Manual

MANUAL FOR USERS OF LIVING FACILITIES

Specialized Veterinary Support Service – SAVE
State University of Campinas (UNICAMP)
2020

Manual for Bioterium Users
1st Edition – 2017
2st Edition – 2018
3st Edition – 2020

Prepared by
Paula
Ana Tada Fonseca Brazil Antiorio
Denise Isoldi Seabra

reviewed by
Paula
Ana Tada Fonseca Brazil Antiorio

This manual is aimed at all personnel directly involved with laboratory animals, whether they are employees or users of any of the vivariums at the State University of Campinas – UNICAMP, and aims to present, in a summarized manner, the main guidelines for the use of its facilities, standardizing conduct and procedures, in order to guarantee the well-being and health of the animals kept, from their birth to their final destination.

The development of this document highlights this University's commitment to technical-scientific, humanitarian and ethical principles, aiming to guarantee the responsible use of animals raised, kept and/or used in research protocols, under its responsibility.

The following text was developed based on the following current national legislation, as well as national and international reference guides:

• Law 11.794, of October 08, 2008 (Arouca Law);
• Law 11.105, of March 24, 2005 (Biosafety Law);
• CONCEA Normative Resolution No. 37, of February 15, 2018;
• CONCEA Normative Resolution No. 33, of November 18, 2016;
• CONCEA Normative Resolution No. 30, of February 02, 2016;
• CONCEA Normative Resolution No. 25, of September 29, 2015;
• CONCEA Normative Resolution No. 15, of December 16, 2013;
• CONCEA Ordinance No. 465, of May 23, 2013;
• CONCEA Ordinance No. 1.332, of December 03, 2014;
• RDC ANVISA nº 222, of March 28, 2018;
• CONAMA nº 358, of April 29, 2005;
• Law No. 12.305, of August 2, 2010 (National Solid Waste Policy);
• Risk classification of biological agents – Ministry of Health, 2nd edition – 2010;
• Guide for the Care and Use of Laboratory Animals, NRC 2014, 8th ed.

More detailed information must be made available in the vivarium itself, in the form of Standard Operating Procedures (SOPs).

• Packaging: act of packaging segregated waste in bags or containers that prevent leaks and resist puncture and rupture.
• Biological agents: microorganisms capable or not of causing some type of infection, allergy or toxicity in the human body, such as: bacteria, fungi, viruses, chlamydia, rickettsia, mycoplasma, parasites and other agents, cell lines, prions and toxins.
• Biosafety: actions aimed at preventing and minimizing risks inherent to activities, aiming to protect the health of humans and animals, and preserve the environment.
• Animal corpse: animal's body after death.
• CEUA: Ethics Committee on the Use of Animals.
• CONCEA: National Council for Animal Experimentation.
• CQB: Biosafety Quality Certificate.
• EPC: Collective Protection Equipment.
• PPE: Personal Protective Equipment.
• Euthanasia: the killing of animals by humane means, under conditions that involve, depending on the species, a minimum of physical or mental suffering.
• Experiments: procedures carried out on live animals, aiming to elucidate physiological or pathological phenomena, using specific and pre-established techniques.
• Phylum Chordata: animals that have as exclusive characteristics, at least in the embryonic phase, the presence of a notochord, gill slits in the pharynx and a single dorsal nerve tube.
• Inputs: products provided to animals such as feed, water and shavings.
• Macroenvironment: secondary physical environment, where microenvironments or primary environments are contained.
• Maravalha: sawdust-type bedding material, used to line rodent cages.
• Microenvironment: environment in direct contact with the animal, i.e. the housing cage.
• Group A healthcare waste: waste with the possible presence of biological agents that, due to their characteristics, may present a risk of infection, listed in Annex I of RDC 222 of 2018.
• Group D healthcare waste: waste that does not present a biological, chemical or radiological risk to health or the environment, and may be equated to household waste, listed in Annex I of RDC 222 of 2018.
• Group E healthcare waste: sharp or scarifying waste, such as: needles, scalpels, glass ampoules, scalpel blades, lancets, capillary tubes, micropipettes, slides and coverslips, spatulas and all glass utensils broken in the laboratory (pipettes, blood collection tubes and Petri dishes), listed in Annex I of RDC 222 of 2018;
• Subphylum Vertebrata: chordate animals that have, as exclusive characteristics, a large brain enclosed in a cranial case and a vertebral column.

The use of animals in research protocols is subject to approval of the project by the CEUA of this University, as provided for in Law 11.794 of 08/10/2008.

Protocols must be submitted for evaluation by completing the “Unified Form for Requesting Authorization for the Use of Animals in Experimentation and/or Teaching” available on the website heaven  In the same place, you will find instructions for submitting projects, meeting calendars and other relevant information.

After analysis, researchers will receive a letter from CEUA indicating the status of the research project, which may be approved or rejected, or clarifications may be requested. The project may only be initiated after the CEUA-UNICAMP approval certificate has been issued. A copy of this document must be sent to the animal facility where the project will be developed, and only then will its initiation be authorized. The document must remain available at the animal facility until the work is completed.

User training is required before starting activities, which includes knowledge of general procedures for using the vivarium facilities, as well as procedures related to direct contact with animals and experimental techniques related to the study to be developed.

The minimum training will consist of completing the online course “Basic principles in animal experimentation".

Teachers/researchers are responsible for passing on information to their students and collaborators.

According to Decree No. 45.781 of April 27, 2001, animals in transit must always be accompanied by transport documentation, which consists of Health Certificate e Animal Transit Guide (GTA). In the case of genetically modified animals (GMOs), the user must follow Law No. 11.105/2005 and complementary CTNBio regulations. The guidelines are detailed in the file “Transportation of animals”, available on the website heaven in the SAVE area.

In order to ensure that the health standards of the colonies are maintained and to avoid compromising ongoing research, it is recommended that animals from CEMIB/UNICAMP be brought into the vivariums, provided that there is adequate physical space for housing.

The entry of animals from other origins may only be permitted when they are of equal or higher sanitary standard to the animals kept in the vivarium.

Prior authorization from the coordinator and the veterinarian responsible for the technical examination is required for the entry of animals from sources other than CEMIB/UNICAMP. This authorization will take into account the analysis of the animals' most recent health report.

As a general rule, animals that have been used outside the vivarium facilities must not return to their original buildings.

For safety and health reasons, access to the vivarium is only permitted to authorized persons: employees, students and teachers/researchers with research protocols approved by CEUA.

The vivarium must maintain access control to its facilities. Preferably, this control should be electronic, accepting, at the very least, a record of access by means of a signed minute book, allowing traceability, if necessary.

It is recommended that there be a spreadsheet for prior scheduling of use of the facilities in order to optimize the execution of activities by different groups.

Personal belongings (including cell phones) must not be taken into the animal rooms and may be stored in lockers provided at the entrance, where available. Access is only permitted with materials strictly necessary for carrying out the activities.

The restrictions covered by this item aim to prevent cross-contamination, safeguarding the health of the animals and the proper development of research protocols. Likewise, they minimize the risk of spreading infectious and potentially pathogenic agents outside the vivarium.

In order to avoid unnecessary traffic, make sure you have all the materials you need to conduct your research on hand. If necessary, bring a roadmap with you to make your work easier.

When entering the vivarium, employees and/or users must disinfect their hands with alcohol and wear PPE (lab coat, cap, mask, foot covers and procedure gloves), following the following order:

• Put on the pro-foot;
• Sanitize your hands with 70% alcohol or 70% alcohol gel;
• Put on disposable procedure gloves;
• Wear the mask covering your mouth and nose;
• Put on the cap covering all of your hair;
• Wear the apron, which must be long-sleeved;
• Put on a second pair of gloves, covering the cuff of the apron;
• Disinfect the surface of the gloves with 70% alcohol or 70% alcohol gel.

Animal species normally used in research are very sensitive to environmental conditions (temperature, humidity, noise and odors), therefore, it is recommended:

• Respect one minimum acclimatization period of at least 10 – 14 days, so that the animals received by the vivarium become accustomed to the new housing conditions before the start of the experimental protocol;
• Carry out activities in small groups to avoid excessive noise that could cause stress to the animals;
• Transit and stay in the vivarium must be as calm and quiet as possible in order to guarantee the well-being of the animals housed and the smooth running of the research protocols;
• Do not use perfumes, colognes and cosmetics in general;
• Do not use cell phones and sound and/or electronic devices in the internal areas of the bioterium, even if they are on vibrate mode;
• For safety reasons, the use of headphones is not permitted;
• In order to avoid accidents, the use of watches, rings and bracelets is not permitted when the user is working directly with animals;
• Disinfect work surfaces before and after use with sanitizers (70% alcohol, quaternary ammonium or VIRKON).

We emphasize that sanitizers, except 70% alcohol, must be prepared at the time of use to maintain their potential action.

we remember that are not allowed the consumption and storage of food and/or drinks in any of the vivarium facilities.

Animals must be kept in air-conditioned environments with controlled photoperiod. The temperature and humidity of the macroenvironment must be monitored and recorded daily, and kept within acceptable values ​​for each species, with the least possible variation.

In order to ensure the well-being of animals throughout the period they are housed in the vivarium, minimum area recommendations must be respected. The occupancy density is defined based on the weight of the animals, floor area per animal and the height of the cage. It is also important to ensure that sociable species, such as rodents, are not housed individually.

The cages must remain properly identified with tokens and these must be fixed using appropriate token holders. The use of drafts or post-its is not permitted and, similarly, tokens must not be stuck to the cage with adhesive tape. Below you can see the standard identification form template recommended for UNICAMP bioteriums (available in the SAVE area for printing), containing the necessary basic information.

Different species (rats and mice, for example) should not be housed in the same room (RN 15, CONCEA).

Air exchanges in animal rooms must be done with 100% renewal, and there should be no exchanges with the air in the room itself. The supply of 15 to 25 air changes per hour in animal rooms is an acceptable recommendation to maintain air quality in the macroenvironment at a constant volume and can also ensure air quality in the microenvironment (RN 15, CONCEA).

The cage should be provided with clean, dry bedding in an appropriate quantity to allow urine absorption, covering the entire bottom. The shavings should be arranged in such a way as to reach, on average, 1,0 cm tall. It is important to note this detail, as an excessive amount of wood shavings can make it difficult for the animal to access the drinker and prevent adequate air flow in the micro-isolators. Likewise, excess bedding can come into direct contact with the drinker spout, causing water to leak and increasing humidity in the internal environment and reducing the available dry area, which compromises the animals' well-being.

A minimum cleaning frequency of: 02 changes/week for mice and 03 changes/week for rats.

Animals must have free access to water and good quality feed. Fasting or water and/or food restriction is only permitted with authorization from the CEUA or under the express recommendation of the veterinarian and in these cases the information must be clearly recorded on the identification form.

Do not feed animals that are part of another research protocol. If they are without food or water, notify the person responsible who will take the necessary measures.

It is also recommended to keep spare materials ready for possible emergencies such as leaking waterers or animal deaths between routine water changes. This measure ensures that animals are always kept in a clean and dry environment, with an adequate amount of water and food.

Whenever possible, materials that will come into contact with animals should be sterilized in order to protect them from potentially pathogenic agents. Special attention should be paid to wood shavings bedding.

It is important to remember that to maintain their original health status, these animals must be housed in environments with an absolute filter (HEPA) and, if possible, in ventilated racks. All material in direct contact must be previously sterilized, including water and feed.

Routine procedures should preferably be carried out in laminar flows.

Maintaining the well-being of animals is of fundamental importance at any stage of their life. Since they remain in a closed environment, environmental enrichment can be an alternative to improving the quality of life of animals, consisting of a factor to be considered and implemented whenever possible.

For rodents, we suggest using igloos or PVC tubes that allow the animals to live and feel protected. The materials for this type of enrichment must be washable and disinfectable, without cracks or sharp edges, and of an appropriate size for the species. The materials can also be made of paper or cardboard, but they must be sterilized before use and discarded weekly. Nesting material (cotton, paper, burlap) can also be provided, but in this case they must also be sterilized before use.
of use and discarded weekly. Rabbits usually accept dehydrated alfalfa, platforms and plastic balls well.

Animals should be removed from the room by users as calmly as possible and taken to the procedure room where the procedures will be performed. Preferably, in order to reduce local traffic, only one person should enter the animal room, preserving environmental conditions as much as possible.

Animals must be transported in their own housing boxes, always after removing the water trough to prevent water leaks.

After completing the procedures, the animals must be returned to their original location, replacing the drinking fountain. The laboratory, equipment and utensils must be cleaned and disinfected after use.

Ensure that procedure rooms and animal rooms are kept tidy so as not to interfere with the work of other teams, as well as ensuring the health and well-being of the animals.

The disposal of corpses, carcasses and waste generated by the use of animals must follow current federal, state and municipal legislation, or substitute legislation such as, for example, the National Solid Waste Policy, Law No. 12.305 of August 2, 2010 (RN 30, CONCEA).

Waste generated in animal facilities is classified as health service waste group A (subgroups A1, A2, A4 or A5), groups D and E. Consult the RDC nº 222, of March 28, 2018 which regulates Good Practices for the Management of Health Service Waste.

Be careful with carcasses, blood, animal tissues, wood shavings, air filters and other materials with the presence of biological agents of risk class II or higher (Salmonella spp., Leishmania spp., Toxoplasma spp., Plasmodium spp., Schistosoma spp., and other bacteria, parasites, fungi and viruses) that, due to their characteristics, may present a risk of infection for humans and animals, and/or environmental contamination. Consult the “Risk classification of biological agents of the Ministry of Health – 2nd edition, 2010".

Health service waste must be deposited in a milky white plastic bag, identified with the infectious symbol, and then properly closed.

Attention must be paid to the amount of material discarded in order to avoid rupture and leakage of the contents stored therein. The bag must be replaced when it reaches 2/3 of its capacity or immediately if degradation and release of odors are intense. Infectious waste cannot be recycled, reused or repurposed, including for animal feed.

When potentially contaminated with pathogenic agents they must be autoclaved before disposal. Mandatory, All waste classified as NB2 or higher must be autoclaved in the vivarium itself before to be forwarded for collection by a specialized company.

Sharp objects must be disposed of in appropriate boxes. This category includes scalpel blades, syringes, needles, etc.

The separation of the syringe-needle assembly is permitted with the aid of safety devices, and manual disconnection and recapping of needles is prohibited.

Researchers, teachers and users of animals for teaching or scientific research purposes are responsible for aspects related to animal welfare. It is their responsibility to plan or conduct projects or protocols, considering that animals are sentient beings and that their welfare is an essential factor during the conduct of teaching or scientific research activities (RN 33, CONCEA).

We remind you that each user is responsible for their group of animals and must carry out daily inspection of the same in order to guarantee adequate accommodation and prompt care. Care should be extended to weekends and holidays, whenever necessary, such as when the animals are in critical periods of post-surgical recovery.

We recommend that surgical procedures not be performed on Fridays or on the eve of holidays, so that the animals remain constantly monitored in the first 48 hours following more invasive interventions.

Any occurrences related to the vivarium must be recorded in Incident Record Book available exclusively for this purpose. Problems with scheduling the use of rooms and observations regarding animals and environmental conditions, such as: housing, sudden changes in temperature and/or humidity, changes in appearance and/or behavior, should be recorded. This book will be used as a direct communication channel between users and those responsible for the location, seeking to ensure and promote the well-being of the animals.

The following documentation must be kept up to date and available at any time, including for verification by inspection bodies:

• Project approval certificates, issued by CEUA-UNICAMP;
• Risk Map;
• Biosafety Manual;
• Standard Operating Procedures (SOPs);
• Animal Transit Guide (GTA) and corresponding health certificate;
• CIBio authorization of the unit for the use of GMO animals;
• Purchase invoice and prescription for controlled-use medications (e.g., anesthetics such as xylazine, ketamine, isoflurane, and others);
• Record of the use and stock of controlled medications (e.g., anesthetics).

We emphasize that animal facilities are subject to inspection by the following bodies:

• CONCEA – National Council for Animal Control and Experimentation;
• CRMV-SP – Regional Council of Veterinary Medicine;
• ANVISA – National Health Surveillance Agency;
• MAPA – Ministry of Agriculture, Livestock and Supply;
• CTNBio – National Technical Commission on Biosafety;
• CIBio – Internal Biosafety Committee.