Ajeesh glp
- 1. GLP PRINCIPLES FOR INVITRO STUDIES. AJEESH RAJENDRAN MSc BIOPHYSICS
- 2. OECD Organization For Economic co-operation and Development. An intergovernmental organization. Representatives of 30 industrialized countries in North America, Europe, Pacific and the European Commission. To co-ordinate and harmonize policies, discuss issues of mutual concern, and work together to respond international problems.
- 3. Environmental Health and Safety Division. The Principles of GLP have been developed to promote the quality and validity of test data used for determining the safety of chemicals and chemical products (Schechtman, 2007)
- 4. Good Laboratory Practice A quality system concerned with the organizational process and the conditions under which non-clinical health and environmental safety studies are planned, performed, monitored, recorded, archived and reported. Its principles are required to be followed by test facilities carrying out studies to be submitted to national authorities
- 5. Common principles for GLP facilitate the exchange of information and prevent the emergence of non-tariff barriers to trade. The issue of data quality has an important international dimension. If regulatory authorities in countries can rely on safety test data developed abroad, duplicative testing can be avoided.
- 6. PRINCIPLES OF GLP There should be unique identification for the study and all of its parts. All original observations in a study should be immediately , clearly and legibly recorded. All records should be in the form of bound notebooks or on continuously numbered sheets. All entries and corrections to them should be dated and initialed. Records related to the test systems itself should be gathered and retained.
- 7. Specimens should be clearly identified so as to allow full traceability. All the end of a study , all raw data should be assembled , catalogued and archived. The possibilities inherent in the applicable control mechanisms might subsequently provide the means for judging the shortcomings of the present activities and for improving the scientific quality of the future work.
- 8. The pillars of good laboratory practices. Good Laboratory Practices is based on four pillars which have to support the implementation and daily observance of its principles: The Management. The Quality Assurance. The Study Director. The National Compliance Monitoring Authority.
- 9. As efforts to decrease the use of animals in safety testing are intensifying, in vitro methods are gaining a more prominent role as alternatives or supplements to in vivo safety testing. Anticipated developments in the fields of toxicogenomics, toxicoproteomics, toxicometabonomics and in various high through-put screening techniques are expected to enhance the importance of in vitro methodologies for safety testing, beyond their traditional use as test systems in the area of genetic toxicity testing. The Application of the Principles of GLP to in vitro Studies.
- 10. The OECD Working Group on Good Laboratory Practice considered it therefore worthwhile to develop further guidance specifically of relevance to the application and interpretation of the OECD Principles of GLP1 to in vitro studies. Studies involving in vitro test systems have long been used to obtain data on the safety of chemicals with respect to human health and the environment. National legislation usually requires that these studies be conducted in accordance with Good Laboratory Practice (GLP) requirements.
- 11. Traditionally in vitro methods have been mainly used in the area of genetic toxicity testing, where the hazard assessment is based to a large extent on data derived from studies using in vitro test systems. As efforts to decrease the use of animals in safety testing are intensifying, in vitro methods are gaining a more prominent role as alternatives or supplements to in vivo safety testing. Furthermore, developments in the area of toxicogenomics, toxicoproteomics, toxicometabonomics and various (e.g., micro-array) high through-put screening techniques will also enhance the importance of in vitro methodologies for safety testing.
- 12. In vitro Studies In vitro studies are studies which do not use multicellular whole organisms, but rather microorganisms or material isolated from whole organisms, or simulations thereof as test systems. Many in vitro studies will qualify as short-term studies under the definition provided by the GLP Principles. For these studies, the OECD Consensus Document on The Application of the GLP Principles to Short-Term Studies should be consulted and used as appropriate, in order to allow for the application of the provisions facilitating the work of Study Director and QA.
- 13. Test Facility Management Most of the responsibilities of test facility management are of a general nature and are equally applicable to in vivo and in vitro studies, such as the requirements that test facility management has to ensure the availability of qualified personnel, and of appropriate facilities and equipment for the timely and proper conduct of the study. However, test facility management should be aware that in vitro testing may influence the execution of some of their responsibilities For example, test facility management must ensure that personnel clearly understand the functions they are to perform.
- 14. In vitro testing may also necessitate the availability of specialized areas and the implementation of procedures to avoid contamination of test systems. Another example is provided by the requirement that test facility management should ensure that test facility supplies meet requirements appropriate to their use in a study. Certain in vitro studies may necessitate the use of proprietary materials or test kits.
- 15. Study Director The general responsibilities of the Study Director are independent of the type of study and the responsibilities listed in the Principles apply to in vitro studies as well. The study director continues to be the single point of study control and has the responsibility for the overall conduct and reporting of the study. In in vitro studies the Study Director should pay particular attention to documenting the justification and characterization of the test system, an activity which may be more difficult to accomplish for in vitro studies.
- 16. Quality Assurance In general, Quality Assurance (QA) activities will not be greatly different between in vitro and in vivo studies. In vitro studies may qualify in certain cases for treatment under the conditions of short-term studies; in these cases, the OECD Consensus Document on The Application of the GLP Principles to Short- Term Studies will be applicable. Thus, such studies may be inspected, if applicable and permitted by national regulations, by QA on a process-based inspection program .
- 17. Since the GLP Principles require QA to inspect especially the critical phases of a study, it is important that, in the case of in vitro studies, QA is well aware of what constitutes critical phases (and critical aspects) of such studies. Corresponding guidance for QA inspections should be developed in co- operation with Study Directors, Principal Investigators and study personnel in the relevant areas.
- 18. Facilities The GLP Principles mandate that test facilities should be suitable to meet the requirements of the studies performed there in, and that an adequate degree of separation should be maintained between different activities to ensure the proper and undisturbed conduct of each study. The GLP Principles require that a sufficient number of rooms or areas should be available to ensure the isolation of test systems, and that such areas should be suitable to ensure that the probability of contamination of test systems is minimized.
- 19. The term “areas”, however, is not specifically defined and its interpretation is therefore adaptable to various in vitro situations. The important aspect here is that the integrity of each test system and study should not be jeopardized by the possibility of potential contamination or cross-contamination or mix-up. In this way it may be possible to incubate cells or tissues belonging to different studies within the same incubator, provided that an adequate degree of separation exists and that no test item is sufficiently volatile so as to contaminate other studies that are co-incubated.
- 20. Separation of critical study phases may be possible not only on a spatial, but also on a temporal basis. Manipulation of cell and tissue cultures, e.g., sub cultivation procedures, addition of test item, etc., Another important aspect is the availability of devoted rooms or areas with special equipment for the long-term storage of test systems. The equipment, including storage containers, should provide adequate conditions for maintenance of long-term integrity of test systems.
- 21. Apparatus, Material, and Reagents While the commonly observed, routine requirements for apparatus used in a GLP compliant environment apply equally to apparatus used for in vitro studies, there are some specific points and issues of particular importance. As an example, it may be of importance for the integrity and reliability of some in vitro studies to ensure that the proper conditions of certain equipment, like microbalances, micropipettes, laminar air flow cabinets or incubators are regularly maintained, and monitored and calibrated where applicable.
- 22. For specific equipment, critical parameters should be identified requiring continuous monitoring or the setting of limit values together with installation of alarms. The requirements in the GLP Principles for reagents with respect to labelling and expiry dates apply equally to those used for in vitro studies.
- 23. Test Systems In vitro test systems are mainly biological systems, although some of the more recent developments in alternatives to conventional in vivo testing (e.g., gene arrays for toxicogenomics) may also exhibit some attributes of physical-chemical test systems, and still others, e.g., toxicometabonomics, may mainly rely on analytical methodology. Test kits, including proprietary test kits, should also be considered as test systems.
- 24. Conditions for Test Systems As for any other biological test systems, adequate conditions should be defined, maintained and monitored to ensure the quality and integrity of the test system during storage and within the study itself. This includes the documented definition, maintenance and monitoring of the viability and responsiveness of the test system, including recording of cell passage number and population doubling times.
- 25. Since maintenance of the proper environmental conditions during the storage of test systems may influence data quality to a greater degree than for other biological systems, these records may be of special importance in the maintenance of data quality and reliability. Records should also be kept for environmental conditions (e.g., liquid nitrogen level in a liquid nitrogen cryostorage system, temperature, humidity and CO2 concentration in incubators, etc.) as well as for any manipulation of the test system required for the maintenance of its quality and integrity.
- 26. Test System Records The GLP Principles require that records be maintained of source, date of arrival and arrival condition of test systems; for cells and tissues these records should include not only the immediate source (e.g., commercial supplier), but also the original source from where the cells or tissues have been derived (e.g., primary cells or tissues with donor characteristics; established cell lines from recognized sources, etc.). The requirements in the OECD Principles of GLP for test items and reagents with respect to labelling and expiry dates apply equally to test kits used as in vitro test systems.
- 27. Test kits, whether used as test system or in any other way, e.g., for analytical purposes, should have an expiry date. Extending this expiry date can be only acceptable on the basis of documented evaluation (or analysis). For test kits used as test systems, such documented evaluation may, e.g., consist of the historical record of observed responses obtained with the respective batch of the test kit to positive, negative and/or vehicle control items, and proof that, even after the expiry date, the response did not deviate from the historical control values. A documented decision of the Study Director as to the extension of the expiry date should provide evidence for this evaluation process.
- 28. Test and Reference Items (including Negative and Positive Control Items) In general, there are no specific requirements for receipt, handling, sampling, storage and characterization for test and reference items that are used in studies utilizing in vitro test systems besides those listed in the GLP Principles. Aseptic conditions may, however, be required in their handling to avoid microbial contamination of test systems. For negative, vehicle and positive control items, it may or may not be necessary to determine concentration and homogeneity, since it may be sufficient to provide evidence for the correct, expected response of the test system to them.
- 29. The expiry date of such control items may also be extended by documented evaluation or analysis. Such evaluation may consist of documented evidence that the response of the respective test systems to these positive, negative and/or vehicle control items does not deviate from the historical control values recorded in the test facility, which should furthermore be comparable to published reference values.
- 30. Standard Operating Procedures (SOPs) There are activities and processes specific to in vitro testing that should be described in Standard Operating Procedures. Such SOPs should therefore be additionally available for, but not be limited to, the following illustrative examples for test facility activities related to in vitro testing. a) Facilities Environmental monitoring with respect to pathogens in the air and on surfaces, cleaning and disinfection, actions to take in the case of infection or contamination in the test facility or area.
- 31. b) Apparatus Use, maintenance, performance monitoring, cleaning, and decontamination of cell and tissue culture equipment and instruments, such as laminar-flow cabinets and incubators; monitoring of liquid nitrogen levels in storage containers; calibration and monitoring of temperature, humidity and CO2-levels in incubators. c) Materials, Reagents and Solutions Evaluation of suitability, extension of expiry dates, assessment and maintenance of sterility, screening for common pathogen contaminants; description of procedures for choice and use of vehicles; verification procedures for compatibility of vehicles with the test system.
- 32. d) Test Systems Conditions for storage and procedures for freezing and thawing of cells and tissues, testing for common pathogens; visual inspection for contaminations; verification procedures (e.g., use of acceptance criteria) for ensuring properties and responsiveness on arrival and during use, whether immediately after arrival or following storage; morphological evaluation, control of phenotype or karyotype stability, control of transgene stability; mode of culture initiation, culture conditions with sub cultivation intervals; handling of biohazardous materials and test systems, procedures for disposal of test systems. e) Performance of the Study Aseptic techniques, acceptance criteria for study validity, criteria for assay repetitions. f) Quality Assurance Definition of critical phases, inspection frequencies.
- 33. Performance of the Study and Reporting of Study Results The GLP requirements for the performance of in vitro studies are identical to those provided for the more conventional safety studies. In many cases, the OECD Consensus Document on The Application of the GLP Principles to Short-Term Studies may be consulted in combination with the OECD GLP Principles in order that in vitro studies may be conducted in a GLP compliant way. There are a number of issues specific to in vitro testing that should be addressed in the study plan as well as in the final study report. These issues, however, are mainly of a scientific, technical nature, such as the (scientific) requirement that any internal controls (appropriate positive, negative, and untreated and/or vehicle controls), carried out in order to control bias and to evaluate the performance of the test system, should be conducted concurrently with the test item in all in vitro studies.
- 34. Storage and Retention of Records and Materials The general retention requirements of the GLP Principles apply to in vitro studies as well. Additionally, it should be considered to retain samples of long-term preservable test systems, especially test systems of limited availability (e.g., special subclones of cell lines, transgenic cells, etc.), in order to enable confirmation of test system identity, and/or for study reconstructability. Retention of samples of test item should be considered also for such in vitro studies which can be categorized as short-term studies, especially in cases where in vitro studies constitute the bulk of safety studies. Records of historical positive, negative, and untreated and/or vehicle control results used to establish the acceptable response range of the test system should also be retained.
- 35. CONCLUSION The purpose of GLP is to assure the quality and integrity of data submitted in support of the safety of regulated products. Protocols , SOPs , adequate facilities , and equipment's ,identification of test substance , proper animal care , accurate recordings of observations and adequate reporting of results are basic necessities for the conduct of high quality valid toxicity study. The results obtained in compliance with GLP are to be mutually accepted by the health authorities of the OECD member states . Therefore , multiple testing can be avoided.