Law: Procedures in Laboratory Animals
Procedures in laboratory animals, which have to be considered as laboratory animal experiment according Austrian Laboratory Animal Act 2012:
`Procedure’ means any use, invasive or non-invasive, of an animal for experimental or other scientific purposes, with known or unknown outcome, or educational purposes, which may cause the animal a level of pain, suffering, distress or lasting harm equivalent to, or higher than, that caused by the introduction of a needle in accordance with good veterinary practice.
This includes any course of action intended, or liable, to result in the birth or hatching of an animal or the creation and maintenance of a genetically modified animal line in any such condition, but excludes the killing of animals solely for the use of their organs or tissues.
This means all procedures and techniques carried out in animals (injections, surgery, application of a substance by water or food, modification of standard housing conditions, breeding, housing and generating impaired genetically modified mice which might be impaired as a consequence of the mutation, etc.) are included.
There is one exception - at least for the moment: killing a mouse by a standard method without anesthesia (e.g. for taking tissue) is not considered as an animal experiment.
According to the Austrian Laboratory Animal Act 2012, the use of animals for scientific purposes is highly regulated (as is the case more or less in every single EU country). This short introduction should help you to find your way through this matter. You are asked to carefully stick to the rules and help the IMP IMBA animal facility to meet all legal requirements.
Sick animals, breeding of sick animals, genetically modified mice:
Sick animals and animals which demonstrate reduced animal wellbeing in our facility have been always a problem when dealing with authorities. Those animals must not be kept outside an authorised project.
When genetically modified mice are sick and this might be a consequence of their mutation, the maintaining of the colony (breeding, generating a mouse line etc.) requires an authorised project too according to the new act. Any course of action intended or liable to result in the birth or hatching of a sick animal or the creation and maintenance of a genetically modified and therefore sick animal line is considered as a procedure, this includes healthy animals, which were taken for breeding.
Inspections by the authorities:
So far our facility was inspected at least once a year by the authorities without prior warning. Now the inspections focus much more on animal wellbeing, detection of sick animals, traces of procedures etc. (see clause “sick animals”).
It is new that the competent authority has to adapt the frequency of inspections on the basis of a risk analysis for our facilities, taking account of the number and species of animals housed, the record in complying with the requirements of the laboratory animal act, the number and types of projects carried out and any information that might indicate non-compliance. This means: the more objections the more inspections!
Suspension and withdrawal of authorisations:
Where mouse users as well as laboratory animal house staff not sufficiently comply with the requirements set out in the new act, the competent authority have to take appropriate remedial action, or require such action to be taken, or suspend or withdraw its authorisation.
So far it was not that easy to withdraw authorisations, now this is easier for authorities and above all explicitly required. This means competent authorities must not be flexible any more here. Exceeding a certain number of objections may lead to the suspension or withdrawal of permissions and authorizations.
Embryos and some fetal forms:
Manipulating mouseembryos (as from the last third of their normal development) has to be considered as procedure. It requires a project authorisation therefore in the same way as work with normal laboratory animals already born. Procedures in mouseembryos at an earlier stage than that referred to above have to be carried out only within an authorised project too if the animals live beyond that stage.
Severity classification of procedures (ANNEX VIII 2010/63/eu):
All procedures are classified as "non-recovery", "mild", "moderate", or "severe" on a case-by-case basis.
“Severe”: a procedure must not be not performed any longer if it involves severe pain, suffering or distress that is likely to be long-lasting and cannot be ameliorated.
The severity of a procedure shall be determined by the degree of pain, suffering, distress or lasting harm expected to be experienced by an individual animal during the course of the procedure.
Non-recovery: Procedures which are performed entirely under general anaesthesia from which the animal shall not recover consciousness shall be classified as ‘non-recovery’.
Mild: Procedures on animals as a result of which the animals are likely to experience short-term mild pain, suffering or distress, as well as procedures with no significant impairment of the well-being or general condition of the animals shall be classified as ‘mild’.
Moderate: Procedures on animals as a result of which the animals are likely to experience short-term moderate pain, suffering or distress, or long-lasting mild pain, suffering or distress as well as procedures that are likely to cause moderate impairment of the well-being or general condition of the animals shall be classified as ‘moderate’.
Severe: Procedures on animals as a result of which the animals are likely to experience severe pain, suffering or distress, or long-lasting moderate pain, suffering or distress as well as procedures, that are likely to cause severe impairment of the well-being or general condition of the animals shall be classified as ‘severe’.
The assignment of the severity category shall take into account any intervention or manipulation of an animal within a defined procedure. It shall be based on the most severe effects likely to be experienced by an individual animal after applying all appropriate refinement techniques.
When assigning a procedure to a particular category, the type of procedure and a number of other factors shall be taken into account. All these factors shall be considered on a case-by-case basis.
The factors related to the procedure shall include:
1.) type of manipulation, handling,
2.) nature of pain, suffering, distress or lasting harm caused by (all elements of) the procedure, and its intensity, the duration, frequency and multiplicity of techniques employed,
3.) cumulative suffering within a procedure,
4.) prevention from expressing natural behaviour including restrictions on the housing, husbandry and care standards.
5.) Examples are given in Section III of procedures assigned to each of the severity categories on the basis of factors related to the type of the procedure alone. They shall provide the first indication as to what classification would be the most appropriate for a certain type of procedure.
6.) However, for the purposes of the final severity classification of the procedure, the following additional factors, assessed on a case-by-case basis, shall also be taken into account:
7.) type of species and genotype,
8.) maturity, age and gender of the animal,
9.) training experience of the animal with respect to the procedure,
10.) if the animal is to be reused, the actual severity of the previous procedures,
11.) the methods used to reduce or eliminate pain, suffering and distress, including refinement of housing, husbandry and care conditions,
12.) humane end-points.
Examples of different types of procedure assigned to each of the severity categories on the basis of factors related to the type of the procedure:
(a) administration of anaesthesia except for the sole purpose of killing;
(b) pharmacokinetic study where a single dose is administered and a limited number of blood samples are taken (totalling < 10 % of circulating volume) and the substance is not expected to cause any detectable adverse effect;
(c) non-invasive imaging of animals (e.g. MRI) with appropriate sedation or anaesthesia;
(d) superficial procedures, e.g. ear and tail biopsies, non-surgical subcutaneous implantation of mini-pumps and transponders;
(e) application of external telemetry devices that cause only minor impairment to the animals or minor interference with normal activity and behaviour;
(f) administration of substances by subcutaneous, intramuscular, intraperitoneal routes, gavage and intravenously via superficial blood vessels, where the substance has no more than mild impact on the animal, and the volumes are within appropriate limits for the size and species of the animal;
(g) induction of tumours, or spontaneous tumours, that cause no detectable clinical adverse effects (e.g. small, subcutaneous, non-invasive nodules);
(h) breeding of genetically altered animals, which is expected to result in a phenotype with mild effects;
(i) feeding of modified diets, that do not meet all of the animals’ nutritional needs and are expected to cause mild clinical abnormality within the time-scale of the study;
(j) short-term (< 24h) restraint in metabolic cages;
(k) studies involving short-term deprivation of social partners, short-term solitary caging of adult rats or mice of sociable strains;
(l) models which expose animals to noxious stimuli which are briefly associated with mild pain, suffering or distress, and which the animals can successfully avoid;
(m) a combination or accumulation of the following examples may result in classification as ‘mild’:
assessing body composition by non-invasive measures and with minimal restraint; monitoring ECG with non-invasive techniques with minimal or no restraint of habituated animals; application of external telemetry devices that are expected to cause no impairment to socially adapted animals and do not interfere with normal activity and behaviour; breeding genetically altered animals which are expected to have no clinically detectable adverse phenotype; adding inert markers in the diet to follow passage of digesta; withdrawal of food for < 24h in adult rats; open field testing.
(a) frequent application of test substances which produce moderate clinical effects, and withdrawal of blood samples (> 10 % of circulating volume) in a conscious animal within a few days without volume replacement;
(b) acute dose-range finding studies, chronic toxicity/carcinogenicity tests, with non-lethal end-points;
(c) surgery under general anaesthesia and appropriate analgesia, associated with post surgical pain, suffering or impairment of general condition. Examples include: thoracotomy, craniotomy, laparotomy, orchidectomy, lymphadenectomy, thyroidectomy, orthopaedic surgery with effective stabilisation and wound management, organ transplantation with effective management of rejection, surgical implantation of catheters, or biomedical devices (e.g. telemetry transmitters, minipumps etc.);
(d) models of induction of tumours, or spontaneous tumours, that are expected to cause moderate pain or distress or moderate interference with normal behaviour;
(e) irradiation or chemotherapy with a sublethal dose, or with an otherwise lethal dose but with reconstitution of the immune system. Adverse effects would be expected to be mild or moderate and would be short-lived (< 5 days);
(f) breeding of genetically altered animals which are expected to result in a phenotype with moderate effects;
(g) creation of genetically altered animals through surgical procedures;
(h) use of metabolic cages involving moderate restriction of movement over a prolonged period (up to 5 days);
(i) studies with modified diets that do not meet all of the animals’ nutritional needs and are expected to cause moderate clinical abnormality within the time-scale of the study;
(j) withdrawal of food for 48 hours in adult rats;
(k) evoking escape and avoidance reactions where the animal is unable to escape or avoid the stimulus, and are expected to result in moderate distress.
(a) toxicity testing where death is the end-point, or fatalities are to be expected and severe pathophysiological states are induced. For example, single dose acute toxicity testing (see OECD testing guidelines);
(b) testing of device where failure may cause severe pain, distress or death of the animal (e.g. cardiac assist devices);
(c) vaccine potency testing characterised by persistent impairment of the animal’s condition, progressive disease leading to death, associated with long-lasting moderate pain, distress or suffering;
(d) irradiation or chemotherapy with a lethal dose without reconstitution of the immune system, or reconstitution with production of graft versus host disease;
(e) models with induction of tumours, or with spontaneous tumours, that are expected to cause progressive lethal disease associated with long-lasting moderate pain, distress or suffering. For example tumours causing cachexia, invasive bone tumours, tumours resulting in metastatic spread, and tumours that are allowed to ulcerate;
(f) surgical and other interventions in animals under general anaesthesia which are expected to result in severe or persistent moderate postoperative pain, suffering or distress or severe and persistent impairment of the general condition of the animals. Production of unstable fractures, thoracotomy without adequate analgesia, or trauma to produce multiple organ failure;
(g) organ transplantation where organ rejection is likely to lead to severe distress or impairment of the general condition of the animals (e.g. xenotransplantation);
(h) breeding animals with genetic disorders that are expected to experience severe and persistent impairment of general condition, for example Huntington’s disease, Muscular dystrophy, chronic relapsing neuritis models;
(i) use of metabolic cages involving severe restriction of movement over a prolonged period;
(j) inescapable electric shock (e.g. to produce learned helplessness);
(k) complete isolation for prolonged periods of social species e.g. dogs and non-human primates;
(l) immobilisation stress to induce gastric ulcers or cardiac failure in rats;
(m) forced swim or exercise tests with exhaustion as the end-point.
Education of scientists:
Persons carrying out procedures on live animals have to demonstrate the requisite competence. So far it is not clear whether an official approved training (FELASA course etc.) is required or whether in-house training will be accepted by the authorities. We shall set up a training course in future but it is not certain that authorities will take the attendance of this course as the required requisite competence. At least for now we recommend that those group members, who teach new colleagues, keep exactly track of this training (date, amount of hours, subject etc.). This can be taken as evidence for the requisite competence and will presented to the authorities. We continue doing the same with our mandatory introduction seminars for new users.
Keeping records of animal records:
The amount of data (quantity and quality) required by the new act is incredible high. The authorities have to be provided with the number and the species of animals bred, acquired, supplied, used in procedures, the origin of the animals, including whether they are bred for use in procedures, the dates on which the animals are acquired, supplied, from whom the animals are acquired, the name and address of the recipient of animals, the number and species of animals which died or were killed (or animals that have died, the cause of death shall, when known, be noted) and the projects in which animals are used. In addition a list of animals which were bred and killed but not used for procedures, genetically modified mice, which were used for the generation of mutant mousestrains as well as animals used for maintaining those colonies, methods of genotyping and the approximate number of animals in which those methods were applied together with a severity classification of the appropriate methods etc. This boils down more or less to a record of every single animal in our facility.
Animal welfare body
According to Austrian Laboratory Animal Act 2012 an institutional animal-welfare body was set up including at least the person (or persons) responsible for the welfare and care of the animals and scientific members. The animal-welfare body receives input from the designated veterinarian. The IMP IMBA animal-welfare body carries out the following tasks:
- advise the staff dealing with animals on matters related to the welfare of animals, in relation to their acquisition, accommodation, care and use;
- advise the staff on the application of the requirement of replacement, reduction and refinement, and keep it informed of technical and scientific developments concerning the application of that requirement;
- establish and review internal operational processes as regards monitoring, reporting and follow-up in relation to the welfare of animals housed or used in the establishment;
- follow the development and outcome of projects, taking into account the effect on the animals used, and identify and advise as regards elements that further contribute to replacement, reduction and refinement; and
- The records of any advice given by the animal-welfare body and decisions taken regarding that advice are kept for at least 3 years. The records are subject to inspections and are made available to the competent authority upon request.
Public information - non-technical project summaries
Information on the objectives of the project, including the predicted harm and benefits and the number and types of animals to be used;
A demonstration of compliance with the requirement of replacement, reduction and refinement.
Specification whether a project is to undergo a retrospective assessment and by what deadline.
Carrying out procedures (laboratory animal experiments)
Procedures may be carried out only within the framework of a project. Only an authorised project justifies the application of one or more procedures.
This is not new but now and in future applications have to focus much more on animal welfare beyond the scientific scope of the application, much more details concerning those issues are required: a harm-benefit analysis of the project has to be carried out, to assess whether the harm to the animals in terms of suffering, pain and distress is justified by the expected outcome taking into account ethical considerations.
There might be a retrospective assessment in certain cases, which has to evaluate whether the objectives of the project were achieved, to evaluate the harm inflicted on animals as well as the severity of the procedures and to evaluate any elements that may contribute to the further implementation of the requirement of replacement, reduction and refinement.
For those who apply for projects: the proposed severity classification (expected highest degree of pain, suffering, distress or lasting harm expected to be experienced by an individual animal during the course of the procedure) has to be included. The case by case classification has to happen when the animal is not used in experiments any more (after the procedures).
Three main requirements have to be met before a scientist may start procedures in live animals:
- A valid project license, approved by the authorities (§ 26 TVG 2012)
- A person, who is permitted to direct laboratory animal projects (§ 27 TVG 2012)
- A laboratory animal facility, which is registered as such with the authorities (§ 16 TVG 2012)
Before carrying out a procedure in live mice:
- Procedures always require a project license. Please make sure, that the procedure or technique you want to carry out in animals is covered and permitted by a project license. If you want to work with animals using a already existing license you have to ask the holder of the appropriate license ("Projektleiter") if she/he agrees. If she/he gives you the permission to use the license, you can do your work officially under her/his supervision and control. Everything you intend to do has to be in accordance with the project licence, no modifications and deviations are possible. In doubt the holder of the project license will be held responsible by the authorities for everything which happens in the name of the license. A project license expires after a certain time (normally 3-5 years). The maximum number of animals permitted by the authorities must not be exceeded. Only persons who have the permission to direct laboratory animal experiments may apply for a license. For all cases you are not the holder of a project license please always consider that you carry out the procedures under control and supervision of the license holder and not on your´s own responsibility.
- Before you may submit a project application to the authorities, you have to get the general permission to direct a scientific project involving live animals (so called “Projektleiter”-license). To obtain this permission, there has to be some evidence, that the applicant already acquired the necessary skills for carrying out procedures in animals and has proper knowledge in the field of laboratory animal science. The authorities will decide, whether you can show the required qualifications. A second precondition is that the animal experiments have to be carried out in a registered and licensed laboratory animal facility. The IMP IMBA animal facility is a registered a licensed laboratory animal facility with the additional permission operate this facility.
- If there is no appropriate project license yet, animal experiments in general start with writing a new project application, which has to meet several legal requirements. Then the project application has to be submitted to the appropriate authority (MA58 or BMWFW, depending on the institute). The authorities check whether the purpose and the expected outcome of the scientific project justifies the use of live animals and if there are no further questions, a ruling will be sent back to the applicant including the permission to carry out the procedures covered by the license (official decision of authorities with an official number referring to the project). Only the procedures and techniques described and implemented in the project application are permitted. In general the validity of the project license will not be longer than 3 years, the maximum number of animals per year cited here must not be exceeded. After expiration a new application is required, or no animals may be used any more. The prolongation of a current license is not possible, but it is possible to apply for the same scientific project (as new license).
Carrying out a procedure or technique in animals under control and supervision of a license holder:
- Whenever you intend to carry out a procedure or technique in animals under control and supervision of a license holder you have to use the IMP IMBA animal experiment form, which may be considered as a daily report of procedures you carried out in live mice based on this specific project license. Above all the name of project license and the number of animals used on that day has to be inserted here. The blank forms differ in a unique experiment number (mouse experiment form number in the upper left corner of the form), which makes it possible to identify every single form.
- Actually the veterinary inspectors want us to use a new form every day (daily report), when procedures in mice are carried out. After some discussions they accepted that one single form can be used as a report covering more days (maximum 5 weeks). This means all mice, which were used within this period may be counted in one form (the authorities may reduce this five weeks period to one day as worst case scenario). Therefore the validity of a form starts on the day it was taken (it will be assumed that the day the form was taken from the file will be the day the mice are used for the first time) and it expires 5 weeks later. After 5 weeks at latest the completed form has to be submitted to the animal house.
- The IMP IMBA animal experiment forms are stored in several files, e.g. in the corridors etc.
- Before taking out a mouse experiment form from a file you have to confirm in a list with the current date, name and your signature that you have taken this form (never take this list away). The authorities have to be able to allocate the form to the scientist. A mouseform, which was taken away without signing for it has to be considered as invalid and "not allocated". Mouseforms, which are not given back in time will not be accepted by the authorities any more and the form automatically gets the status "not allocated" as well. All procedures in live mice, which are reported in these forms are considered automatically as illegal.
- The animal experiment form number has to be cited in your laboratory journal together with the results of your experiments. In addition it has to be cited on the cage card belonging to the cage (upper margin), where the experimental mice are housed. When completing the form you have to allocate it to the name and to the number of the project license, which covers the procedures you carried out. Please complete the form carefully. The Austrian veterinary authorities will check your laboratory journal from time to time. They will check whether the results you obtained from the use of the animals are in accordance with the appropriate license, they will check what happened to the mice, euthanasia, results etc. Authorities may enter the facility whenever they want and as often they want.
- A mouse experiment form must be allocated to one single project license only. This means that a mouse cannot be used for two or more project licenses at the same time.
- Every cage containing experimental mice has to be marked with a label on the cage card holder saying that the license holder is a member of IMBA or a member of IMP or of a different institution (IMBA experiment or IMP experiment etc.).
- There has to be a list in every animal room containing the mouse form numbers of all cages with experimental mice in this room as well as number of the ruling to which the mouse form numbers were allocated.
- Make sure that there is a valid project license already.
- Get a mouse experimental form on the day you want to carry out a procedure in live mice (do not take away a form in advance).
- Officially confirm that you have taken this special form (date, name, signature). The validity of this form expires 5 weeks after you have taken it.
- Carry out the procedure and complete the form (number of mice, date, name of license, license holder, anesthesia etc.)
- Mark the cage with a label saying to which institution the license holder belongs.
- Write the mouse experiment form number on the cage card.
- Fill in the mouse form number in a list as well as the number of the ruling belonging to the project license (BMWFW..., MA 58.....). This list has to remain in the animal room.
- Submit the completed mouse experiment form to the animal house on the same day or after 5 weeks at latest (starting with the date the form was taken, which should be the date when the mice were used for the first time.
After the animal experiment:
- The completed forms will be stored in the animal house office and are subject to the controls of the Austrian veterinary inspectors.
- The animal house staff will take care that the completed mouse experiment form will be checked and signed by the license holder after you put it back (again: you should have asked her/him before you started).
- Please inform the animal technician, who is in charge of the room, on the humane endpoints cited in the project license.
- Before you leave IMP or IMBA (because your work is finished here) you have to inform the license holder on the results obtained from the laboratory animal experiments you carried out in the current year and in the last year so that he can give this information to the official veterinarians. If it is you who holds the license, please inform the animal house that you are going to leave soon.