. Medical devices in each class has to undergo a set of Toxicological tests to pass the safety or nontoxic criteria. We have earlier noted about the different types of test that are applied to the Testing. For carrying out these tests samples are required. Candidate materials that go into the fabrication are of different size and shapes varying from powder, granules, strips, sheets, tubes, wires etc. Likewise the manufactured devices are also of different size, shapes, tubes, powders, pastes etc. Further medical devices are designed, fabricated and tested in various countries by many different companies. Therefore standardization of tests and test sample requirements for each test is a necessity and these are drawn up and published by International organization for Standards generally known as ISO. This is a group formed by members drawn from various organization of Standards of different Countries, especially European. Food and Drug Administration, USA is another organization which sets standards for design, fabrication, testing and certification, safety and hazard notification of Medical Devices, American Society for Testing of materials is yet another organization for setting up Standards for materials that go into the fabrication of medical devices as well as the testing of safety of medical devices, Bureau of Indian Standards is the Indian counterpart setting up standards. Therefore to get the details of sample preparation one can refer to the particular standard he wants to follow. Depending on the test to be carried out test samples could be used directly as in the case of Implantation test or sensitization test or indirectly as extracts of test materials as in the case of Systemic toxicity or Pyrogen tests. The various Standards specify the appropriate amount of test materials or amount and nature of extraction mediums and the temperature at which extractions should be carried out. The untoward response of test samples arise from leachable ingredients contained in that sample. When samples are directly tested these ingredients if present may leach into the test system to express untoward response. In the indirect system the untoward ingredients will leach into the extraction medium which in turn will express toxic response in the test system. The nature and site of use of medical device, the temperature at which it is manufactured, sterilization methods it has to undergo, the recommended storing conditions etc. etc. of each medical device have to be considered while selecting test methods and extraction conditions. Sodium chloride for injection(SCI) (USP,IP) Vegetable Oil (USP, IP,)1/20 alcohol in SCI, Dimethyl sulfoxide ( DMSO), Polyethylene glycol 400(PEG) are the common extraction media used. Other clinically relevant media may be used along with reasons for the choice. 37,50,70,120 degree centigrade are the commonly used temperatures for carrying out extraction depending on the nature of the test samples and the selected medium for extraction. Test samples are generally taken by surface area for extraction tests. 6 cm2 area if the thickness is 0.5 mm and 3cm² if the thickness is more than 0.5 mm. per ml.You may note that material requirement for each test vary and the International and National Standards give detailed picture of sample requirement and kindly look at them carefully. Cytotoxicity, blood compatibility, mutagenicity, genotoxicity tests are carried out the sample requirement and test conditions are very specific and may change with the nature of the sample to be evaluated.

11 – Invitro Cytotoxicity Tests for Medical devices

Medical devices are life saving devices and must be free of any kinds of hazards. Each device has a specific place to be used and serve the life supporting function. The nature of service may be in direct contact with the body tissues or indirect contact. Here the device has to be neutral to the biological action elicited by the body or body tissue and carry out the intended function. This nature of the device is termed as Biocompatibility. Each and every medical device has to be biocompatible and therefore has to undergo a battery of tests as we have noted in the last week’s class. Generally Cytotoxicity test is carried out as a primary screening test to weed out test samples which may turn out to be toxic in standard  biocompatibility testing in animals. International standards are available for Cytotoxicity testing.  National standards have to be streamlined with International standards and it will need time, money and  co operation among Nations around the World to carryit out. It is yet to be ascertained about the correlation of the Cytotoxicity test results with other Toxicological tests. There is difference of opinion that Cell culture systems used for carrying out cytotoxicity test does not mimic the living tissue systems in metabolism, scavenging and energy supply systems. Further there is no unanimity in methods of assessing the Cytotoxic response.

 In Cytotoxicity tests the test samples are prepared as per the test method followed. For direct method these samples are sterilized and placed on the surface of tissue  or cells in the petridishes. For indirect contact the test samples are extracted as per the method chosen to follow and these extracts are placed over the cell layers prepared for the test. Then these cells in culture dishes are observed as per the test method for cell growth /damage, cell counts, changes in morphology, biochemical changes are noted and recorded to calculate the result of the test.

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