Rules and Responsibilities About Animal Usage in Medical Researches

 

For usage, humanistically cure and appropriate care of experiment animals that are used on the purpose of research, test and education; experiment animal has to be known and also every personnel that approaches to animal with this purpose has to be trained. Binding rules in usage and caring of experiment animals are reported in this part.
In the way that authority for operating the program is given to person that is experienced at medicine and generally laboratory animals or to any trained veterinary surgeon, it can be given to qualified personnel as well. In experiment animals center, anyone who is trained for used animal kind or anyone who is experienced or at least any veterinary surgeon has to be assigned.

Intended Purpose of Experiment Animal

1. Protection of human, animal and plants from diseases or prevention of disease. Therefore, experiment animal can be used for testing of activity, safety, quality and production of products or materials,
2. diagnosis and treatment of diseases at human, animal and plants,
3. diagnosis, detection, regulation and modification of psychological conditions of human, animal and plants,
4. protection of the environment,
5. education and practice,
6. forensic investigation
7. and other scientific researches that Ethical Committee will approve.

Animal Caring and Usage Protocols

Main purpose of medical research is improvement or maintenance of human health. Researchers have to avoid from using experiment animal unless it is really necessary and have to show reasonable and scientific grounds for using of these living creatures. The reason why there is no option has to be clarified. Researcher has to accurately define the aims of research .
All relevant individuals that use animals in research,

• think the fact that animals kept in cage,
• the fact that experiment animal has to be killed in case of need and
• the fact that there can be pain in animals and uneasiness during the research. All researchers have to know the fact that good health of animals

affects the results of research positively.
Research has to be planned by this means;

• applicability of research;
• using of physiologically most favorable kinds for research and simple organism for research;
• using of minimum number of animals that is enough for calculating statistically in experiment;
• the fact that operation on animals is kept in low levels, the fact that experiment has to be taken short as possible and providing the analgesia-anesthesia-sedation that minimizes the pain
• the fact that there is no tautologies operation;
• usage suitability or probability of lesser invasive applications, other kinds, isolated organ preparations, cell or tissue cultures or -if available- computer simulations;
• removing the experiment animals from research if experiment is expected to be painful or stressful or criterions of euthanasia -if it is performed in timed intervals;
• animal care after the application;
• method of euthanasia and annihilating of animal;
• experience of personnel in experiment and sufficiency of practice;
• ordinary care and care requirements;
• and suitability of work environment for personnel has to be researched.

Animal Usage Options In Biomedical Research

Due to the fact that main purpose of medical research is improvement of maintenance of human health and due to the fact that performing most of needed researches on humans is not ethically right, animals must be used in researches instead of human. Biomedical research, learning, testing or basics of experiment animal using options focuses on 3R philosophy:

1. Replacement ,
2. Reduction ,
3. Refinement .

Replacement is using animals that are in the bottom of phylogenic scale

1. instead of developed animals,
2. using in vitro methods such as organ, cell, tissue culture,
3. using inorganic systems that are not animal (retroviral gene transfer) or
4. using unanimated systems (for instance computer system).

Reduction is to aim minimizing total required animal number in order to provide statistical materiality.
Refinement is simplifying the process or method to use. Thus, the pain or stress that animal feels is minimized. The aim in this modification is to control pain or stress much better, to use noninvasive method and minimize invasive process.

Research Conducted On Experiment Animal

Medical research requires animal using for unique purposes. Kind of animal that is used in medical research can vary from simplest organism such as protozoan parasite to cold-blooded vertebrate -for instance frog- and mammals (especially rat, mouse, cat, dog, monkey, guinea pigs, and rabbit). Attempts that are made on animal relatively change:

• Animals can be conscious throughout the experiment (for instance, injection or medicine application)
• Application can be completely performed under anesthesia, animal will rally at the end of process yet,
• Application is completely performed under anesthesia, animal does not remain alive at the end of process yet,
• Animals can be used on the purpose of tissue support for in vitro culture,
• Animals can be used for material (for instance; antibody, enzyme and blood components) support.

Points To Take Into Account In Experimental Process

Experiment has to be performed under general or local anesthesia. During and after the experiment, animal should not be led to any trouble, pain or ache.
Painful or stressful applications

1. has to be performed with appropriate sedative, analgesic and anesthetics(SAA). SAA has to be used academically at necessary intervals.
2. Veterinary counseling has to be demanded at planning process.
3. Paralytics should not be used without anesthetic.

If using of analgesic or anesthetic in experiment is not fit for purpose -as experimental algesia- permission has to be obtained from local Ethics Committee and experiment has to be terminated as soon as possible and if the trouble continues euthanasia has to be performed. That kind of operation should not be performed unless it is necessary.
The fact that animal will survive or will be murdered with a humanly method has to be reported.

1. If animal will survive, surgical glove, mask, sterile tool and aseptic has to be used for surgical attempt. Before and after the surgical operation, care has to be carried out in surgical applications. The fact that animal will suffer after surgical operation has to be prevented. If suffering cannot be prevented, animal is always murdered humanistically and without pain.
2. If animal will not be able to survive at the end of experiment sterility is not necessary in experiment. Animal has to be murdered humanistically and with a painless method after the application.
3. Murdered or dead animal has to be kept in cold room with its own products until annihilation. Annihilation has to be performed by burning or burying solely in indicated special place.
4. Any animal that is used in an process causing acute or chronic pain and trouble should not be used in final experiment to be held in later if it does not get well.

As final experiment can be a process that continues until the death of animal, it can be a small intervention.
If applied material or medicine on animals is toxic, animals have to be kept in different packs.
Any extensive surgical operation should not be performed suddenly if animal survives after the experiment. But:

1. If there is any scientific reason,
2. operation can be performed if it is necessary for veterinary care.

Restriction of Physical Activities of Anaesthetized Animals

In order to restrict some or whole normal movement of animal in accordance with aim of experiment, physical activity is restricted by manual or mechanic methods. Physical activity can be restricted for specimen handling, giving medicine, treatment or experimental operation as well. Animal can be kept at short notice expressed as minutes in several research applications.
Activity of experiment animal is restricted by either manually or by holding tool. In order to minimize uneasiness that animal feels and prevent physical injuries, form and size of this holding tools has to be appropriate with operation. Several dogs, monkey or other mammals has to be accustomed to stand inactive for a short time or has to be accustomed to positive force. Restriction of physical activity for a long time might be stressful for animal, therefore try to avoid if it is not main purpose of research. All physical restriction except for restriction in routine operations has to be reported in research protocol. If restriction lasts in more than 4 hours, it has to be defined particularly in protocol. The fact that extended physical restriction is appropriate is not sufficient reason on its own. In case of the fact that restriction lasts in more than 24 hours, the idea of using merest restriction method should recommended to researcher even if it is expensive and technically difficult. When extended physical restriction is needed, following rules has to be obeyed:

• Animals that are put onto restriction tool has to be accustomed to tool by restriction until the necessary restriction time.
• Restriction time has to be limited with minimum required time for researcher's aim.
• Fastening of non-human primate to chair is not pleasant in operations that generally last in more than 24 hours. If there is no any appropriate option for extended chair restriction, animals has to be detached from chair frequently.
• Pay attention to lesions or diseases (contusion, deceits ulcers, loss of weight) that are caused from restrictions. In situations like this, veterinary care has to be performed. This needs temporary or constant removal of animal from restriction tool. If the health problem of animal seems serious according to veterinary, improvement of animal health becomes priority in research.
• The fact that there is a normal way of harboring animal in holding tools should not be thought.
• Animals that are put onto holding tools has to be trained in order to accustom to personnel and tool.

Restriction of Water And Food

When experimental researches need to restrict water and food, minimum amount of water and food has to be served in order to development of young animals and good condition of animals that have been kept in center for long time. Restriction for the aim of research has to be approved scientifically. Water and food restriction of laboratory animals causes stress in animals and leads animal to behave badly and differently. If the operation needs food and/or water restriction, behavior of animal and the place where animal is kept have to be observed frequently. Daily record of body weight has to be used for evaluating the development. Body weight should not be under than 80% of normal. Homogeneous and contemporary controlled animals that can eat their food independently have to be used in detecting of body weight index. This is important for animals in development period. If the decrease in body weight is aimed, it has to be performed with progressively decrease of caloric intake in long time. Complete food restriction in order to reach target weight is unacceptable. The fact that weight loss is lower than expected is a reason for temporary or constant removal of animal from experiment. The restriction is measured as daily food intake percent or weight change of animal percent.
Water cannot be restricted completely for more than 24 hours. Water restriction (dehydratation) should not grow to a point that clinical symptoms -such as elasticity loss of skin, hair bulking, embedding of eyes to socket- seem. In order to prevent from acute and chronic dehydratation, fluid intake and bodyweight has to be recorded at least one time -in case of need two times- in a week.

Experiment Animal Immunization and Freud's Complete Adjuvant Injection

Using of Freud's Adjuvant (CFA) for raising antibody in animals causes ache. Therefore, the fact that whether there is an option for CFA or not has to be researched. In order to decrease the number of animals that are exposed to unexpected immunological reactions, following rules have to be obeyed.
Freud's Complete Adjuvant is used
to make mouse and rabbit polyclonal antibodies according to amount of desired serum and antigen. CFA can be used only when there is small amount of immunogenic. If the amount of immunogenic is excess or if there is supernumerary immunogenic, other adjuvant have to be considered. CFA can never be used as intravenous. Antigen and CFA are mixed at the rate of 1:1. Immunization method can vary subject to animals.

1. Intra-dermal method: This method is used for only rabbits. Instead of a injection, inoculums that are above 0.5 mL cannot be given. The localization of site has to be detected meticulously in order to prevent injuries. Spaces between scapulas can be chosen as injection site. Injection have to used in small numbers and scapulas have to be distant from each other. Injection can be painful frequently and injected skin causes drainage of granulomas. Additionally, antigen-adjuvant emulsion cannot reach its aim when it is lost due to drainage of injected site and throwing away the scab. Laboratory personnel who injects has to be careful so that not to pricking the pinpoint to himself/herself. This causes pain and long-acting pus on human.
2. Subcutaneous (s.c.) Method: This method can be used on rabbits, guinea pigs and mice. Injection site on rabbits has to be between scapulas on dorsi; 0.1 mL injection has to be given to everywhere and number of injections has to be kept low as far as possible. More than ten places should not be used. Several sites in the dorsal region of neck on guinea pigs have to injected 0.1 mL. More than 0.1 mL cannot be injected to mice and it is only injected to neck region.
3. Intramuscular (i.m) Method: This method can be used on rabbit, guinea pigs and big rodents. It is not recommended for small rodents and mouse For rabbits; antigens at CFA are given to biceps femoris muscle, 0.5 mL has to be injected to every site and number of sites has to be kept low as far as possible. For guinea pigs or big rodents, injection of 0.1 mL to biceps femoris muscle is recommended.
4. Intraperitonea (i.p.) Method: It is only used on mouse; more than 0.1 mL injection is not given.
5. Paws: These regions are not used for rabbits. This method can be used for rat and mouse if other methods do not work. Only a paw is allowed to be injected and animal has to be put onto soft place.

Injection place has to vetted 3-4 times in a week after every injection. Number of injection is determined by antibody titer. Three or four injections are adequate for rabbits or other animals.
Due to the fact that a new injection of CFA to sensitive animal might be painful, it causes more serious hypersensitive reactions. Therefore, any process that uses more than a CFA injection has to discussed in advance. In Incomplete Freud Adjuvan process, dosages that take to next stages should be thought.

• After the sensitization, animals which watery solution of antigens is injected have to be observed in terms of symptoms of anaphylactic shock and given appropriate treatment has to react.
• Serious urethritis reactions in injection has to be reported to veterinary for vet and treatment.

Making Hybridom-celles and Monoclonal Antibody

There is no other option except mouse for this purpose. The protocol that minimizes the uneasiness of animal is given:
Mouse Immunization: Generally Balb/C mice are used and they have to be 43-78 days old. CFA has to be used at the start of immunization if there is any immunogenic that can melt in small amounts. Emulsifier watery antigen -with an equal volume of adjuvant- is used and 0.5 mL is injected with appropriate protocol. If the number of antigen is high or if immunogenetical antigens are used, other adjuvans (Freud's Incomplete Adjuvan) have to be used. CFA can never be used as intravenous.
Intradermal method is used for inducing the answer which cell intercedes. More than 0.05 mL can not be given to single site. Injection mostly causes to ulceration in the site where it is applied. This method is not suitable for mouse.
The suitable region for subcutaneous method is neck region on mouse and more than 0.01 mL can not be injected.
Intramuscular method is not suitable for CFA on mouse. More than 0.1 mL can not be given to injection site. More than two biceps femoris muscles should not be used.
İntraperitoneal (i.p.) method can be used for only CFA. Injections should not be over o.1 mL.
CFA can not be applied to paws. This method can be used when the other methods do not work. Only one paw of mouse can be used. Animals must be kept in soft beds.
Injection place has to be vetted 3-4 times in a week after every injection. The number of injection is determined by plasma titration. Several protocols find three or four intraperitoneal injections adequate. If titration is at the rate of 1:100 or more than this, it is approved as adequate. The blood is drawn to the heparinized tube by cutting tail of mouse with sharp scissors. This process should not be applied more than 3 times.
Prepare for fusion: Mouse is murdered by cervical dislocation. Spleen is removed for cell fusion.
Forming and Gathering of Acid: If overdose of CFA or pristan intraperitoneal is injected, it causes acid forming on periton. Meanwhile weight loss, humped image and lethargy seem. When the clinical symptoms are observed, number of injections to form acid has to be minimize. Mouse has to be prepared for using with only a pristan in principle. If pristan injection needs to be repeated, this has to be carried out four weeks after the first injection. After the injection of hibridoma cell in both process, injection has to be kept waiting before preparing. 0.2 mL pristan (2,6,10,14-tetrametilpentadekan) is recommended; maximum dose of injection is 0.5 mL. Hibridoma cells which are prepared in balanced salt solution at the rate of 3.2x106 has to be given 0.1-0.2 mL. "ip" has to be carried out at the rate of 0.5 mL at least 14 days before the pristan cell injection.
Mouse begins to produce acid 4-8 days after the injection and acid continues to form in time. Acid producing velocity is pretty variable. Acid fluid is emptied from venter of mouse with injection no.22. In order to prevent coagulation, small amount of heparin has to be added to tube. Experiment animal is vetted daily in order to detect forming of acid fluid; the fact that whether there is an swelling in venter or not is checked. Acid fluid is emptied when the venter of mouse grows as pregnant. Accumulation of fluid by sedation or -ideally- under the effect of anesthesia. Emptying of fluid can be carried out three times totally. Euthanasia is performed in last process.
Animals always have to be kept comfortable and respiration, activity and appetite of animal have to be pursued daily. The fact that abdominal distension is in level that incommodes has to be prevented. If any uneasiness is detected, the last fluid is emptied and euthanasia is performed. The fact that animal goes into shock is prevented by intraperitoneal or giving physiologically subcutaneous serum after the gathering of acid.

Acute Toxicity Test

In order to determine the acute toxicity of chemicals/medicines in mammals, such an information can be obtained only from animal researches due to the fact that other methods are not secure Rational and specific reason to research has to be presented to ethics committee. Minimizing the number of animals used has to be regulated according to minimizing the velocity and size of animal production. The protocol has to be presented to ethics committee and has to be approved by the before the start of test.
Three basic approaches are commonly used : 1) "limit" or secure test, 2) space finding test that gives approximately LD50 or 3) LD50 which is statistically valid

1. Limit Test is suitable for determination of the fact that whether the test compound is lethal or toxic at the rate of determined dosage. Determined dosage is given to 10 animals from sensitive kind or 5 or 10 animals from all kinds. Toxic answers that seem in a short time are recorded. Ancillary test is made according to results. One or two animals are exposed under normal circumstances in advance and if any toxicity is not observed same dosage are used with different animals to prove the reliability of same material.
2. If the lethality of compound is not known, space finding tests have to be used in determination of appropriate dosage levels in order to minimize the number of used animals. Generally one or two animals are used in the rage of dosage that permits estimate of LD50 for every dosage level.
3. LD50 test evaluates the acute lethality on exposure of a material or a product. LD50 value is a dosage that is expected to kill half of experiment animals. This method typically is used on 100 or more animals. Five or more dosages are given to ten female and male animals on dosages that test material generally increases. Mortality of a short time is recorded; LD50 is detected thanks to statistical methods. This test is carried out for various purposes:
   • testing products or materials for legal obligations in labeling and secure safe transportation,
   • providing mutual information in chemical dosage-answer schedule,
   • providing information for acute intoxication treatment,
   • standardizing of some biological products,
   • adjusting dosage levels for toxicity researches,
   • providing data for evaluating alternative test methods.

Animals have to be observed pain-stress symptoms by following the dosage in all of these tests. If any unexpected stress or pain occurs and test cannot be evaluated for this reason, ethics committee has to be kept informed immediately. The rest of test might requires modification of protocol and it might evaluated once again by ethics committee.
Test agent has to be known thoroughly before the animal is exposed. On the purpose of observing for a time that is coherent with the aims of research, animals have to be used according to dosage. The most standard acute toxicity tests require at least 14 days, sometimes require more than this -for the determination of delaying symptoms of toxicity-. If there is any pain or stress symptom primarily, animals which are about to die have to be killed as soon as possible.

Experiment Based On Pain

In painful or stressful experiments of animal, animal is eased or sedated by using analgesics, anesthetics or tranquilizers only when analgesics, anesthetics or tranquilizers do not react to experimental objects. Animal studies protocols has to state used medicines (or medicine) and the dosage. If pain or stress is avoidable and if these medicines are contraindicate to structure of research, responsible researcher has to obtain permission from local ethics committee by showing reason. Essential pain level that requires the using of chemical relaxant is generally more intense than the pain that is caused by injection. If attempt causes abnormal escape behavior and serious tissue damage of animal, it is accepted as painful and if it causes same things on human it is accepted as painful again Attempts that are made on animal relatively change are,

1. as painless and stressless,
2. might cause pain and stress that is medicated by anesthetic, analgesic or tranquilizer medicines.

Implementations Causing No or A Trace of Pain And Stress

When these operations are carried out by expert individuals that use defined method, they can be considered as causing no or a trace of pain or else stress.

1. The implementation of the following ones,
   • anesthetic, analgesic and tranquilizers,
   • fluid and electrolyte,
   • immunization,
   • oral treatment
2. acute cauterization,
3. depletion (intra-cardiac and except periorbital in some kinds)
4. gastric gavage;
5. some routine application that are used by veterinaries for diagnosing and treatment -such as injections, palpation, rontgen, skin pressing(?) -,
6. euthanasia that is carried out according to recommended method;
7. intra-cerebral inoculations in neonatal rodents. Intra-cerebral inoculations of several neonatal rodents are carried out by trained staff. This operation causes only temporary ache or stress. In the event that the result of any above mentioned operations is ache or stress, that operation has to be evaluated with following category.

Implementations Causing Ache or Stress Salved With Appropriate Treatment

The operations that cause ache or stress and anesthetic, analgesic or tranquilizer implementations might be carried out are:

1. all surgical implementations that contain placing of biopsy, gonadectomy, electrode and recorder,
2. burnt, freezing and branding implementations,
3. bone fractures,
4. electric shocks that is accepted to cause ache in human body,
5. using of an agent that causes severe pus and necrosis,
6. test which are carried out for the determination of LD50 of medicine or radiation toxicity,
7. restriction of movement for more than 24 hours on animals which are not entered into adaptation transaction,
8. test of skin or cornea corrosion,
9. intra-cardiac depletion,
10. peri-orbital depletion from any kind except mouse and hamster. Peri-orbital depletion from unanesthesized animals such as mouse and hamster which have not real orbital sinus is not suitable.

General Care and Organization of Animals

There has to be freedom of action of experiment animal in the place where it is throughout the research, food and water have to be provided. All kind of care has to be carried out in order to keep animal healthy. Physiological needs (peeing, defecation, maintaining its body temperature, normal action and posture adjustment) of animal should not be restricted as possible as. Experiment animals have to checked daily; if there

The Reusing of Animals

The reusing of animal is performing an different second implementation after the first implementation.

• Even if used animal gets healthy again, it cannot be used in different experiment.
• Healthy, used animals can only be benefited from with the purpose of training.

Using of Animal Products

When the researchers study with materials that are obtained from animal products, researchers should make a point of minimizing the number of animals.

Responsibilities of Staff

Everyone using experiment animal by experimenting in research, testing or providing the tissue is responsible for providing the protection and good condition of animal at the highest level. Transportation and care of animal after the transportation have to be carried out meticulously.

Education, Practice and Authorization

Researcher, anyone that supports the experiment and anyone that cares for animals have to be trained at the level of needed responsibility. Education, practice and advanced practice have to be carried out by someone who knows caring of animal or under the custody of that person. If researcher is not experienced, s/he has to go training in order to learn appropriate using method of experiment animals from competent institutions.

Providing of Animals

Animals have to obtained from centers making sales. These animals should never be put next to your own other laboratory animals. Birth date and stock of every chordate that are used in research have to be registered.

• Wild kinds of animal cannot be used in research. But in special conditions and in circumstances that there is no alternative option, wild kinds of animal can be used with the special permission of ethics committee.
• Production has to be organized in the way providing the research requirements such as number, sexuality, weight, health etc. Especially over birth and unnecessary separation should not be performed.
• Surgical attempts on animals that are not rodent have to be carried out only in Research Center. Animal experiments on rodents have to be performed with the permission and custody of local ethics committee in the place where is determined beforehand.