quality management for chemical safety

Environmental Health Criteria 141

Quality management for chemical safety testing

Please note that the layout and pagination of this web version are not identical with the printed version.

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 141

QUALITY MANAGEMENT FOR CHEMICAL SAFETY TESTING

This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization.

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization

World Health Orgnization Geneva, 1992

The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals.

WHO Library Cataloguing in Publication Data

Quality management for chemical safety testing.

(Environmental health criteria ; 141)

1.Hazardous substances - toxicity 2.Laboratories - standards 3.Quality control 4.Toxicology - methods I.Series

Quality management for chemical safety testing (EHC 141, 1992)

Page 1 of 76

ISBN 92 4 157141 1 (NLM Classification: QV 602) ISSN 0250-863X

The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available.

(c) World Health Organization 1992

Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved.

The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

CONTENTS


INTRODUCTION

1. GENERAL QUALITY MANAGEMENT APPROACH FOR QUALITY ASSURANCE

1.1. Organization and personnel1.1.1. Introduction1.1.2. Organization1.1.3. Test facility management1.1.4. Study director1.1.5. Support personnel1.1.6. Quality assurance function1.1.7. Personnel selection and development1.1.8. Orientation and training of new personnel

1.2. Quality assurance programme1.2.1. Introduction1.2.2. Quality assurance and quality control1.2.3. Organization and personnel1.2.4. Inspections and audits1.2.5. Records and reports1.2.6. Quality assurance SOPs

1.3. Facilities and equipment1.3.1. Introduction1.3.2. Facilities for handling test, control

and reference substances


Page 2 of 76

1.3.3. Field study facilities1.3.4. Equipment

1.4. Study plan1.4.1. Introduction1.4.2. Study plan preparation1.4.3. Format and content of study plan1.4.4. Use of study plan

1.5. Standard operating procedures1.5.1. Introduction1.5.2. Format and content of SOPs1.5.3. Preparation of SOPs1.5.4. Typical SOPs1.5.5. Use and availability of SOPs1.5.6. Adequacy of SOPs1.5.7. Maintenance of SOPs

1.6. Test, control and reference substances1.6.1. Introduction1.6.2. Test, control and reference substance

characterization 1.6.3. Handling1.6.4. Storage1.6.5. Distribution

1.6.6. Mixtures of substances with vehicles (carriers)

1.6.7. Stability1.6.8. Labelling1.6.9. Facilities and equipment

1.7. Quality control1.7.1. Introduction1.7.2. Level of quality control1.7.3. Pre-analytical quality control1.7.4. Analytical quality control1.7.5. Statistical considerations1.7.6. Analytical performance evaluation

1.8. Documentation and record keeping1.8.1. Introduction1.8.2. Manual data records1.8.3. Computer data records1.8.4. Indirect computer data records

1.9. Final report1.9.1. Introduction1.9.2. Contents1.9.3. Indexing

1.10. Archiving and retention of data1.10.1. Introduction1.10.2. Facilities1.10.3. Responsibilities for an archive1.10.4. SOPs for archiving1.10.5. Receiving, indexing and identification1.10.6. Filing and storage1.10.7. Access and security1.10.8. Retrieval of data and control of access1.10.9. Retention of information

2. QUALITY MANAGEMENT APPLIED TO TOXICITY STUDIES

2.1. Introduction2.2. Procedural requirements2.3. Phases of animal use2.4. Obtaining animals2.5. Shipping and receipt of animals2.6. Animal care facilities


Page 3 of 76

2.7. Animal husbandry supply facilities2.8. Facilities for handling test, control and

reference substances 2.9. Pre-study evaluation of animals2.10. Allocation of animals to a study2.11. Exposure of animals to a test or control substance2.12. Control of laboratory environment2.13. Evaluation of in-life animal responses to test

and control substances

2.14. Removal of animals from a study2.15. Transfer of animal tissues and specimens to

archives

3. QUALITY MANAGEMENT APPLIED TO HUMAN AND ENVIRONMENTAL MONITORING STUDIES

3.1. Introduction3.2. Procedural requirements3.3. Selection of sampling strategies and study design3.4. Sampling procedures and documentation3.5. Handling of samples3.6. Analytical performance evaluation3.7. The regression method3.8. Practical application of the regression method3.9. Other analytical performance evaluation programmes3.10. Analytical performance criteria3.11. Quality control samples

REFERENCES

APPENDIX I

WHO TASK GROUP ON QUALITY MANAGEMENT FOR CHEMICAL SAFETY TESTING

Members

Professor W. Almeida, Department of Preventive Medicine, State University of Campinas, São Paulo, Brazil

Professor E.A. Bababumni, Department of Biochemistry, University of Ibadan, Ibadan, Nigeria

Dr A.W. Choudhry, Kenya Medical Research Institute, Nairobi, Kenya (Chairman)

Dr C. Morris, International Chemical Consultants, Alexandria, Virginia, USA

Dr M. Ruchirawat, Chulabhorn Research Institute, Bangkok, Thailand (Vice-Chairman)

Dr A. Strik, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands

(Rapporteur)

Dr K. Kanagalingam, Office of Compliance Monitoring, United States Environmental Protection Agency, Washington, DC, USA

Professor M. Vahter, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden

Dr Z. Xing-Quan, Institute of Environmental Health


Page 4 of 76

Monitoring, Beijing, China

Mr R. Zisa, Office of Compliance Monitoring, United States Environmental Protection Agency, Washington, DC, USA

Representatives of other organizations

Dr R.F.M. Herber, Coronel Laboratory, University of Amsterdam, Amsterdam, The Netherlands, representing the International Union of Pure and Applied Chemistry (IUPAC)

Professor D. de Wied, Rudolf Magnus Institute of Pharmacology, University of Utrecht, Utrecht, The Netherlands, representing the International Union of Pharmacology (IUPHAR)

Dr H. Könemann, Public Health Inspectorate, Ministry of Welfare, Health and Cultural Affairs, Rijswijk, The Netherlands, representing the Organisation for Economic Co-operation and Development (OECD)

Dr R. Länge, Schering A.G., Berlin, Germany, representing the European Chemical Industry Ecology and Toxicology Centre (ECETOC)

Secretariat

Dr E.M. Smith, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland ( Secretary)

Dr D.T. Mage, Prevention of Environmental Pollution, Division of Environmental Health, World Health Organization, Geneva, Switzerland

NOTE TO READERS OF THE CRITERIA MONOGRAPHS

Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are kindly requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda.


The WHO Task Group on Quality Management for Chemical Safety Testing met in Bilthoven, The Netherlands, from 28 May to 1 June 1990. Dr E. Smith welcomed the participants on behalf of the heads of the three IPCS cooperating organizations (UNEP/ILO/WHO). The Task Group reviewed and revised the draft monograph and extended its scope.

The first draft of the part of the monograph dealing with quality assurance of toxicological studies was prepared by Mr E.A. Brisson (US Food and Drug Agency, Rockville, Maryland, USA) and the part dealing with quality control in human health monitoring was prepared by Professor M. Vahter (Karolinska Institute, Stockholm, Sweden) with contributions from Professor R. Herber (Coronel Laboratory, University of Amsterdam, The Netherlands). Additional text on quality assurance for environmental monitoring was prepared by Dr R. Länge (Schering AG, Berlin, Germany). Dr H. Könemann (Ministry of Welfare, Health and Cultural Affairs, Rijswijk, The Netherlands) reviewed and integrated the text. Finally, Dr Könemann, Professor R. Herber (Coronel


Page 5 of 76

Laboratory, University of Amsterdam, The Netherlands) and Professor M. Vahter acted as an ad hoc editorial group and gave valuable assistance in the preparation of the final text. Dr E.M. Smith and Dr P.G. Jenkins, both members of the IPCS Central Unit, were responsible for the overall scientific content and technical editing, respectively, of this monograph.

Support for the meeting was provided by The Netherlands National Institute of Public Health and Environmental Protection (RIVM).

The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged.

ABBREVIATIONS

EQC External Quality Control

HEAL Human Exposure Assessment Locations

IQC Internal Quality Control

MAD Maximum Allowable Deviation

MRBIS Mean Running Bias Index Score

MRVIS Mean Running Variance Index Score

NEQUAS National External Quality Assessment Scheme (UK)

QA Quality Assurance

QAP Quality Assurance Programme

QC Quality Control

QCP Quality Control Programme

SOP Standard Operating Procedure

INTRODUCTION

Chemical safety is a world priority. Considerable effort is being devoted by governments and industries to ensure that the manufacture and use of chemicals will not have an adverse effect on human health or the environment. Many governments have introduced laws, regulations, and guidelines designed to prevent human health risks and environmental degradation.

Concerns about the potential of chemicals to have adverse effects on human health and the natural environment has led the World Health Organization (WHO), together with the International Labour Organisation (ILO) and the United Nations Environment Programme (UNEP), to cooperate actively in the International Programme on Chemical Safety (IPCS). The objectives of the IPCS include the evaluation of the effects of chemicals on human health and the environment and the development of methodology and testing methods in order to produce internationally comparable results. The importance of the quality of data in achieving these objectives is self-evident. The need for quality of data generated in laboratory and field toxicological studies is parallelled by the need for quality of analytical data on tissue concentrations, environmental exposure, and exposure surveillance and monitoring studies. It is important to realize that chemical risk assessment utilizes not only data from studies carried out for regulatory purposes, such as notification, but


Page 6 of 76

also data from many types of research studies, both pure and applied. In the process of risk assessment, biological dose-effect and dose-response data are integrated with analytical data. Thus, overall quality management of data generation and application of quality assurance and quality control are crucial. Data quality is inherent in the IPCS evaluations of the risk to human health and the environment of chemicals, published as Environmental Health Criteria (EHC) monographs. The relevance of quality assurance and quality control for the generation of sound data is a key element in the monographs dealing with principles and methods for the evaluation of toxicity, e.g., the monograph on Principles of Toxicokinetic Studies (WHO, 1986a).

The validity and usefulness of the results from experimental studies, whether these relate to basic research or tests carried out to meet regulatory requirements, are critically dependent on the way in which they are designed, managed and performed. In risk assessment, the quantity and quality of data are both important. Limited or inadequate data, even where there are no doubts on their quality, cannot result in a balanced evaluation, and the overall conclusions on risks to human health and the environment are inevitably limited. An extensive data base that is of poor quality also gives rise to poor assessments and, probably, erroneous conclusions.

The quality of data is considered to be an objective matter. Data should be meaningful and reliable for use in the assessment of the safety of chemicals. Of course, quality of data depends to a large extent on the quality of the scientists and other individuals involved in the production of these data, but many aspects of quality can be verified, measured or assessed objectively, and therefore, also improved systematically.

Promoting quality is a management responsibility. Quality management is a broad approach, using all possible tools to carry this responsibility. Studies are complex activities involving people, facilities, equipment, test systems, chemicals and materials, often over a considerable period of time. Therefore activities must be carefully coordinated so that specific events occur when scheduled and in the way intended.

In the field of safety testing of chemicals and preparations, quality management was introduced in the 1970s after the discovery of some cases of fraudulent tests and increasing concern over the careless way in which many tests supporting the registration of drugs were being carried out. The United States Food and Drug Administration responded to these concerns by developing regulations on Good Laboratory Practices (Lepore, 1979; FDA, 1987a,b). The philosophy and many of the requirements were based on the quality management systems developed for industrial production, such as that of military equipment. Experience had already been gained in applying similar principles, e.g., the application of Good Manufacturing Practices to the production of pharmaceutical products.

The International Organization for Standardization in ISO 8402 (ISO, 1986a) defines quality as "the totality of features and characteristics of a product or service that bear on its ability to satisfy stated or implied needs", quality management as "that aspect of the overall management function that determines and implements the quality policy", and quality policy as the "overall quality intentions and direction of an organization as regards quality, as formally expressed by top management". Quality management focuses on the organizational process. It is directly relevant to the conditions under which laboratory studies are planned, performed, monitored,


Page 7 of 76

recorded, reported and archived. Quality management principles have been laid down in national legislation and in documents from international organizations. Major examples are the Principles of Good Laboratory Practice produced by the Organisation for Economic Co-operation and Development (OECD, 1982), publications of the International Standardization Organization, such as ISO 8402 (ISO, 1986a) and ISO 9000 and 9004 (ISO, 1987a,b) and quality assurance principles for analytical laboratories of the Association of Official Analytical Chemists (AOAC, 1984). The philosophy followed in these various documents is broadly similar and has served as guidance for developing this monograph.

A prerequisite for producing data of good quality is the availability of adequate facilities, equipment, and personnel who are both well educated and well trained. Studies need to be adequately designed and planned. Routine laboratory procedures need to be well defined, so that they can be carried out in the best possible way and in a consistent and reproducible manner. Other key elements in performing and reporting studies are the test, control and reference substances, the documentation and record keeping, the final report and, lastly, the archiving and retention of data. This monograph provides guidance for these activities.

An important feature of quality is that it should be verifiable and indeed be verified. For that purpose the quality assurance approach has been developed (Burck, 1979). Quality assurance includes independent study monitoring that assures laboratory management and users of data that facilities, equipment, personnel, methods, practices, records and controls conform to accepted quality management principles. An effective quality assurance system provides confidence that a study report meets the pre-established quality standards of accuracy, integrity, completeness and clarity. Quality assurance should be integrated within the entire study process to ensure that the results are valid and that the final report accurately reflects these results. This monograph therefore deals with the organization of a quality assurance programme.

An important quality assurance tool is known as quality control. Quality control is the quality assessment of quantitative, routine laboratory determinations. Many techniques are used for this purpose. Quality control plays a major role in monitoring studies, but in addition, it is frequently applied in toxicity studies, for instance, to assess the quality of biochemical and haematological data.

The application of modern quality management approaches to the two fields of safety assessment of chemicals is crucial. These fields are a) quality management of laboratory toxicity studies and b) studies to monitor the extent of exposure and effects and the presence of chemicals in man and the environment.

Quality has its price. Considerable investment, especially in terms of human resources, has to be made in a laboratory before a quality management system is operational. Furthermore, it requires continued effort to keep the system functioning. On the other hand, quality assurance is a major help for management in organizing the work, planning it well, ensuring continuity and increasing productivity. In addition, an efficient quality assurance system reduces considerably the risk that erroneous data will lead to non-acceptance of studies and rejection of reports or the need to repeat studies.

The following guidance is not directly based on legal precepts but follows the same philosophy, and it describes quality assurance considerations involved in the production of reproducible and reliable


Page 8 of 76

test data. It addresses basic elements of a quality management programme for chemical safety testing with the aim of promoting the quality assurance concept in test facilities throughout the world and of facilitating the global sharing of useful information on chemicals. It is not intended to deal with the details of quality management of toxicity and ecotoxicity testing and chemical analysis, but chapter 2 describes quality assurance as applied to biological testing in some detail because this is an area that is not familiar to all scientists working in this field. Even though certain countries may not have extensive testing facilities, the national and international acceptability of their research data can be greatly enhanced by utilizing these basic quality assurance and management principles. In addition they can apply the principles of quality assurance and quality control to assure the quality and acceptability of safety data presented to them for risk assessment purposes.

1. GENERAL QUALITY MANAGEMENT APPROACH FOR QUALITY ASSURANCE

1.1 Organization and personnel

1.1.1 Introduction

To ensure that studies are valid and properly conducted, an organizational structure should be established that will best serve the needs of the testing facility and will employ an adequate number of personnel that are well qualified by education, training and/or experience to perform their assigned tasks.

1.1.2 Organization

Regardless of the specific organizational structure, facility personnel include test facility management, study directors, quality assurance programme personnel and support personnel. The responsibilities for successfully running a laboratory are shared among these groups.

The typical composition and responsibilities of each of these groups are discussed below.

1.1.3 Test facility management

In general, duties that are more administrative than scientific are the direct responsibility of management. The primary duty of management is to ensure that all studies are properly planned, conducted and reported, and that sufficient staff and resources are made available for the successful conduct of studies. Before any studies are started, management must make various administrative decisions concerning the nature of the studies that will be conducted. Management must decide which controls will be in place to ensure the successful conduct and completion of studies, and decide the organizational coordination within the test facility to carry out the studies. It must establish a quality assurance programme that assures compliance with the study plan test facility procedures, and, where necessary, government regulations.

Properly trained personnel are crucial to the conduct of a quality study. It is management's responsibility to see that staff, both professional and technical, receive adequate training to ensure that they are competent to carry out their duties. Management must make available to all personnel the opportunity for external and on-the-job training to maintain and improve necessary laboratory skills.

Management must implement organizational and personnel directing


Page 9 of 76

and coordinating mechanisms that will adequately serve the facility. It must also be certain that an adequate number of qualified employees

are available who clearly understand the functions they are to perform, as well as any health and safety precautions that need to be taken during the conduct of a study.

In addition, management must apply proper managerial skills in dealing with the staff in order to promote a high level of job performance. Positive feedback to the personnel for recognition of good work performance should be practised continually by management. All personnel should view their work as a positive contribution to a high quality end product, the final study report.

The most fundamental decisions of management concern the types and numbers of studies that will be conducted at the testing facility. Taking into account the experience of personnel both within the test facility and outside, such as consultants, management must take decisions on a number of aspects, e.g., what type of test systems will be maintained, whether studies will be of a short-term or long-term nature or a combination of both, whether other external test facilities will be required to conduct certain portions of a study, and how many studies can be properly handled considering the personnel, equipment and facilities available in the test facility.

Management must establish procedural controls that will assure that all study activities will be properly conducted. Typically, these include the requirement that written Standard Operating Procedures (SOPs) exist for all routine functions conducted within the test facility or by other external facilities, and that the test facility management is kept informed of study progress or set-backs.

The designation of job responsibilities and the assignment of these responsibilities to the various entities within the organization should be documented. Management must appoint a study director to be responsible for overall study management and the single point of study control. A qualified person must be designated to assume the study director's responsibilities on a temporary basis in his absence. If a study director needs to be replaced on a permanent basis, this should be done promptly.

Management delegates much of its responsibilities and authorities to the appropriate divisions and personnel within the organization. Each division must be held accountable to laboratory management to accomplish its assigned tasks. This requires open communication between the various organizational personnel and laboratory management.

1.1.4 Study director

Experience has shown that unless responsibility for the proper conduct of a study is assigned to one person, there is a potential for personnel to receive conflicting instructions, which can result in a poor study. Therefore, it is recommended practice, before a study is

initiated, for management to designate one individual who will serve as study director and be considered the chief scientist in charge of a study. It is essential for the study director to have a strong scientific background, as well as proven managerial abilities, strengths in communication and problem solving, and the ability to organize the day-to-day and long-term objectives of a study.

Specific responsibilities of a study director include assuring that:


Page 10 of 76

* the study plan is agreed;

* the procedures specified in a study plan are followed by personnel engaged in the conduct of that study;

* all revisions to a study plan are brought to the attention of appropriate personnel;

* test systems are appropriate for the study;

* personnel involved in the study clearly understand their functions and are qualified to perform them;

* data are accurately and promptly recorded and verified;

* health hazards associated with the test system are recognized and controlled, including hazards to personnel as well as to the integrity and quality of the test system;

* studies are conducted in a manner that is safe for personnel;

* all raw data, samples and specimens are archived promptly at the completion of the study, as appropriate;

* the reported results of the study accurately reflect the raw data;

* unforeseen circumstances that may affect the quality and integrity of a study are noted, corrected and documented.

The study director must use the advice, education, experience and assistance of other scientists participating in the study.

Concerning the study director's workload, both the types and lengths of the studies have a significant impact on the number of studies for which a single study director may be responsible. The maximum number of studies assigned to one study director should be very carefully assessed by management.

1.1.5 Support personnel

A variety of specialists is required to conduct a study adequately and to provide the administrative support to ensure its proper conduct. In addition to clerical and administrative support to staff, each study requires the participation of such specialists as toxicologists, biochemists, clinical pathologists, veterinarians, chemists, histologists, statisticians, equipment and maintenance specialists, computer specialists, and animal caretakers. The personnel needs of each facility will be guided by the particular studies to be conducted.

1.1.6 Quality assurance function

In many laboratories the quality assurance function is run by quality assurance programme (QAP) staff carrying out quality assurance duties on a full-time basis. The primary functions of quality assurance personnel are to conduct facility inspections and data audits. Through inspections they ensure, in general, that studies are conducted in accordance with the quality assurance principles; and in particular, that the study plans and SOPs are followed. Through their data audit function they assure that the final reports of studies are verifiable from the raw data, and that these data were collected, recorded and maintained according the quality assurance principles.


Page 11 of 76

QAP staff or those exercising a quality assurance function should have a scientific or technical background. They should be well versed in:

* basic management skills;

* communication skills;

* understanding of intra-organizational relationships and functions;

* good record keeping;

* negotiation skills.

Generally, quality assurance personnel are drawn from experienced study personnel and are qualified for their quality assurance responsibilities through formal training courses and on-the-job training exercises. It is often helpful for the test facility to structure the on-the-job training so that personnel are certified proficient as they successfully accomplish each duty listed in the job description. For example, when laboratory staff demonstrate that they can review a study plan for adherence to acceptable laboratory practices, this would be indicated in their personal records.

1.1.7 Personnel selection and development

All personnel engaged in the conduct of a study should have sufficient education, experience and training to perform their assigned duties. The careful and systematic recruitment and employment of laboratory personnel should be considered an important management function. The job requirements should be delineated in written job descriptions established for each position available at the laboratory. Job descriptions should specify the duties and responsibilities, the reporting relationships (i.e. to whom the employee should routinely report the study status and unexpected emergencies) and the reporting requirements expected from the employee.

1.1.8 Orientation and training of new personnel

The need for adherence to the principles of quality assurance and to safety procedures, personal sanitation, and clothing and health restrictions should be discussed with each new employee. Copies of the SOPs defining the laboratory's personnel health and safety regulations and copies of relevant SOPs for their areas of responsibility should also be provided to employees. Before beginning any work, employees should be instructed by their immediate supervisor on their particular job duties, responsibilities and working conditions. Employees should be required to review SOPs and quality assurance manuals pertinent to their specific jobs.

This orientation process is followed by a period of on-the-job training under careful and direct supervision. The amount of training required for each function will be determined by the nature of the job function. When a supervisor believes that an employee can proficiently perform a specific job function, the employee should be authorized to perform such functions independently. Documentation of this proficiency and authorization should be maintained by the appropriate department (e.g., personnel department and/or division where the employee is directly employed). Supervisors should hold additional training sessions as often as needed to emphasize a procedure or explain a change in a procedure. These sessions should


Page 12 of 76

also be appropriately documented.

The laboratory should have documentation that explains how employees should be oriented and trained for their new positions, as well as how their training will be documented.

1.2 Quality assurance programme

1.2.1 Introduction

All testing facilities (e.g., laboratories, field operations), regardless of size, that generate data for assessing the impact of chemicals (e.g., industrial chemicals, pesticides or pharmaceuticals)

on human health or the environment should have an efficient management system. The establishment of an independent quality assurance programme (QAP) is an essential mechanism for accomplishing these goals.

A QAP does not necessarily require a separate organizational entity consisting of personnel permanently assigned to this task. However, it is critical that an organizational separation exist between quality assurance inspection and study personnel if a laboratory or field operation is to expect an impartial analysis of the accomplishments and operations encountered in the conduct of a study.

1.2.2 Quality assurance and quality control

It is important, at the outset, to distinguish between the related concepts of quality assurance and quality control. Quality control is a valuable quality assurance tool. Assessing study quality periodically is an essential aspect of quality assurance. Thus, the quality of an analytical measurement may be validated, for example, by comparing analytical results against a known standard taking into consideration the sensitivity, accuracy, precision, calibration and maintenance of the analytical equipment. These measurements would be part of a quality control system.

1.2.3 Organization and personnel

Management assures adherence to the principles of good laboratory management practices, authorizes study plans and standard operating procedures, and corrects operational defects that are reported by the quality assurance programme staff. A quality assurance programme must be established by management so that impartial inspections can be conducted and observed defects can be corrected promptly. Most laboratories and field operations generating data for regulatory purposes have a quality assurance programme unit as a distinct organizational entity reporting to management. If scientists carrying out testing are also to conduct quality assurance inspections and audits, they should have no scientific or personal involvement in the studies being quality assured and must be impartial.

It is important for a quality assurance programme to be implemented by highly motivated, qualified, and trained individuals who possess sufficient knowledge of experimental procedures to permit an adequate impartial assessment.

1.2.4 Inspections and audits

General inspection of facilities and critical activities and auditing final reports are very important tasks in a quality assurance programme. The purpose of inspection is to verify that the study is


Page 13 of 76

being conducted in accordance with the study plan, the SOPs (see

chapter 4) and the applicable principles of good laboratory management practices. The goal is to detect and correct systematic or unintentional flaws in the study process before the flaws have an adverse impact on the quality of the study. It is very important to assure that a study is conducted as directed by the study plan. Auditing has two purposes. The first is to confirm that the results presented in the final report accurately reflect the raw data that were collected during the study and the second is to highlight any circumstances that would adversely affect the study. SOPs should be developed to cover procedures of study conduct and for the audit of the final report. The QAP must compare these audit findings against the study plan and the supporting raw data to ensure that the report accurately reflects the results of the study in accordance with the study plan. The SOP should also indicate which raw data are to be reviewed during the audit and how they are to be reviewed.

Prior to initiating an inspection of the facilities and equipment, the designated quality assurance person should be familiar with the experimental operation to be inspected. This requires a review of the study plan and the SOPs that relate to the laboratory or field operations. Personnel files and equipment logs should be reviewed as well as the report of the previous inspection of the same operation. These inspections should be conducted in such a manner as to minimize any disruption of normal operations. The quality assurance person should explain the purpose of the inspection to laboratory or field staff. Any deviations should immediately be made known to the responsible personnel. The kinds of items to be examined include reagent preparation and labelling, personnel files, presence of authorized SOPs, equipment logs, recorded data entries, environmental controls, storage for specimens and test substances, and general cleanliness and orderliness of the laboratory or field operation areas.

Quality assurance staff may interview laboratory or field personnel whenever information on a study-related process is needed. Most importantly, quality assurance staff must witness the actual operations to assure that they are being conducted in accordance with the study plan and SOPs. It is not possible to estimate how much time should be spent in observing each operation, because this depends on the complexity of the operation. In any event, enough of the operation should be observed to conclude that the operations are in control and are being conducted in accordance with the SOPs.

Large amounts of data are often collected during studies, which are summarized in the final report. It is important, therefore, to assure that the summarized data in the final reports are accurate and complete. When large amounts of raw data are to be audited, several approaches can be used to select data points to evaluate. One approach is a pragmatic sampling plan which is based on the experience of the quality assurance staff with certain operational units. For example, activities associated with receipt and handling of the test

or reference substances generally result in very few recording errors, and it may be necessary to sample only 10-20% of the recorded information. In contrast, the recording of test system observation may frequently be a problem; 75-100% of these observations should be audited and the activity inspected on a regular basis. Another approach used is a random sampling plan. These plans are developed with the aid of statisticians and they provide a statistical confidence level for verifying the data in the final report. In accordance with these plans, a certain fraction of the data is examined, and if this fraction is found to be accurate, it is assumed


Page 14 of 76

that the whole report is accurate. If the fraction examined is shown to contain errors, then a larger number of data entries is examined, and so on. One should take into account, however, that errors may not occur at random and that statistical sampling plans may give misleading results. Auditing final reports occurs prior to the archiving of the study records. The quality assurance auditor assembles the raw data and all other study records for the study to be audited. All of these records are read for completeness and those that contain raw data are checked against the data in the final report in accordance with a random sampling plan. It is important to locate missing records and it is equally important to confirm the presence of all specimens and reserve samples of test and reference substances. All errors in the final report should be brought to the attention of the study director for resolution.

1.2.5 Records and reports

As with other activities in the laboratory or field operations, it is important that timely and accurate reports on quality assurance inspections are made and careful records kept.

There are three kinds of reports:

a) A routine, periodic report to management and to the study directors of all quality assurance work that was conducted during a defined period (e.g., one month). This report should give an overall summary of the inspections and audits that were conducted. It should highlight emerging problem areas, if any, and should forecast projected quality assurance activities.

b) A report made to the study director and to management whenever significant problems are found that can have an adverse effect on a study's quality. The report should describe completely the observed deficiencies.

c) The report that accompanies the final study report. This report lists the dates and phases of inspection conducted in the course of the study, and the dates when the inspectional findings were reported to management.

It is necessary for a quality assurance programme to keep copies of the following records: the master schedule sheet of all studies being conducted by the facility; study plans; periodic reports to management; inspection and audit reports; an index of quality assurance records and a compendium of all the test facility SOPs; and quality assurance programme SOPs.

1.2.6 Quality assurance standard operating procedures (SOPs)

In order to accomplish its purpose, a quality assurance programme must continually review study records for ongoing and completed studies, and inspect ongoing studies and all related facilities and equipment. This requires a number of SOPs specifically for quality assurance in order to describe the methods for accomplishing these tasks.

The content of the SOPs will vary depending on the organization, but generally the following areas should be covered: structure and organization of the quality assurance programme; to whom and how the quality assurance staff report findings; procedures for study inspection; procedures for auditing the final report; scheduling of inspection and audits; and procedures for indexing and maintaining records.


Page 15 of 76

An SOP describes the organization of the quality assurance programme and how it relates to the organizational structure at the testing facility. This is important for a number of reasons. It should include the following issues. Firstly, a quality assurance programme must provide reports directly to management to be effective. The SOP should describe the reporting procedure. It should also address the internal organization of a quality assurance programme. Secondly, a quality assurance programme must be composed of an adequate number of trained individuals to carry out its responsibilities, and the required qualifications for each position must be known. The SOP should include procedures for training personnel so that the personnel meet the stated qualifications.

The SOP needs to include how often reports are made and the format in which the reports are to be prepared. Internal reporting within the quality assurance programme should be in writing. There are two important aspects of reporting that should be addressed in the SOP: the manner in which reported deficiencies are corrected, and the manner in which any corrections are to be documented.

The SOP covers procedures for the in-process inspection of critical laboratory operations and their scheduling. It is necessary for the quality assurance personnel to visit the various areas regularly and to observe ongoing study-related activities to assure compliance with the study plan and relevant SOPs.

1.3 Facilities and equipment

1.3.1 Introduction

A test site must have suitable facilities and equipment to ensure the proper conduct of studies. These must be of adequate design, construction, capacity and location for their intended purpose. In addition, they must be properly used, maintained, and cleaned. Equipment used for generation, measurement or assessment of data must be calibrated and/or standardized with maintenance of appropriate records.

1.3.2 Facilities for handling test, control and reference substances

The facilities must allow separation of areas involved in the storage, handling and distribution of test, control and reference substances. This is necessary to prevent contamination of facilities, equipment, personnel and test systems, as well as to prevent confusion of substances. To accomplish this, there should be separate areas for:

a) receipt and storage of test, control and reference substances;

b) mixing of test, control and reference substances with a carrier (e.g., feed);

c) storage of the test, control and reference substance mixtures.

In addition, storage areas for the test, control and reference substances should be separate from areas housing the test systems. The storage facilities should be adequate to preserve the stability of the test, control and reference substances. For example, adequate refrigerator or freezer storage space should be available for test, control and reference substances requiring low temperature storage.

There needs to be separate space for the performance of the routine and specialized procedures required by study plans. This


Page 16 of 76

would include specialized areas for performing activities such as necropsy, histology, X-rays, and analytical and clinical chemistry.

The extent of space needed and the degree of separation required will vary widely depending upon the nature and amount of work being conducted. Larger laboratories may require a complete array of facilities capable of carrying out every phase of a study, whereas smaller laboratories may hire contract testing laboratories to perform some phases of the study that they do not routinely perform, such as histopathological slide preparation, clinical chemistry tests, or analytical chemistry for test substance characterization. In either case, laboratory operations must be performed under conditions that provide an adequate degree of separation to preclude confusion and interference between areas performing different aspects of the study.

1.3.3 Field study facilities

Adequate facilities should be available for the receipt, handling and storage of chemicals, such as herbicides, pesticides, and fertilizers, necessary for the conduct of good agricultural practices. These areas should be separated from the areas containing the test, control and reference substances. Adequate space should also be available for processing soil and plant residue samples and there should be adequate refrigerator and freezer space for interim storage before shipment to analytical residue laboratories.

1.3.4 Equipment

Along with adequate buildings, a test facility or field operation must have equipment of appropriate design and adequate capacity to function according to the study plan. This applies especially to equipment used in the generation, application, measurement or assessment of data. It also includes equipment used for laboratory environmental control, such as refrigerators, freezers, and air conditioning for test system areas and test substance preparation and storage areas. All equipment should be suitably located for ease of operation, inspection, cleaning, maintenance and safety.

Proper maintenance and calibration of equipment is a fundamental quality assurance management practice. Equipment should be inspected, cleaned and maintained. In addition, equipment used for the generation, application, measurement or assessment of data should be tested, calibrated and/or standardized.

A testing facility should have SOPs that describe in sufficient detail the methods, materials and schedules to be used in the routine operation, inspection, cleaning, maintenance, testing, calibration and/or standardization of equipment, and there should be appropriate documentation. These procedures should specify remedial action to be taken in the event of failure or malfunction and should designate the person responsible for performing each operation. Copies of these SOPs should be readily available to personnel in the vicinity of the equipment. In order to assure that all equipment associated with a study was operating properly at the time of its use, SOPs must be followed and records maintained for all equipment inspection, testing, calibrating, standardizing and maintenance. The records should contain the date the operation was performed and, in the case of maintenance operation or repairs, the type of function or malfunction, how and when it was observed, and, as appropriate, any remedial action taken.

1.4 Study plan

1.4.1 Introduction


Page 17 of 76

A clearly written, comprehensive study plan is an essential element of all chemical safety studies. A study plan should state the objectives, schedules and all methods for the conduct of a study. The plan, in combination with the SOPs, should provide the complete specifications and instructions for the execution of the study.

There is a practical difference between the role of the SOPs and the role of the study plan. The SOPs describe how specific routine operations are to be conducted. The study plan contains the design specifications for a single study or a group of related studies. Its role is to describe what the study objectives are and what methods are to be employed to achieve the stated objectives. In short, the plan describes what is to be done and when, while the SOPs address how specific study events are to be accomplished.

As the design specification for a study, the plan has an important quality assurance function: it serves as the reference for measuring study performance. If the plan of a well-designed study is followed, the study objectives should be achieved. However, if the plan is inadequate or is not followed, the intended objectives of the study may not be met. In this case, the study plan cannot serve as a suitable reference against which the quality of the study can be assessed.

The study plan is a useful management tool; it helps the long- term planning of, for instance, workload, manpower implications and the necessary facilities and equipment.

Test facilities should have SOPs covering the preparation and approval of study plans to help assure an orderly, complete and consistent review process. This is especially important in the preparation of the study plan, because errors or omissions can jeopardize the entire study.

Generally, SOPs for study plans should address the persons who will be responsible for preparation and review, how the preparation process begins and ends, what elements of format and content should be included in the plan and to whom it is to be distributed.

1.4.2 Study plan preparation

The study director has the primary responsibility for preparing the study plan and is generally responsible for designing the plan scientifically to assure that the study will achieve its intended purpose. Senior scientists who will participate in various aspects of the study should be involved in the preparation and/or review of the plan. This may include writing portions of the plan that involve heir

area of expertise. For example, the pathologist for the study may prepare the pathology section of the plan. Involvement of other scientists may include reviewing what the study director has prepared, correcting errors and omissions, and commenting on its adequacy. Technical personnel should be involved in plan preparation to ensure that the functions specified in the plan can be performed.

Management is also involved in the preparation and review of the study plan since it is its responsibility to assure that personnel, resources, facilities, equipment, materials and methodologies are available as scheduled in the plan. It is also useful for the study plan to be reviewed from the quality assurance perspective to assure that it contains the basic elements required in the applicable SOP.

For studies conducted under contract, the sponsor of a study


Page 18 of 76

should also be involved in the preparation and review of the study plan and must approve it prior to the initiation of the study. This is necessary to permit a clear understanding between the study director and the sponsor of the requirements and objectives of the study.

1.4.3 Format and content of study plan

The specific contents of each study plan will vary according to the objectives of a given study and the particular methods and procedures that will be employed. However, there are basic elements common to most chemical safety testing studies that should be included in the study plan:

a) A descriptive title and a statement of the purpose of the study. Stating the title and purpose of the study in the study plan clearly establishes the study objectives, and the study performance can later be assessed to ensure that the stated objectives were achieved.

b) The identification of the test, reference and control substances by name, chemical abstract number or code number. If code numbers are used to identify the test substance, such numbers should be unique to this substance.

c) The test system, i.e. any human, animal, plant or microbial system, as well as other cellular, subcellular, chemical or physical systems, or a combination thereof, which is used for the test or control substance. The selection of the appropriate test system is an important feature of study design, and should be described fully. The failure to do so could result in the inadvertent use of a test system other than that intended by the study director or the sponsor.

d) The specific procedure to be used for the identification of components of the test system. This most commonly refers to the method of identifying test animals within their respective test groups. Proper identification of the test system is necessary to prevent confusion of animals, samples and/or specimens.

e) A description of the experimental design, including the methods for the control of bias.

f) The type and frequency of tests, analyses, and measurements to be made. The plan should specify which parameters are to be measured and how often such determinations are to be made.

g) The records to be maintained. Because written records and electronically captured data are the tangible results of any study, the study plan should identify which records are to be kept. Many test facilities have developed SOPs that describe the identity, use, and retention of study-related records. In identifying records to be maintained, reference to the SOPs is appropriate.

h) A statement of the proposed plan for choosing the appropriate methods for statistical analyses of the data set. Although the study plan may assume that a particular test is applicable, a statistical test may show that it is not appropriate and that another test (e.g. nonparametric) must be chosen.

The above elements are not the only items that can be addressed in the study plan. For example, in the case where a study is modelled on an established procedure published in the scientific literature,


Page 19 of 76

the study plan may include literature references. The study plan may also include such items as the proposed starting and completion dates of the study and the name and address of the testing facility.

The study plan should be written in clear, concise language with adequate reference to the SOPs involved in the application of the test methods described. The pages should be numbered consecutively and the total number of pages should also be given. The body of the plan may be divided into clearly identified sections for ease of reference.

1.4.4 Use of study plan

A study plan must be used properly to achieve valid study results. Adherence to the study plan requirements, documenting adherence, and controlling and documenting any deviations or changes to the study should be monitored by the study director.

Although all personnel associated with the study refer to the study plan, the individuals most involved on a daily basis are the personnel performing study operations and the study director. Personnel use the plan to guide the performance of their duties. The

study director uses it as a means of informing study personnel of the study requirements. This individual has the overall responsibility for assuring that the personnel are aware of the current study plan requirements and that the study is carried out as directed by the study plan.

Once the study has started, a quality assurance programme also uses the study plan, e.g., to determine that no deviations from approved plans or SOPs are made without proper authorization and to audit the final report. To accomplish this, a quality assurance programme must maintain a copy of all study plans and current amendments. Quality assurance staff use these to compare actual practices against those specified in the plan for a given study. The plan often contains a schedule of study events that is useful to a quality assurance programme in scheduling its inspection of a study. In this regard, the quality assurance unit must verify that raw data are available to document that the approved study plan was followed.

To be used effectively, the study plan should be available, familiar to personnel and in a convenient format. However, the copying and distribution of the study plan should be controlled to prevent unauthorized copying and distribution. A copy of the study plan should be located in the immediate area of the test facility where a study event is being performed.

A final requirement for the effective use of the study plan is that it must be current. Although a well-designed and well-prepared plan should require little change after the study has begun, changes will almost always be required as the study progresses. The reasons for the planned and unplanned changes are many. For example, after the study has begun, an unusual or unexpected toxic effect may be observed that requires additional tests or changes in the frequency of observations. The study plan should be updated to reflect these changes in the form of appendices. The study director must approve, by means of a dated signature, the change or revision and document the reason for the change. In addition to dating, it is also desirable to number the amendments sequentially so that the most recent amendments can be quickly identified and study personnel can be sure they have received all amendments. Also, if study plan amendments are themselves revised in subsequent amendments, the current amendment should reference the superseded amendment.


Page 20 of 76

Complete records are important for documenting adherence to the study plan. These include proper recording of study data and of records related to study performance. When the plan directs the performance of a study event, records should be kept to document that the study plan was followed.

1.5 Standard operating procedures

1.5.1 Introduction

Quality assurance involves the development and use of standard operating procedures (SOPs). SOPs are written procedures which describe how to perform certain routine laboratory tests or activities, normally not specified in detail in study plans or test guidelines. SOPs set forth in detail how specific routine laboratory operations are to be carried out and complement the study plan by describing all routine study methods. SOPs are not described in detail in this monograph. There are no universal SOPs, although those detailing the same operation will have many close similarities from laboratory to laboratory. The act of developing SOPs, taking account of the operation and how its conduct is directly influenced by laboratory organization and structure, is a key function of laboratory management.

The importance of effective SOPs in conducting a study must be emphasized. SOPs are management directives designed to ensure that all personnel associated with a laboratory study will be familiar with and use the same procedures. If different individuals perform important study functions, such as dosing animals, preparing solutions, archiving documents or receiving test articles, these operations should be performed in the same manner. By standardizing procedures for the conduct of studies, SOPs have a valuable quality assurance function. They prevent the introduction of possible errors in the generation, collection and reporting of data.

The development of SOPs includes the following aspects: who should prepare, review and authorize SOPs; which laboratory operations require SOPs; and what information the SOPs should contain. The nature of the laboratory work being done and the training and experience of the laboratory personnel at a particular facility will determine exactly how extensive the content needs to be.

1.5.2 Format and content of standard operating procedures

SOPs should be easy to use. They should be identified by a descriptive title and a number. The individual pages of an SOP should be numbered, and each page should note the total number of pages contained in each individual procedure. This practice is especially valuable where SOPs are loosely bound, because it enables individuals using the procedure to assure themselves that pages are not missing.

In addition to the descriptive title an SOP should have a short statement of its specific purpose or objective. This should be presented under a separate heading at the very beginning of the SOP. A section listing cross-references to related SOPs or other literature is another format element. For example, an SOP for the operation of a piece of laboratory equipment should reference maintenance and use

of the equipment on the basis of the manufacturer's literature. An SOP for determination of animal body weights or organ weights may reference the SOP covering calibration and use of the scales to measure the weights. The appropriate use of cross-references can reduce the SOPs volume by eliminating redundancy and can assure that users are aware of all other important procedures related to the


Page 21 of 76

operation that they are performing.

SOPs should have a section listing all materials and equipment required for performance of the indicated operation. This is especially true for highly technical laboratory procedures where the unavailability of required equipment or omission of required materials may result in the improper performance of the procedure.

The exact content of SOPs will vary accordingly to the specific procedures with which they are concerned. SOPs concerning the use of laboratory equipment should describe the methods, materials and schedules to be used for the routine inspection, cleaning, maintenance, testing, calibration and/or standardization, and the way to document these operations. They should also specify remedial action to be taken in the event of failure or malfunction, and should designate who is responsible for the performance of each of the operations mentioned above. Depending upon the complexity of the equipment, the SOP should contain sufficient detail to permit trained laboratory personnel to operate the instrument. For example, the use of equipment logbooks or forms should be described completely. SOPs should provide procedures for handling emergency situations, such as the spillage of chemicals or fires.

1.5.3 Preparation of standard operating procedures

Laboratory management is primarily responsible for the development of adequate SOPs.

Scientists, laboratory technicians and quality assurance staff play a significant role in the preparation and review of SOPs. They should be written by scientists and laboratory personnel who actually perform the studies and should be approved by management. SOPs that are prepared by a single department should be reviewed centrally by someone outside that department. This will assure that newly prepared SOPs are not in conflict with existing SOPs of other departments. Central review may be performed by a quality assurance programme or by representatives from each of the departments in the laboratory (i.e. a standard operating procedures review committee).

1.5.4 Typical standard operating procedures

It is important to identify which specific routine operations require SOPs. Although specific examples of operations requiring SOPs exist, there is no single list that includes all operations that require these procedures. Different laboratories will require the

existence of different SOPs depending upon such considerations as the nature of the work, the kinds of equipment and facilities, and the qualifications and training of personnel. Laboratory management should decide which SOPs are required to assure the quality of the laboratory's work. At any rate, there should be SOPs to cover those areas and operations that are routinely involved in the conduct of a study and that involve or impact on the generation, collection or reporting of data.

To assure proper preparation and maintenance of SOPs, the laboratory should have an SOP covering the format, initial preparation, review, and approval of SOPs, changes, continuing review, update, and distribution. The SOPs should also identify who is responsible for each of the steps in the preparation process. The SOP covering changes to these procedures should identify what types of changes will be made (i.e. minor, major, etc.), who will be responsible for making changes, and the documentation required to make changes.


Page 22 of 76

In a toxicological laboratory, the following SOPs are typically needed:

* preparation, review and distribution of SOPs;

* receipt, identification, labelling, handling, sampling, usage and storage of test and reference substances;

* maintenance, cleaning and calibration of measuring apparatus and environmental control equipment;

* preparation of reagents and dosing formulations;

* record-keeping, reporting, storage and retrieval of records and reports;

* data collection;

* preparation and environmental control of areas housing the test systems;

* receipt, transfer, location, characterization, identification and care of test systems;

* handling of the test systems before, during and at the termination of the study;

* disposal of materials;

* use of pest control and cleaning agents;

* quality assurance programme operations;

* safety and emergency procedures.

1.5.5 Use and availability of standard operating procedures

SOPs are used by laboratory management, scientific and technical personnel, a quality assurance programme, and other interested parties for several different purposes. Management uses SOPs as a means of directing and instructing personnel in procedures that are in accordance with the requirements of quality assurance. Scientific and technical personnel use the SOPs directly in the performance of their duties and as a means of training new personnel in the conduct of study operations. A quality assurance programme uses SOPs both directly and indirectly: directly to carry out its operations and indirectly to monitor the operations of every other laboratory section. Part of quality assurance inspections and reviews must include a comparison of the procedures that are being used against those specified in the SOPs to determine if these are being used and are adequate. Since it is the role of a quality assurance programme to assure management that the relevant SOPs exist and are being followed, it is desirable for a quality assurance programme to maintain a current copy of all of the laboratory's SOPs so they can be available for ready reference. The SOPs may also used by parties outside the laboratory, such as inspectors from a regulatory authority, and during study audits.

Every laboratory should have a formal procedure for training employees in the use of SOPs, and there should be documentation verifying the completion of such training. Some laboratories accomplish this by making the laboratory supervisor responsible for providing new personnel with copies of the SOPs and reviewing them


Page 23 of 76

with the employee. The employee is then supervised over a period of time to ensure that the relevant SOPs are fully understood and the procedures are being followed correctly. This may be documented in the employee's training file by a record signed by the laboratory supervisor indicating the procedures in which the employee is proficient. Some laboratories also require employees to sign a statement that they have received and read the SOPs pertinent to their responsibilities. Of course, employees should be aware of all SOPs that may relate to them, not just the ones they use daily in their own limited area. For example, a laboratory animal technician collecting specimens of blood for the haematology laboratory needs to be aware of the haematology laboratory's SOPs with respect to the labelling, storage and processing of specimens.

Management should identify who is responsible for distributing new or revised SOPs and who is responsible for removing obsolete procedures from use. It should also establish a standard distribution pattern to assure that all involved personnel and departments receive copies of updated SOPs and properly remove obsolete copies. In

addition, it should address procedures to prevent unauthorized copying of SOPs. Some laboratories have approached this by the use of an official stamp or other mark on each page of the SOPs that will not photocopy. Thus, when there is a quality assurance inspection, any copies of the SOPs not bearing this mark will be recognized as unauthorized copies and can be removed from use.

Copies of the SOPs should be located in the immediate area where they are to be used, preferably at each work station. For example, SOPs covering the calibration and use of a piece of equipment should be located near the equipment, those covering procedures performed in animal rooms should be located in the animal rooms, and those covering the performance of necropsy or preparation of tissues for histopathological evaluation should be located in the laboratory performing these operations.

Copies of all the SOPs should be kept together in a manual, and, depending upon the size of the laboratory, there may be one or more such manuals. In laboratories that have SOPs divided into individual manuals by department or operation, a general index listing the various department's manuals is often useful. In order for laboratory personnel to refer easily to those SOPs that apply to the operations for which they are responsible, a table of contents for each individual manual is also useful. In smaller laboratories, where all the SOPs are contained in one manual, a single table of contents is adequate.

A complete set of all current SOPs should be kept by a quality assurance programme and by management.

1.5.6 Adequacy of standard operating procedures

The first evaluation of the adequacy of an SOP is made during preparation and review. Once an SOP has been developed and approved, its adequacy still needs to be continually evaluated, because of the changing nature of laboratory operations. The continuous evaluation of the adequacy of an SOP is the responsibility of the laboratory personnel using it and of quality assurance programme personnel. During its inspections of laboratory operations, the quality assurance personnel should examine SOPs and compare them against actual operations. If there are discrepancies, the existing SOP may be inadequate and revision warranted. Also, if there are continuing problems in particular operations, this could be an indication of an inadequate SOP. Obsolete SOPs should be kept and not discarded since


Page 24 of 76

they may be necessary to reconstruct certain aspects of completed studies.

1.5.7 Maintenance of standard operating procedures

The responsibility for maintaining currently active SOPs is shared by laboratory management, technical and scientific personnel,

and a quality assurance programme. Management is responsible for reviewing and authorizing new SOPs or significant changes to existing ones that are required to keep them current. Technical and scientific personnel who use the SOPs daily are primarily responsible for identifying those that have become obsolete. These individuals should identify required changes and seek appropriate action from their supervisors to update the SOP. Laboratory personnel, especially the first line supervisors, are also responsible for seeing that current SOPs are physically maintained in the laboratory and available for use, although a quality assurance programme is primarily responsible for verifying that SOPs in use are current, approved, adequate, and available.

The SOPs covering continuing review and update should indicate how often SOPs will be reviewed and who is responsible for review and update. There are two approaches to continuing review. Some laboratories have no programme for regular review of SOPs but simply rely on the users to initiate updating as needed. In this case, the SOP covering review and update of SOPs would not specify how often they are reviewed but should still address how the frequency of review and updating is established. Other facilities have a programme that requires a regular scheduled review of all SOPs regardless of apparent need. Either approach is acceptable provided it assures that the procedures are maintained up-to-date.

Maintenance of historical files of SOPs should be a central responsibility. This could be part of a quality assurance programme or be located in the unit responsible for maintaining data archives. Alternatively, the various departments could be responsible for their respective SOPs. The historical files of SOPs should include all revisions of the SOPs and the dates of the revisions.

If historical files of SOPs are not maintained by a central unit but are instead kept by each laboratory department, the individual within each department responsible for maintenance of the historical file should be identified. Also, because there will be a number of different individuals involved, there should be a uniform laboratory-wide index to facilitate the retrieval of historical SOPs from each department by the quality assurance programme or other personnel outside of the specific department. The location(s) of historical files should be identified in SOPs covering their maintenance.

1.6 Test, control and reference substances

1.6.1 Introduction

The term "test substance" refers to a chemical or a mixture which is under investigation for the purpose of evaluating its effects on the test system. A test substance can be any class of product, e.g., drugs, biological products, food additives and pesticides. Control

and reference substances are chemicals that are administered to the test system as a positive or negative reference for the purpose of establishing a basis for comparison with the test substance. For example, in a mutagenicity assay such as the Ames test, a known


Page 25 of 76

mutagen is used as a means of comparing the response of the test system to the test substance. The known mutagen is the reference substance. The test, control and reference substances and the test system are the major components of a nonclinical safety study. Unless the laboratory has adequate procedures, facilities and equipment for proper characterization and handling of these substances and their mixtures, a valid safety assessment of the test substance cannot be made.

There should be adequate procedures covering the characterization of test, control and reference substances, the handling and distribution of test and reference substances, and the preparation and handling of mixtures of substances with carriers such as feed. Characterization includes determining the identity, strength, purity and composition or any other characteristics that will appropriately define the test or reference substances. Stability determination is considered to be a part of the characterization process. The importance of this process in the study is paramount because it is essential to know what is being tested before a meaningful evaluation can be made of study findings.

Proper handling of test, control and reference substances is also required to preclude the possibility of contamination, deterioration, damage or misidentification during the testing process. Proper handling includes adequate study records that document the receipt and use of the test and reference substances to establish their administration, in known quantities, to the specified test system.

Laboratories should have the necessary equipment and facilities for handling test and reference substances. They should have separate areas for receipt and storage of test and reference substances, mixing of test and reference substances with carriers, storage of test and reference substance mixtures, and for carrying out experiments, in order to prevent contamination and mix-ups. Laboratories should also have necessary equipment to test, prepare, store and administer test and reference substances. Equipment must be maintained, cleaned, calibrated and inspected to assure adequate performance.

1.6.2 Test, control and reference substance characterization

The identity, strength, purity and composition or other characteristics (such as impurities) that will appropriately define the test, control or reference substance should be determined and documented prior to study initiation. Methods of synthesis, manufacture or derivation of the test and reference substances should be documented by the sponsor or the testing facility. It is necessary in any study to characterize fully discrete quantities of these

substances. To do this, specific batches and lots must be obtained and used in the test. The sponsor or the laboratory conducting the test must have information such as laboratory analytical reports and/or batch records documenting the manufacturing and testing of these materials to show that they conform to the specified standards of identity, strength and purity.

The test, control and reference substance containers must carry identifying information. This is facilitated by the use of a batch number. A batch is a specific quantity of substance that has a common origin and defined physical/chemical characteristics. For a study, the same batch of test substance should be used for the entire study, after being appropriately characterized. If more than one batch is used, each should be fully characterized. Complete documentation must be maintained. It is not a preferred practice to use different batches of the same substance without prior characterization.


Page 26 of 76

When the test, control and reference substances are received for the test, the name of the substances and the lot number must be recorded on all study records relating to any analysis of the substances. For example, chromatographs from analysis of the test substance for purity or identity should include the chemical or code number identifying the compound analysed and the specific batch or lot number.

When marketed products are used as reference substances, full characterization testing is not usually necessary because these products can be characterized by their labelling. However, the laboratory should have procedures for ensuring adequate label review and procedures for accepting shipments of reference substances. Also, label information such as lot numbers, expiration dates, and the name of the manufacturer should be documented in the study records so that the substances used in the study can be traced. Again, one batch should be used for the entire study, if possible.

The stability of each test, control or reference substance should be determined by the testing facility or by the sponsor before initiation of a study. It is necessary to know the stability of the test and reference substances to ensure that the test system is exposed to substances of adequate potency and concentration over the full course of the study.

Although laboratory management should assure that the stability has been adequately determined, the sponsor may assume this responsibility under the same conditions as specified above for characterization of the substances; namely, this assumption of responsibility should be described in the protocol and/or contractual agreement.

If the stability of the test, control and reference substances cannot be determined before initiation of a study, schedules should be

established and followed to provide for periodic re-analysis of each batch. For test substances whose stability cannot be determined prior to study, SOPs or the study plan should specify procedures and schedules for sampling and testing the substance for stability. The study plan can be amended as the results of the stability test become known.

There must be ample storage facilities for any stability samples retained. The storage facilities must provide the same environmental conditions as those facilities used to store the test and reference substances during the study. The storage conditions, i.e. temperature, humidity and other conditions as required, must be documented.

Each storage container for a test, control or reference substance must be labelled by name, chemical abstract number or code number, batch number and expiry date (if any). Where appropriate, storage conditions necessary to maintain the identity, strength, purity and composition as well as safety information, if available, of the test or reference substances must be indicated. If safety information is not available, this should also be noted on the label. The laboratory should have procedures for labelling the test substances. These SOPs should require that this information be clearly indicated on the label. They should also address who will be responsible for labelling the containers and/or verifying the label of any containers received by the laboratory. The SOPs should also address how labels will be applied. It is generally unacceptable to place the only identifying label on the lid of the container. This practice may lead to


Page 27 of 76

confusion between containers when lids are removed during use. SOPs regarding labelling may include procedures for assuring that labels will remain affixed and legible during conditions of storage and use. For example, test substance containers may be exposed to moisture or solvents that could alter the labelling.

Storage containers should be assigned to a particular test substance for the duration of the study. This is necessary to preclude the possibility of cross-contamination, which may occur when the same container is used subsequently to store a different test substance. It is good practice to dispose of empty test substance containers after a study has been completed rather than attempt to decontaminate and reuse them.

Retention of samples of test, control and reference substances is useful should a question arise regarding the quality or identity of the test substances actually administered to the animals.

1.6.3 Handling

A laboratory should have adequate procedures for a system of handling the test, control and reference substances to ensure that there is proper storage, that distribution is made in a manner

designed to preclude the possibility of contamination, deterioration or damage, that proper identification is maintained throughout the distribution process, and that receipt and distribution of each batch is documented. These procedures should include a description of the records to be maintained in order to document that procedures are being followed.

1.6.4 Storage

Handling procedures should include proper storage. Storage requirements vary with the specific test, control and reference substances used. For example, some test substances may require storage at temperatures below 0 °C, while others are stable at room temperature. Some may require storage in the dark or in special containers and cabinets due to high volatility. In all cases, the laboratory must have procedures that will require each test or reference substance to be evaluated upon receipt for special storage and handling conditions, and to assure that these conditions are met. For example, there may be a standard form that is filled out upon receipt of a substance that requires the recipient to record any special storage conditions required, to ensure that these conditions are recorded on the label, and that the substances are stored accordingly. Laboratory procedures for receipt of the test and reference substances should also require the person receiving them to determine, if possible, the storage conditions of the substances during shipment and at the time of receipt. For example, if the substances are to remain frozen until used, they should be inspected upon receipt for thawing and this should be documented in the study records. The storage requirements for the test, control and reference substances should generally be provided by the sponsor or manufacturer responsible for initially providing the substances.

Procedures for ensuring proper storage need, at a minimum, for required storage conditions to be specified in the labelling and study records, necessary facilities to be identified and available to meet required storage conditions, storage conditions during transport and use to be monitored and documented, and deviations from the required conditions to be investigated and documented. Where deviations occur, the study director should determine their impact on the study and record in the final report any that may seriously affect the study.


Page 28 of 76

1.6.5 Distribution

Procedures covering test, control and reference substance handling should ensure that distribution is made in a manner designed to preclude the possibility of contamination, deterioration or damage. Such procedures might include the types of containers used to transport articles to the laboratory areas where they are to be used and the designation of specific storage areas. Laboratories dealing with large numbers of test substances should have SOPs identifying where the substances are to be stored. Procedures should also

identify where and how containers of test and reference substances are to be opened for dispensing. This usually takes place in an area specially designed for this purpose with an isolated environment. Many laboratories have a specially designated room or laboratory bench top under a laminar air flow to prevent the possibility of cross-contamination of equipment and facilities, as well as to protect employees from potentially hazardous test substances. Special gowning and decontamination procedures are generally required for the test substance dispensing areas and are included in the SOPs for test and reference substance handling. Adherence to these procedures must be documented. Such documentation will include records of cleaning the areas between use for different compounds.

A systematic process for receipt and distribution of test, control and reference substances should be fully described in laboratory SOPs for test, control and reference substance handling. Ideally, there should be a centralized procedure responsible for receiving and logging in test, control and reference substances. This should ensure that proper identification is made initially and then maintained throughout the distribution process.

Documentation should specifically include the date and quantity of each batch distributed or returned. An inventory record is often used to record the receipt of all test and reference substances. Entries include the name or identifying number of the substance received, the batch or lot number, the date of receipt, the amount received, and the signature of the individual who received the substance and completed the record. Many laboratories record the weight of the container and its contents as received. When a portion of the test substance is subsequently dispensed, the amount is weighed and recorded in the inventory record. The record should also reflect to whom and for what purpose the substance was dispensed. As an additional quality assurance measure, many laboratories not only weigh out the amount to be dispensed, but also weigh and record the remaining substance in the bulk container. This provides a continual inventory of the test substance and allows detection of any dispensing or record-keeping errors that may indicate a potential misdosing problem.

It is clear that there should be procedures covering every aspect of the handling of test, control and reference substances to ensure proper storage, distribution, identification and accountability. Records should be maintained to document that these procedures were actually followed. All study records pertaining to use, testing and distribution of the test or reference substances should be directly traceable to the specific lot or batch of materials issued, and accountability records should document that the quantities and lot number used were consistent with study plan requirements. Periodic checks should be made that the amount of test substance in the inventory matches with the amount used in the study.

1.6.6 Mixtures of substances with vehicles (Carriers)


Page 29 of 76

A vehicle (or carrier) is the material with which the test, control or reference substance may be mixed for administration in the test system. It can be feed, water, solvents and/or excipients, depending on the form of dosage and route of administration. A common example of a carrier is the rodent feed used to mix a test substance for a chronic feeding study. However, the phrase "mixture of substance and vehicle" also refers to solutions and suspensions, e.g., a solution of test substance in distilled water.

A laboratory should have procedures for the preparation, analysis, storage, labelling, distribution and return or disposal of mixtures of substances with vehicles. The procedures should accomplish the same goals of maintaining proper identity and ensuring proper storage and use as those procedures already discussed for the test and reference substances alone. In addition to the concerns already mentioned, mixtures of substances with vehicles present special problems. For example, there must be procedures to assure that the mixtures used are uniformly mixed, stable, and provide the proper concentration of the test or reference substance. To accomplish this for each test, control or reference substance mixture, appropriate analytical methods should be used to determine the uniformity of the mixture and to determine, periodically, the concentration of the test or reference substance in the mixture. The standard of uniformity should be determined before the start of the study.

Each time a batch of substance carrier mixture is made, it should be recorded that the batch was properly formulated and mixed according to procedure. For liquid preparations, uniformity determinations need not be made on true solutions, but should be made for suspensions. In laboratories that perform numerous short-term assays (e.g. mutagenicity studies) by using liquid dosage forms, it may not be practical to analyse dosing solutions from every assay. In this case, it is imperative for the laboratory to have well-documented dilution and dosing procedures and SOPs for determining and documenting solubility of the test and reference substances.

1.6.7 Stability

In addition to analyses for uniformity and concentration, tests should be conducted to determine the stability of the test, control and reference substances in the substance carrier mixture. If the stability cannot be determined before the study is initiated, the study plan should provide for periodic re-analysis of the test and reference substances in mixtures. The laboratory must determine the stability of the mixtures over their periods of use. This would include the period between the day the batch is prepared and the day the last portion of the batch is used. Stability should be determined under actual conditions of storage and use. Once the stability of a

given concentration of a test substance carrier mixture is substantiated for one batch, no further stability testing is necessary for each subsequent batch of that concentration. However, periodic re-analysis to determine concentration must be carried out, as discussed previously.

A sound practice related to stability testing is the use of expiry dates. Where any of the components for the test or reference substance carrier mixture has an expiry date, that date should be clearly shown on the container. If more than one component has an expiry date, the earliest date should be shown. This requirement is necessary to assure that outdated or unstable mixtures are not used.


Page 30 of 76

1.6.8 Labelling

The importance of proper labelling cannot be overemphasized. This is especially true for the labelling of test, control or reference substances, as well as for their mixtures. Misapplied or inadequate labelling can lead to major problems that can invalidate study results. For this reason, every laboratory should have SOPs for labelling test, control or reference substances and their mixtures with carriers. These SOPs should address how labelling will be applied and what its content will be.

Labelling for a mixture container should include the expiry date of the mixture; the use of expiry dates is intended to preclude the use of mixtures that may have deteriorated. Procedures should exist to assure that outdated or deteriorated batches of test and reference substance mixtures are not used.

1.6.9 Facilities and equipment

There should be a separate area for the receipt and storage of test, control and reference substances. Laboratories may have a centrally located facility for this purpose. These areas are usually supplied with storage cabinets, freezers, refrigerators, balances and related equipment required for the orderly receipt, storage and distribution of the substances received. These areas are sometimes equipped with special environmental equipment, such as laminar air flow and biohazardous hoods, depending on the extent to which these areas may be used for opening and dispensing test and reference substances for use in the mixing areas. If containers of test and reference substances are opened in the receiving/storage area, SOPs should prescribe the necessary precautions to be taken to avoid contamination of the area and equipment with the various substances.

Employee safety precautions in handling chemicals are essential. These should include decontamination procedures for spills and other emergencies, as well as procedures for routine cleaning. The nature and extent of the facilities and their use will dictate what procedures and equipment will be required. The area should be

physically secured and only authorized personnel permitted to store or remove materials. In smaller laboratories, there may not be room for a separate facility for this purpose. In this case, test and reference substances may be stored in areas used for other purposes; however, the same general requirements apply as stated above. In particular the area, such as a cabinet, should be isolated as much as possible from other areas and activities and should be made physically secure with authorized access only. It should not be used for any other purpose. Limited access to test substances is required to prevent unauthorized use and mix-ups. Accountability records should be maintained, documenting receipt and dispersal of test and reference substances. Such documentation for all substances is often maintained in the receipt and storage area. The entry or removal of substances from the designated areas should be documented.

In addition to separate areas for receipt and storage of test, control and reference substances, the laboratory should provide an isolated area for preparing and mixing these substances with carriers. The extent and nature of these facilities will depend upon the scope and type of the laboratory. Larger laboratories may have elaborate equipment, facilities and procedures for mixing test, control and reference substances with carriers. In a typical laboratory, engaged routinely in conducting safety studies, there will usually be a separate area or areas where test substances are stored and weighed. The pure substances are then transferred to the separate mixing and


Page 31 of 76

dosage preparation areas.

Another major consideration in the design of a mixing area is cleaning and decontamination. The area should be easily cleanable. The walls, ceiling, and floor should be smooth and impervious to cleaning agents used. Cleaning and decontamination design considerations also apply to personnel who work in the area.

There must be specific SOPs to cover the cleaning and/or decontamination of equipment. This is especially important for equipment, such as mixers, that may come in direct contact with test substances. In specifying cleaning agents, the SOP should take into account the solubility of materials to be removed from the equipment and also the removal of any cleaning agent residues. In studies where the test material may present unusual equipment cleaning problems, the procedures for cleaning may be included in the study plan. It is also very important for the cleaning of important equipment to be monitored and documented.

Once the test, control or reference substances have been mixed or otherwise prepared, they should be moved to a separate area designated for the storage of these mixtures.

SOPs should also deal with the disposal and/or reuse of containers used to store test and reference substances and their mixtures. In laboratories where containers are reused to store these

substances, there should be strict references and documentation covering decontamination and labelling. Procedures for final disposal of hazardous waste should be defined by national or local governmental regulations. Laboratories should ensure that they conform to these procedures.

1.7 Quality control

1.7.1 Introduction

Quality control is applied to routine laboratory biological, chemical and physical analyses in order to assure reliability and comparability of test data. It involves statistical approaches designed to demonstrate the constancy or variability, and precision of analytical data.

When the concentration of a chemical is determined repeatedly in the same laboratory a certain scatter is always seen. The scatter is due to variation in the analytical step as well as to changes that may take place during the collection, preparation and storage of the samples.

Quality control depends, among other factors, on the type of compounds involved. A major issue is "to keep the compound as it is" and it is reasonable to make a distinction between inorganic and organic compounds. Since over ten million organic compounds exist, a further distinction must be made between simple organic compounds, such as solvents and metabolites, more complex compounds that may have a low vapour pressure, such as some pesticides, compounds that are capable of being adsorbed, and very complex biological compounds such as proteins and enzymes.

In the case of inorganic compounds, contamination of the test substance with the same compound from an external source is a major concern. Some elements are volatile, e.g., elemental mercury, and special care is needed to avoid loss of analyte. Certain elements are readily reduced to a different oxidation state even within the


Page 32 of 76

container used. Examples are the reduction from Cr(VI) to Cr(III) and the reduction from Hg(II) via Hg(I) to elemental mercury, which disappears from the test material. There may be effects due to compounds already present in the test substance, to compounds added for preservation or to wall effects of the container. The latter category can be separated into chemical effects, e.g., reduction and oxidation, and physical effects, e.g., adsorption to the wall or stopper, and precipitation/ coprecipitation of the analyte alone or together with other compounds.

In the case of simple organic compounds, a well-sealed container is needed to prevent vaporization loss. The container or stopper can be responsible for adsorption or leakage. Reduction as well as oxidation of metabolites in biological material may occur.

Ultraviolet light, as occurs in sunlight, may decompose organic compounds by photochemical reactions, and the infrared radiation in sunlight may cause thermal degradation of organic compounds. Thermal degradation may also occur due to other infrared sources such as heat radiators or laboratory apparatus. Another factor which may lead to thermal degradation is the temperature of the environment of the test substance from sampling until final analysis within the apparatus.

In the case of more complex compounds with a low vapour pressure, such as some pesticides, vaporization of the analyte is unlikely. Certain pesticides may undergo reduction or oxidation. Photochemical and thermal degradation may occur in this class of compounds.

Proteins and enzymes are extremely vulnerable to changes in pH and redox potential, heat and exposure to ultraviolet light. Moreover, as many proteins can bind easily to heavy metals, they need special care.

Another important item is the concentration of the compound of interest. Concentrations of compounds in biological materials range in general from parts per million, e.g., essential elements in plant and animal, to parts per trillion for very toxic compounds such as dioxins. This enormous range of concentrations is also seen in the soil, surface water, indoor air, and, to a lesser extent, in sea water, drinking-water and outdoor air. As precision is often inversely related to concentration, it is evident that the determination of sub-ppm concentrations of compounds is always problematic, regardless of the method of choice.

Analytical variation may be divided into two major categories: accuracy and precision. Accuracy refers to the agreement between the measure and the true amounts of the analyte, and precision refers to the random variability or reproducibility of the method.

In order to assure the reliability and comparability of the test data, an extensive quality assurance programme should be implemented. This should cover the sampling and sample handling (preanalytical quality control), as well as the analytical procedures (analytical quality control). The purpose of quality assurance is to identify different types of errors that may invalidate the test data and to make sure that the total of all errors is below certain established limits.

1.7.2 Level of quality control

A basic question in quality control is how accurate and precise the different measurements need to be in order to provide reliable exposure assessments. This decision may be based on prior information on the inherent variability in the quantity being measured, and this


Page 33 of 76

is likely to differ for each pollutant and medium. Existing information from measurements already conducted in the locality in

question, or in similar areas or groups of people, can be used to provide a guide. The variability can then be assessed as the relative standard deviation, expressing the standard deviation as a percentage of the mean, assuming that a normal distribution basis is adequate for the present purpose.

An important issue in biological test substances is the biological variability, which can be separated into the intra-individual variability, i.e. the variability within an individual, and the inter-individual variability, i.e. the variability

between individuals. The variability may be expressed as a relative standard deviation. The allowed standard deviation for a determination used in clinical and environmental chemistry is half the standard deviation of the inter-individual variability at a maximum, or, alternatively half of the sampling error (Youden, 1967).

As another example of how to deal with precision, the procedure in the UNEP/WHO (1984) and WHO (1986b) studies may be used. Within these studies a relative standard deviation of 20% was set arbitrarily.

The error in precision within a laboratory is thought to be of the order of one-half to two-thirds that of the inter-laboratory error.

1.7.3 Pre-analytical quality control

It is essential to ensure correct sampling, i.e. that the collected air, particles, water, food or other samples really represent the whole sampling period and the subjects concerned, and that contamination of the samples is avoided. It is important for the field personnel and the laboratory staff to be properly trained. Detailed guidelines for the sampling procedures should be prepared prior to the sample collection and discussed with all the people concerned.

In the case of many pollutants, it is necessary to check their content in containers and other equipment used for sampling and storage before sampling in order to avoid contamination.

Audit procedures are useful to control the reliability of the sample collection, transport and storage. The major steps to be audited during sampling would be:

* (preventative) maintenance

* calibration of sampling and analytical measurement equipment

* procedural control checks

* tick-off chart

* cleanliness during sampling, sample transport and storage

* sample deterioration, temperature control and stabilizers

* time lapse before analysis

* data recording, calculations and record keeping

Sampling should be representative, and the number of samples


Page 34 of 76

should be adequate. Sampling can be random or selective, depending on the goal of the study. If sampling should be random, all subsequent steps should also be carried out in a randomized manner. For example, if an "exposed" population of animals or humans is compared with a reference group, sampling should be performed randomly. Also the pre-analytical steps, such as the distribution of the samples in the containers and the time lapse before determination, should be performed carefully to avoid any systematic influence.

Although sampling errors are often the greatest source of variability, especially in environmental measurements, these errors are mostly beyond the control of the laboratory, unless the laboratory is also responsible for the sampling operation (Horwitz, 1989). Regardless of who collects the samples, this source of variability must be considered individually for each lot or population to be examined. The magnitude of this source of variability is assessed by taking a number of random samples from the population and examining them individually (see also section 1.7.5).

In occupational toxicology and sometimes in environmental health, the timing of specimen collection is important. Some industrial chemicals have a long biological half-life in various body compartments, and the time of sampling is not critical (Herber & Schaller, 1986). For other chemicals the timing of sampling is critical because after exposure the compound and/or metabolites may be rapidly eliminated from the organism.

In the case of urine samples, 24-h or 6-h urine samples are preferable to spot samples. Preservation against bacterial growth and fungi can be achieved by adding a solution of sodium azide. Determination of enzymes should generally be performed within 24 h. However, some stable enzymes may be determined after a longer time interval, but preferably within a week when stored at 4 °C.

In the case of blood samples, clotting is a major source of errors and this should be avoided at all costs. Clotted blood samples cannot be used for analysis and must be rejected. Many urine samples have a precipitate but generally this can be redissolved by acids without influencing the determination, by carefully heating up to 37 °C, or by ultrasonic treatment. Another possibility is to remove the precipitate by adequate centrifuging. When a precipitate forms during cooling, care must be taken that the samples are stirred so that any

deposits are dissolved or evenly distributed. All pre-analytical procedures must be checked comprehensively before starting routine determinations.

Storage of samples is optimal at -80 °C. At this temperature samples may be stored for years. A more practical temperature is -20 °C. At this temperature proteins can be stored for months. Inorganic compounds maybe stored at +4 °C, provided that no vaporization takes place.

1.7.4 Analytical quality control

Validation of analytical procedures with respect to accuracy and sensitivity should be accomplished by appropriate quality control studies. The accuracy of the methods used by the different laboratories should preferably be established by an external quality control programme, in which the reference values of the quality control samples are unknown to the laboratories. If this is not possible, comparisons with reference methods or the analysis of certified reference samples may be used. It is also important to establish the reproducibility of the routine analytical procedures


Page 35 of 76

used. Acceptable limits of variation for control samples should be set primarily by considering the data quality requirement rather than the analytical characteristics of the procedure. The stringency of the limits depends largely on the purpose for which the test data are intended.

In case of random sampling, the complete analytical procedures should be randomized. Thus, destructions or extractions must be performed for an approximately equal number of "exposed" and "control" test specimens within the same run. This applies to instrumental techniques including spectrophotometry, chromatography and atomic absorption spectrometry. Calibration and reference specimens must be distributed randomly between the test specimens.

A major item is the influence of the matrix and, especially in urine, the matrix variability; an example is the variable salt concentration in urine which may cause problems in the determination of metals.

1.7.5 Statistical considerations

According to Taylor (1988a), it is essential to define the degree of uncertainty of the data. This is necessary in order to provide evidence that samples are "representative" for the environment or population to be sampled. Two factors influence the uncertainty of the data, i.e. measurement uncertainty and sample uncertainty. The uncertainty of the measurements can be estimated by replicate measurements on a sufficient number of samples. Taylor proposed that the measurement uncertainty should not exceed one-third of the total uncertainty tolerance.

The sample uncertainty is based on the variability of the population or environment to be sampled. Sampling strategies based on statistical considerations address this factor. During sampling a number of items can represent components of the sampling uncertainty. These are, for instance, systematic components like the properties of sampling equipment and the variability within the actual sampling process. All these components add up to the total sampling uncertainty. It is practical to define acceptable total uncertainty, and then estimate the different components contributing to this uncertainty.

Smith et al. (1988) stated that quality assurance procedures of sampling data require the definition of the precision, the bias, the representativeness, the completeness and the comparability. Precision in this case is the agreement of individual measurements of the same property under the same conditions. It is best expressed in terms of standard deviations. In environmental sampling, it means that the total variance of the measurements is the result of the precision of the analytical measurements and the precision in sampling. The analytical precision is obtained by determining the standard deviation of individual samples, whereas the precision of the sampling as such is determined by analysing a number of replicated field samples. The presentation of results from environmental measurements should include regression equation coefficients, when appropriate, means and standard deviations, and ideally a graph containing the actual data points, the best-fit curve and confidence intervals, in order to provide information for a quality assessment.

Bias is the degree of agreement of a measurement or an average of measurements having an accepted reference or true value. It is usually expressed as the difference between these two values or as a percentage of the true or reference value. Bias is frequently expressed as recovery (100% bias) in environmental monitoring


Page 36 of 76

programmes, and can be estimated by using spiked field samples for the bias in the field sampling phase, and by the repeated analysis of field samples or reference material for the analytical phase.

Representativeness is the degree to which data represent precisely and accurately a characteristic of a population or environmental condition. This can be assured by the use of appropriate sampling techniques.

Completeness is a measure of the amount of valid data obtained from a measurement system compared to the amount that was expected to be obtained to fulfil the objectives. Various conditions, e.g., meteorological conditions, accidents, during sampling and analysis could result in the incompleteness of data, particularly during field sampling. In any case there should be an evaluation of the influence of missing or lost data on the overall result of a study.

Finally, comparability expresses the confidence with which one data set can be compared to another. For this purpose, there should be descriptions of the comparabilities of sites elected for sampling, the calculation and statistical analysis, and the sampling and analysis protocol.

1.7.6 Analytical performance evaluation

There are various ways of performing analytical quality evaluation. Since it is impossible to produce errorless analytical data or to obtain a measure of the accuracy, it is important to estimate the limits of uncertainty of produced data.

Some general statistical approaches to evaluating the quality of chemical measurements have recently been reviewed (Taylor, 1987, 1988b). A well-established procedure for comparing the analytical performance of several laboratories is the round-robin test using a Youden plot (Youden & Steiner, 1975). Two similar samples, designated X and Y, are sent to each collaborating laboratory for analysis. The results provide a pair of coordinates which are plotted in a diagram. If random errors predominate, the results will fall within a circle with the center showing the "true" value. Due to systematic errors most of the observations are usually found along a 45° axis.

Evaluation of the regression line of reported versus reference values for a set of quality control samples is a useful method of guarding against systematic errors in the likely concentration range (Vahter, 1982; UNEP/WHO, 1984; Friberg, 1988). Further discussion of this method is given in section 3.7.

1.8 Documentation and record keeping

1.8.1 Introduction

Any study report must be capable of being validated before it can be fully relied upon for accuracy and completeness of findings and before any scientific conclusions can be derived from it. This means that the information and conclusions stated in the report must be fully supported by raw data documented in the laboratory records covering the conduct of the study. It requires the existence of a complete data trail from the initiation of the study to the time when the last data point is recorded. The data trail should be detailed enough to allow an independent party to trace every aspect of the study. Validation of a study by an independent party is sometimes necessary to assure that all the provisions of the study plan and SOPs were followed.


Page 37 of 76

"Raw data" covers all of the original observations of the study. For example, the term "raw data" may mean any laboratory worksheets, records, memoranda or notes (or exact copies of these) that are the result of original observations and activities of a study and that are

necessary for the reconstruction and evaluation of the final report. Exact copies of raw data may be valid, provided that they are verified as accurate by the dated signature of the person making the copies. Other examples of raw data include photographs, microfilm or microfiche copies, computer printouts, magnetic media, including dictated observations, and recorded data from automated instruments.

There are three common processes for capturing data. These processes include manual recording of data, direct computer entry of data, and entry of written data into a computer.

1.8.2 Manual data records

Many testing facilities find it useful to have standard data collection forms for recording items such as health status information, body weights, body length, clinical observations, test system care, necropsy, clinical chemistry, haematology, histological processing, and environmental condition. These forms should be designed with care and should be readily available to personnel conducting study functions. Procedures for proper use of data collection forms should be described in SOPs or the study plan. When these forms are used during the course of the study, they should be identified by study number, when applicable, test substance and species, and they should carry entry points not only for the collected data but also for dates and initials of the person or persons entering the data.

All data should be recorded in indelible ink, preferably black, and corrections in the data should be made so as not to obscure the original entry. The use of white-out, correction tape, erasers, overwriting or any other means to obscure the original entry is not acceptable. Changes made in records of original observations should ordinarily be made by the individual responsible for the original entry. All corrections should be initialled and dated at the time the correction is made and a justification should be given in the record. The record should be corrected in a timely manner. Some laboratories use codes or abbreviations to indicate common reasons for corrections. Such codes should be defined in the record or the associated SOP and should be used consistently by all employees making corrections.

Standard data collection forms should be designed so that they are unambiguous and so that they require a notation to indicate whether an activity has been performed or an observation has been made. Leaving data entry spaces blank is an unacceptable means of documenting an observation. For example, a standard form to document the collection of tissue at necropsy may list all possible tissues and provide a space to document the collection of specific tissues. If a technician leaves a blank space to record that no tissue was taken, an individual reviewing the record can never be sure if the tissue was, indeed, not taken or the technician merely forgot to fill in the space when the tissue was collected. Similarly, a line drawn through all

data entry spaces on a record can be ambiguous, unless its meaning is clearly defined in the record or SOP and is understood by all individuals completing the record.

A study notebook in which narrative notes may be kept of significant events, such as animal deaths, changes in batches of test substances, clinical examinations, disease outbreaks and animal


Page 38 of 76

sacrifices, supplements the standard data collection forms. Actual data elements required by the study plan for the test system can be recorded in the study notebook.

1.8.3 Computer data records

Although there is still a need for written records to document important daily events, computers may now be used to save time and labour in data collection, validation and report generation. Direct entry of data may be accomplished by use of keyboards, optical bar code readers, touch screens, optical scan sheets, and direct digital input from laboratory instruments or sensors. In order to capture and correlate effectively diverse data from multiple sources, most laboratories collecting data in this manner use a distributed data processing system, where remote terminals and microcomputers collect data, which are later transmitted to the central computer for permanent storage and/or processing.

In systems such as this, data should be verified at the time of collection prior to transfer to the central computer. This can be done by review of the terminal screen at the time of data entry and/or review of data print-outs. As with data collected manually, computer-entered data must include the identity of the individual making the observation and the date the observation was made. Any correction made later at the local level require that the change be verified in the central computer to check that it has been recorded correctly. Experience has revealed cases where legitimate corrections were made at a local level but not submitted to the central computer or included in the final report. The same principles apply for correcting computer-collected data as for paper data entries, i.e. the original entry must not be lost, and the change must be documented to show who made the correction, and when and why the correction was made.

A computer system must be designed to meet the requirements of quality assurance. It must be tested prior to initial use and whenever significant changes are made to the computer hardware or software. The testing process must include a documented review of the system design, development, and acceptance measurements to assure proper system performance. This should be done prior to testing by laboratory personnel. Testing often involves the checking of a previously validated array of hand-collected data against the same data collected concurrently and reported by computer for one or more

studies. For each study, the acceptance testing needs to cover each of the data points the system is designed to collect and process.

For the purposes of retaining raw data, the magnetic media containing the raw data (i.e. disc, tape) and/or a verified printout should be kept. However, manufacturers of magnetic media advise that data retention is not guaranteed beyond a limited time, and, if discs are erased, revised periodically or reused, some errors in the discs can arise. Thus, a paper print-out of the data for data review and storage as original data is essential. However, any print-out retained as original raw data must be verified. This means it must be reviewed for accuracy and this review must be documented by the signature and date of the individual(s) performing it. Proper verification would include review by the supervisor and/or study director and quality assurance personnel.

Any computerized data collection system must include SOPs to describe operations, specification, security, validation and maintenance of both the local data acquisition systems and the central computer system. These documents should be in the appropriate work


Page 39 of 76

stations and should contain such information as how the data are to be entered and what remedial action is to be taken in the event of system failure.

1.8.4 Indirect computer data records

The principles outlined above also apply to manual data that are subsequently entered into a computer. In addition, evidence must exist that the manual data were correctly and completely entered. Frequently, this is a quality assurance programme function. The extent to which the "raw data" are compared with those of the initial paper print-out (or screen display) needs to be defined in SOPs.

1.9 Final report

1.9.1 Introduction

The final report is the end-product of a carefully planned and conducted study. The report must be well organized and reflect accurately all the experimental data. It must contain a detailed account of the study including, where relevant, unexpected deviations in the controls, study plan and environmental conditions.

1.9.2 Contents

The final report should include the following information:

a) a descriptive title;

b) clearly defined objective(s) and study plan;

c) an informative summary of the results of the study;

d) an identification of the test and reference substances by chemical name, code or chemical abstracts number;

e) a description of the characterization of the test and reference substance including purity, stability and homogeneity;

f) name and address of the test facility, and the name and address of any facility that may have performed parts of the study;

g) the name of the study director and any other principal scientists who contributed reports included in the final report;

h) the dates on which the study was initiated and completed;

i) a description of the methods, procedures and materials used, highlighting any changes in the study plan;

j) all information and data called for in the study plan, including "outliers";

k) a description of the test system, and the procedure used for identification;

l) exposure conditions;

m) an accurate presentation of results, including calculations, and a description of the statistical methods used to analyse the data;


Page 40 of 76

n) an evaluation and discussion of test results and any conclusions drawn from the results;

o) the dated signature of the study director.

1.9.3 Indexing

An index is essential for each volume of data comprising the final report. Appropriate references to appended tables and figures are necessary to facilitate the peer review of the validity of the conclusions drawn from the study. Appendices containing graphs, tables and statistical evaluations of all the data and observations are as important as the narrative report. A glossary of terms and abbreviations used in the report should also be included, since this will facilitate an understanding of the findings of the report.

1.10 Archiving and retention of data

1.10.1 Introduction

Records, specimens and reports constitute lasting proof of the validity of a study; facilities and procedures for their archiving should be available. The following basic points should be observed:

* all raw data, documentation, study plans, appropriate specimens and final reports generated as a result of a study should be retained;

* there should be archives for the orderly storage and retrieval of all raw data, documentation (e.g., training and qualification records), equipment maintenance, master schedule of studies and quality assurance reports, study plans, specimens, and interim and final reports;

* material retained or referred to in the archives should be indexed by test substance, date of study, test system and nature of study;

* an individual should be responsible for the archives;

* only authorized personnel should be permitted to enter the archives.

1.10.2 Facilities

Storage facilities required for archiving data and specimens must be adequate to preserve specimen quality and to control access. The storage conditions in the archives should be monitored to prevent accelerated deterioration of data and/or specimens. Archives should be designed to have a controlled access area, i.e. with access limited by doors that lock, and the area should be isolated from operational laboratory areas but still conveniently accessible. Environmental conditions should avoid extremes of temperature and humidity. For example, tissue blocks should be kept cool and dry enough to prevent melting, sticking or mould growth. Paper records should be protected from fire, water, rodents, etc. Environmental conditions in the archives should be monitored to assure that appropriate conditions are maintained. Finally, proper storage cabinets are needed to organize and store data and specimens for easy retrieval.

1.10.3 Responsibilities for an archive

Normally a testing facility has the responsibility for providing


Page 41 of 76

an archive facility and authorized archivist. However, when there is a sponsor/contractor relationship involved, there can be problems over the allocation of responsibility for archiving material between the laboratory and the sponsor. During the course of the study, the

original data remains with the test facility conducting the study. When the study has been completed, there are several ways that data may be archived, depending on the contract between the sponsor and the test facility. Firstly, the test facility may archive the data. Secondly, it may transfer all archival material (e.g., data, slides, tissues) to the sponsor for archiving. In this case, there should be a record of transfer, and documentation identifying the storage location should be maintained in the contract facility's archive. The third option is for the test facility to send the originals to the sponsor and maintain copies in its archive.

1.10.4 Standard operating procedures for archiving

There must be SOPs for each activity performed in operating an archive. Some of the relevant SOPs are:

* receiving, indexing and identification

* filing and storage

* access to archives

* security

* data retrieval

* retention of material

* removal and return of records and raw data (as appropriate)

1.10.5 Receiving, indexing and identification

Generally, the central archive of a test facility is utilized for the paper and computer data from a study. This includes all raw data in notebooks, forms and/or computer print-outs, approved study plans, amendments, and final reports generated. These materials should be received and accepted by an authorized person (the designated archivist or his replacement).

Prior to accepting the data, the archivist should be given an index, signed by the study director, of the submitted data listing the items being archived. When the items have been checked against the index and the archivist is satisfied that the package is complete, the index should be signed and dated by the archivist. After acceptance, the total study package should be archived using a classification system (e.g., a unique number).

1.10.6 Filing and storage

Material files should be filed and cross-referenced for easy access and retrieval of data. Index cards are often used for cross-referencing, but data are increasingly being indexed on computers.

Archived material should be paginated and indexed by test substance, date of study, test system and nature of study. Other titles used for cross-referencing are unique study numbers, the study title and the name of the study director.


Page 42 of 76

In the case of specimens, slides, blocks, tissues, test substances and other materials that are not in the central archive with the raw data and final report, there should be a method for indicating where these articles are archived. One method is the use of a form to be filled out by the person maintaining the items. For instance, if the teratologist maintains an archive for fetal specimens and the pathologist maintains a separate archive for wet tissue, blocks and slides, they should complete and sign a form certifying the location and storage of the specimens. This information would be maintained in the central archive with the other paperwork generated as a result of the study.

1.10.7 Access and security

There should be control of access to any archive where data, specimens and/or slides from studies are stored. The door to the archive should be locked at all times when it is unattended. Only the archivist should have keys for access to the archive records. Ideally, the archive should be designed with a separate area for people to wait while the archivist is retrieving the requested data in order to help maintain the limited access environment.

1.10.8 Retrieval of data and control of access

Although stored data and specimens should be easily identified and retrievable, their removal from the archive should be discouraged.

For the retrieval of data specimens, there must be a standard operating procedure. The approval required may vary, but in many testing facilities, the authority to release data is given to the archivist. For records management and to maintain a tracking system, a "Data and Specimen Request Form" is useful because it becomes a permanent record for the study file and helps make the person requesting data aware of the need to ensure the security and integrity of the items removed from the archive.

The title of the study, name of the borrower, purpose, organization and items requested are entered on the form. When the request is completed, the archivist will sign and date the form and

the borrower will sign, indicating that the items were received and that the security and integrity of the data will be ensured. This form remains in the archive.

When the borrower returns the data, the archivist must check the items returned against those listed on the request form. If all items are present and the archivist is satisfied that the integrity of the study has not been compromised, the form is signed by both the borrower and archivist and becomes a permanent record for the study file.

1.10.9 Retention of information

The length of time that study records must be retained will vary, this depending mainly on legal considerations. It is the responsibility of test facility management and/or the study sponsor to ensure that records, specimens and reports are retained for the required time.

For archived material, such as wet specimens, plates for mutagenicity testing, test and reference substances, haematology slides and histochemical and other specimens that are relatively fragile and that vary in stability and quality during storage, the retention period can be only as long as the period during which the


Page 43 of 76

material is of a quality that affords a valid and meaningful evaluation. Management should develop an SOP establishing the retention time for these types of specimens.

2. QUALITY MANAGEMENT APPLIED TO TOXICITY STUDIES

2.1 Introduction

Laboratory animals are the test system for many studies. In order to provide meaningful safety data, the animals must be properly selected and cared for to ensure that any response observed in the animals is caused by controlled exposure to the test, control or reference substances and not by uncontrolled variables such as disease and adverse environmental conditions. Appropriate laboratory practice needed to assure proper care and use of animals includes providing proper facilities and employing necessary procedures to eliminate or control factors that could interfere with the response of animals to the test and reference substances. The care, housing and treatment of animals used in research and testing is regulated by law in many countries.

Animal care facilities can comprise a large portion of the physical plant of a laboratory because sufficient animal rooms or areas are needed to ensure separation of species or test systems, and provide for quarantine requirements and routine or specialized housing of animals.

2.2 Procedural requirements

* There should be SOPs for housing, environmental conditions, feeding, handling and care of animals.

* Animals recently received from outside sources should be appropriately isolated and their health status should be evaluated before they are used in a study.

* At the beginning of a study, animals should be free of diseases that might interfere with the purpose or conduct of the study.

* Animals that become diseased while under study should be appropriately isolated to prevent infection of other animals.

* Such animals may be treated if treatment is authorized and documented, and if the treatment will not interfere with the interpretation of the test.

* Animals should be identified individually as appropriate (e.g., tattoo, ear tag) to assure that they can be identified with their respective dose regimens and the in-life and postmortem observations. Their individual housing units (e.g., cages) should be labelled with all the information needed to identify specifically each animal within the unit.

* Animals of different species and/or projects should be appropriately separated to preclude inadvertent exposure to test or reference substances, disease transmission or other uncontrolled stress to the animals.

* Animal cages, racks and accessory equipment should be cleaned at appropriate intervals.

* Food and water used for animals should be analysed periodically for potential interfering contaminants, as specified in the study plan.


Page 44 of 76

* Bedding used in animal cages and pens should not interfere with the purpose or conduct of the study, and should be changed as needed to keep animals clean and dry.

* The use of any pest control materials should be approved by the veterinarian/study director and be documented. Cleaning and pest control materials that interfere with the study should not be used.

2.3 Phases of animal use

The procedures listed in section 2.2 must be applied in the various phases of animal use, which include:

* obtaining animals

* shipment and receipt of animals

* pre-study evaluation of animals

* allocation of animals to the study

* exposure of animals to the test substance

* evaluation of in-life animal response to test and reference substances

* removal of animals from a study

* transfer of animal tissues and specimens to archives

2.4 Obtaining animals

Some laboratories obtain their animals from commercial breeders, while others maintain their own breeding colonies. In either case, the laboratory should have SOPs for animal procurement. These SOPs should address the source of supply as well as the responsibility for initiating and authorizing animal orders.

With respect to the source of animals, the SOP should specifically identify acceptable suppliers for each of the different species of animals used at the laboratory. Ideally, the list of suppliers should be developed by a laboratory veterinarian or other animal health care professional with responsibility for directing the laboratory's overall animal health programme. Animal suppliers should be evaluated for their ability to provide healthy animals of known genetic and health background. The laboratory should keep records of its animal health evaluations or should ask to review such records at the animal supplier in order to establish which suppliers can provide suitable animals consistently. Some laboratories require that their veterinarian or health care professional inspect the facilities of animal suppliers as part of the evaluation of the supplier's suitability. Once a list of acceptable suppliers has been developed, only animals from this source should be used. This practice, coupled with an effective programme at the laboratory for evaluating the health status of incoming animals, will help assure the quality of incoming animals.

If animals are from the laboratory's own breeding colony, there should be SOPs for the maintenance and testing of the colony to assure the production of animals of consistent quality. Breeding colonies should be kept separate from the areas housing animals being tested. This helps to preclude inadvertent exposure of the breeding colony to


Page 45 of 76

test substances and disease. Records of health examinations, treatments and breeding should be kept for review by a health care professional responsible for assessing the suitability of animals for use in tests.

In addition to addressing the source of supply, the SOPs for ordering animals should define the person(s) with responsibility for ordering animals, the information required to order animals, and the documentation to be maintained. A single individual or department should be assigned responsibility for ordering animals. It is good practice to have a single authority for ordering animals to assure that the animals are obtained only from an acceptable source. This is often the animal husbandry department, which is headed by the laboratory's veterinarian. However, the individual or department charged with ordering animals cannot assume full responsibility for this function alone. The study director is responsible for assuring that the test system (i.e. the laboratory animals) used is the one specified in the study plan. One means of ensuring this is to require that the study director provides a written request to the animal husbandry department specifying the species, strain and number of animals required for a given study.

The SOP for ordering animals should specify the information to be included in the request. Normally, this will be the same as the information on the test system in the study plan. For example, when requesting animals, the study director should specify at least the following: the species, strain or substrain; number and sex of

animals; weight range and/or age of animals; and any special requirements such as timed-pregnant animals or surgically altered animals. The request should also state the proposed starting date of the study so that animals will be available on time and within the specified age/weight range. The number of animals ordered should also take into account the loss of animals due to shipping. The written request for animals should be signed by the study director indicating that the authorization and animal specifications conform to the study plan. The written request for animals should be retained with the raw data to document the involvement of the study director in this phase of the study. The SOP for ordering animals should also specify what documentation is to be retained. In addition to the request for animals, some laboratories retain purchase orders, shipping tickets and/or other records to document the date the order was made, that the order conformed to the request for animals and that it was placed with an acceptable animal supplier.

2.5 Shipping and receipt of animals

When animals are shipped from an outside source, the receiving laboratory may not have much influence over the handling of animals during shipment. Most reputable animal suppliers will select the shipper and provide adequate housing, feed and water to accommodate the animals' needs during shipment. However, the testing laboratory should be familiar with the supplier's normal means of shipment and inform the supplier of any special shipping requirements. For example, a supplier may normally make deliveries to a laboratory on certain days of the week. However, if for some reason, there will be no one at the laboratory to receive the animals on the normal day, the supplier/shipper should be advised. Otherwise, animals may be left unattended in an unsuitable environment.

In laboratories that breed their own animals, transport of the animals from the breeding colony to the areas where the animals are received and used should be defined in an SOP for transfer of animals. The SOP should include a description of the housing to be used during


Page 46 of 76

transport as well as the precautions to assure that animals are properly fed and watered from the time they leave the breeding facilities until the time they arrive at their point of use. The procedures should clearly identify the individuals responsible for transfer of the animals and should provide for direct communication between the breeding colony and the department that is to receive the animals. The SOP should include any procedures required to preclude unnecessary stress to the animal during transport. Animals must not be allowed to remain in an uncontrolled or a stressful environment any longer than necessary. It is also important that animals transported in-house be clearly identified on their housing as to their origin and destination in order to assure proper routing and use.

Unlike shipping, the receipt of animals ordered by the laboratory from its own breeding colony is under its full control. The

laboratory should have SOPs covering the receipt of animals, which define, at a minimum, who will be responsible for receiving animals, where the animals are to be received, which tests should be done immediately upon receipt, and what documentation should be kept.

At the time of receipt, animals are usually counted and assigned temporary numbers or other means of individual identification. They are placed in standard housing units for transfer to the area where their health status is to be checked (i.e. quarantine). There should be an SOP that covers identification and housing of animals during this pre-study evaluation period. Animals should be identified so that observations of their health can be clearly documented for the individual animal.

Documentation of animal receipt is important information and should be kept with the other study records. It should cover the following details: where the animals were shipped from; the date and time they were received at the laboratory; who received the shipment; the general condition of the shipment, including a description of the number of containers; and the number of live, dead and moribund animals in each container. The assignment of animals to cages for transfer to the pre-study evaluation area should be documented and should include the actual number of animals transferred. Any other information obtained for each animal during the receipt phase should also be recorded. For example, animals may be weighed or, in the case of some large animals, vaccinated or otherwise treated. This treatment should be recorded. It is useful to retain a copy of the shipping tickets and animal container labels in the study records in order to document the receipt of the animals.

2.6 Animal care facilities

Depending on the nature of a laboratory's work, animal care facilities may comprise a large portion of the physical plant. The laboratory should have a sufficient number of animal rooms or areas to assure proper separation of species or test systems, isolation of individual projects, quarantine of animals, and routine or specialized housing of animals.

The need for separation of species should be evaluated by a veterinarian or animal health professional to assess where separation by species is required to prevent disease or stress in the animals. Although not ideal, it is possible to house different species together where it has been determined that such housing arrangements will not potentially affect the health of the animals or otherwise adversely affect the study. This is not a desirable practice, however, and should be avoided.


Page 47 of 76

Individual projects should be isolated to avoid potential confusion and cross-contamination of animals. As a general rule, animals being used in separate projects should be housed in different

rooms. This is especially true in feeding studies where environmental contamination may be present due to dust from handling dosed feed and spillage of feed by animals. Although it may be permissible to house two different projects together under some conditions (e.g., same test substance, same species), it is generally inadvisable to house projects together that use different test substances. In any event, precautions should be taken to minimize or eliminate exposure of laboratory personnel to the test substance.

Facilities for quarantine or isolation of animals are essential. Laboratories should have space available to isolate animals effectively, when necessary, to avoid adverse effects on the conduct of studies. For example, many laboratories quarantine incoming animals in the room where the study will be conducted. They are held in that room until their health status is evaluated and then they are placed on study in the same room. Thus, they are effectively quarantined from other animals at the laboratory, even though there is not an animal room strictly dedicated for quarantine.

Areas for routine or specialized housing are required. Routine housing of animals may include the maintenance of a breeding colony or the housing of animals in a routine study, such as a chronic feeding study. Specialized housing should be available where required. For example, studies requiring collection of metabolism or behavioral data may require special caging or environmental controls. Also, animals used in inhalation studies may be housed in special inhalation chambers. The laboratory management should assure that these and all other facilities are available as required.

In addition to the animal facilities discussed above, a toxicological laboratory should have a number of animal rooms or areas separate from these to ensure isolation of studies being done with test systems or test and control substances known to be biohazardous, including volatile substances, aerosols, radioactive materials, and infectious agents. These areas should include facilities and equipment for disposal of waste and other contaminated material from this area, and special equipment or clothing to protect laboratory personnel.

Separate areas should also be provided, where appropriate, for the diagnoses, treatment and control of laboratory animal diseases. These areas should provide effective isolation for the housing of animals either known or suspected of being diseased or of being carriers of disease from other animals. The extent of, and the need for, these facilities will depend upon the testing laboratory's policy regarding the handling of diseased animals. Many laboratories using rodents have a policy of immediately removing and discarding any animal that develops disease. Such a policy would essentially eliminate the need for any facilities dedicated to diagnosis and treatment of disease in these animals. However, when primates, dogs or other larger animals are used, they frequently are treated for

disease. In this case, special areas should be provided for such diagnosis, treatment and isolation. The extent to which diseased animals need to be isolated to prevent adverse effects on other test animals will require the judgment of the laboratory veterinarian or animal health care professional. However, should isolation be required, facilities must be available. The need for dedicated treatment facilities will also vary depending on the nature of the treatment. Treatment that is highly stressful to the animal or which


Page 48 of 76

includes aseptic procedures, such as surgery, should not be performed in the animal room. On the other hand, oral administration of medication and monitoring of body temperature are examples of diagnostic and treatment procedures that may be carried out in the animal room.

Adequate facilities for animal care must include facilities for collection and disposal of animal waste. In testing facilities that do not dispose of the waste directly, there must be facilities for safe sanitary storage of waste until it can be removed from the laboratory for disposal. Disposal facilities should be designed and operated to minimize vermin infestation, odours, disease hazards and environmental contamination. Facilities for storage and disposal of waste should be adequately separated from animal areas to preclude contamination of the animals, and schedules must be established, followed and documented to assure the timely performance of waste removal activities (e.g., animal cage changing).

A schedule for routine maintenance of buildings should be established. Although many laboratories have such schedules for equipment, many do not have building maintenance schedules. The need for such schedules is most easily illustrated in the area of animal facilities. These facilities receive much wear from both animals and personnel. Walls, floors and ceilings may crack, making these surfaces uncleanable or exposing underlying surfaces that may produce dust. Paint may also crumble. Light fixtures and doors wear out. All of these conditions may have an adverse effect on the animals housed in the facility. It is difficult to repair some of these conditions after a study is underway without adversely affecting the animals' environment. Consequently, maintenance should be scheduled to eliminate the need for repairs while studies are underway. This is especially true for rooms used to house animals in chronic studies. Of course, consideration must always be given to the potential impact on the study of chemical agents used in building maintenance and cleaning. Similarly, attention must be paid to the possible environmental contamination of the building during construction, renovation or repair of laboratory facilities, e.g., the use of certain plastics that leach out toxic chemicals or asbestos used in ceilings or to protect pipes. A schedule of routine preventive maintenance for buildings will ensure proper cleaning and maintenance of all areas.

2.7 Animal husbandry supply facilities

Storage areas should be supplied, as needed, for feed, bedding, supplies and equipment related to animal care and use. Storage areas for feed and bedding should be separated from areas housing the test systems and should be protected against infestation or contamination. There should also be facilities, as needed, for the preservation of perishable supplies, e.g., certain feeds requiring refrigeration.

Most large testing facilities maintain central storage areas for bulk storage of feed and bedding. In the case of feed, the individual responsible for running the bulk feed storage facility will dispense small lots of feed to the various animal areas for short-term storage and immediate use. These small lots are often stored in separate rooms in the immediate animal area under the responsibility of animal husbandry personnel working in the area. Feed may then be further dispensed to relatively small storage containers in the actual animal rooms. Feed is dispensed from these containers into feeders for the animals. Despite the difference in size and location of feed storage facilities at the testing laboratory, they must be protected against infestation or contamination. This is generally not a problem where feed is stored in designated areas outside of the animal rooms. In


Page 49 of 76

these designated areas, feeds are bagged and stored to facilitate stock rotation and to preclude contamination with cleaning agents. The chance of vermin infestation is also reduced. However, if feed is to be stored in animal rooms, special precautions must be taken. Firstly, only small amounts of feed should be permitted to be stored in the room, i.e. amounts of feed necessary for two weeks or less. Secondly, the feed must be stored in sealed vermin-proof containers that can protect feed from contamination during cleaning of the room. Thirdly, the feed must be clearly identified and stored in an area separate from the animal housing unit and designated for no other purpose. Finally, there should be procedures in place to assure the proper rotation of feed. Often, new feed is dumped into a container in the room without emptying it of the previous feed. This practice, if continued, may lead to contamination of the feed with mould and accompanying mycotoxins.

There should be facilities for the storage and handling of bedding so as to prevent contamination and vermin infestation. Some laboratories store substantial amounts of bedding in the area where cages are cleaned. This bedding is sometimes placed in the cages prior to their transport to the animal room. In this case, the facilities must be adequate to protect the bedding from contamination by equipment cleaning agents and dirty equipment brought to this area for cleaning.

Finally, animal supply facilities should provide areas for the storage of clean and dirty equipment, as well as facilities for cleaning it. Such equipment would include cages, racks, water bottles, feeders, and similar items. Some facilities have corridors

and rooms dedicated strictly to either clean or dirty equipment. The laboratory and flow of work are designed to prevent the mingling and/or mixing of clean and dirty equipment. Although designated clean/dirty facilities are ideal, an adequate degree of separation can be maintained by scheduling work so that corridors and cleaning areas are not occupied by clean and dirty equipment at the same time.

2.8 Facilities for handling test, control and reference substances

The facilities must allow separation of areas involved in the storage, handling and distribution of test, control and reference substances and of mixtures containing them. This is necessary to prevent contamination of facilities, equipment, personnel and test systems, as well as to prevent confusion of substances. To accomplish this, there should be separate areas for:

a) receipt and storage of test, control and reference substances;

b) mixing of test, control and reference substances with a carrier (e.g., feed);

c) storage of the test, control and reference substance mixtures.

In addition, storage areas for the test and control substances and test and control mixtures should be separate from areas housing the test systems. The storage facilities should be adequate to preserve the stability of the substance and mixtures. For example, adequate refrigerator or freezer storage space should be available for substances requiring low temperature storage.

2.9 Pre-study evaluation of animals

A fundamental laboratory practice is to place all newly received animals in quarantine until their health status has been evaluated.


Page 50 of 76

The procedures for doing this vary from laboratory to laboratory. Some maintain animal colonies under barrier conditions with extensive programmes for the evaluation of animals entering the barrier. These include thorough screening for bacterial, viral and fungal pathogens. Sentinel animals may also be maintained in the room housing the animals under test. These sentinel animals are evaluated in the same manner as the animals sampled during the pre-study health evaluation.

Whatever pre-study evaluation is employed, a laboratory should have SOPs to assure that the animals' health status is fully evaluated prior to the test. This evaluation may be performed by the laboratory veterinarian, animal care employees or the study director. These individuals should have documented training and experience that qualifies them to recognize symptoms and to diagnose disease problems for the animal species involved.

A laboratory should have facilities that can adequately separate newly received animals from those already housed, and a designated quarantine area is desirable. Laboratories may quarantine newly received animals in the room where they will be housed during the study. This is acceptable provided that employees follow adequate procedures to prevent cross-exposure of other animal rooms with equipment, waste or other materials from the rooms where the animals are housed for pre-study evaluation. The use of the same room for pre-study health evaluation and conduct of the study also permits the acclimatization of animals to the actual environmental conditions in the room prior to the beginning of the study. The facilities required for housing animals during quarantine are generally the same as for housing animals being tested. There should be an adequate number of cages of appropriate design, construction and size for the species to be housed. There should also be facilities for the removal and disposal of animal wastes, and adequate environmental controls for temperature, humidity and lighting. Technical guidance regarding facilities, equipment and proper animal husbandry procedures may form part of government regulations for animal welfare.

SOPs for the pre-study health evaluation of animals should define the parameters to be monitored for each species during the pre-study evaluation period and the frequency of observations. For example, the SOP might require rodents to be observed daily for mortality and clinical signs of illness such as rough hair coat, diarrhoea, laboured breathing and weight loss. A very important SOP topic is the length of the pre-study evaluation period. Animals should be isolated long enough to provide reasonable assurance that the most common diseases affecting that species will have ample time to manifest themselves clinically. This time varies from species to species and should be based on acceptable veterinary medical practice. The SOP should identify who will make the observations, define the circumstances under which a veterinarian or the study director should be called, and what, if any, treatment is to be given to the animals. Any signs of illness observed or treatment rendered should be documented in study records as described in the SOP or study plan. Records of clinical signs of illness should include entries specifically identifying individual animal(s) involved, with a detailed description of the clinical features, the date and time of onset and duration. The records should identify who made the observation.

Records of treatment should include the name of any medications used and the complete details of their administration, including the date of each administration and the amount given. Records should clearly indicate when treatment began and ended for each animal and who authorized treatment; the laboratory veterinarian, in consultation with the study director, should authorize all treatment. These basic requirements apply whether treatment is administered during the


Page 51 of 76

pre-study evaluation period or during the study itself. In any case, no treatment should be administered that will interfere with the purpose or conduct of the study. All records of animal observations

and treatment made during the pre-study evaluation period should be retained with the study records.

The SOP covering pre-study evaluation must reference the SOP for housing, feeding, handling and care of the animals. It should specify the types and sizes of cages to be used, feeds and feeding schedules, bedding schedule and procedures for changing and cleaning cages and equipment, procedures for watering animals, and procedures for monitoring and documenting environmental conditions such as lighting, temperature and humidity. The SOP should specify acceptable ranges for these parameters and remedial action to be taken when readings are outside these ranges. Like the SOP for treatment of animals, the SOPs for the housing, feeding, handling and care of animals is general and required for all animals whether the animals are in quarantine, on test or in the breeding colony.

2.10 Allocation of animals to a study

Most laboratories document the suitability of the test animals at the end of the quarantine period by means of a form signed by the veterinarian in charge of quarantine, which authorizes the release of the animals for use in the study. The study director is notified and the animals are transferred to the room where the study will be conducted if they are not already housed there. At this point, animals are ready for allocation to the study.

Three important events occur during the allocation to study phase. These are:

* Animals are evaluated to determine if they meet study plan requirements such as age, weight and physiological condition.

* Animals are permanently identified so that they can be specifically related to any data or specimens generated during the test. Animal housing units are also identified so that all information needed to identify each animal within the housing unit is located on that unit.

* Animals are allocated to the respective test groups in a manner that precludes bias (i.e. they are randomized).

Related procedures and documentation should exist to assure that each one of these vital study functions is properly performed. In the animal evaluation, some overlap may occur with the quarantine phase of the study. For example, it may be necessary to use animals that have specific blood chemistry values. To establish these values, several measurements may be required over a period of weeks. Blood samples may have already been drawn and analysed for several weeks prior to release of animals from quarantine. Now, however, the results of those blood analysis must be compared against the study plan requirements, and the study director must decide which animals qualify

for study. Also, weight requirements and other parameters that must be met at the time of study initiation should now be evaluated for conformance to the study plan. These are some examples of pre-study evaluations that are made during the allocation phase of the study. They are dictated by the study plan, and the study director has the final say as to whether the criteria are met. The most important record-keeping requirement at this point includes all records of laboratory reports and other observations made to determine if the


Page 52 of 76

animals conform to study plan requirements. There should also be a record of the individual reviewing this documentation and authorizing the animals for use in the study.

At this point, animals should be individually identified in such a way that they can be traced for any in-life or postmortem observation. This is often done by the use of tattoos or ear notches. Whatever method is chosen, it should be thoroughly described in the SOP for identifying animals. The method selected should not interfere with the study, should not separate from the animal, and should not be obliterated over the course of the study. It should also be easily read by animal technicians and all other laboratory personnel handling animals. It is especially important to provide adequate instruction to animal technicians on the proper reading of the identification code used. For example, a system of ear notches may be used to represent animal numbers.

Such a code should be fully explained in an SOP maintained in the animal room for ready reference. The SOP should also cover how numbers are assigned for a given study to assure their traceability. Study records must very clearly document the assignment of permanent animal numbers in such a way that they can be accurately cross-referenced with any temporary number assigned during quarantine. This is necessary to maintain data integrity for a given animal from receipt to disposal.

In addition to identifying individual animals, housing units should be marked to indicate which animals are housed in the unit. Most laboratories utilize cage cards or labels for this purpose. The cage identification should include the number, sex, dose group and study number for the animals housed in the cage. This practice helps assure that the animals are selected from and returned to their proper cage during cage-changing operations and during removal of the animals for required study observations.

The allocation of animals to their assigned test groups is another important study event. Once animals have been appropriately evaluated and approved for use on the study, they must be divided into treatment groups. In a typical study, there are four treatment groups of animals for each sex: control, low-dose, mid-dose and high-dose. Additional groups may be required as dictated by the study plan. Animals should be placed in these groups in a manner that is designed to eliminate bias. This is accomplished by using a procedure that

randomly assigns animals to a given treatment group. The procedure for randomizing animals should be described in a written SOP or the study plan. The allocation of animals using the specified procedure should be fully documented.

In addition to randomizing animals, many laboratories employ procedures to eliminate bias in the housing of animals. This is necessary because, even in the best animal facilities, environmental conditions within the room will vary. For example, there may be areas of lower air circulation or uneven heating and cooling.

The microenvironment of the animal cages in a single rack housing multiple cages may also vary. For example, animals in higher cages may be exposed to more intense light and higher temperatures than animals in lower cages. To overcome this, most laboratories have established SOPs for the placement and rotation of animal housing units within a rack and racks within a room. Commonly, in rodent studies, cages housing animals of a single dose group are placed together in a structured fashion to facilitate easy identification of the group during observation, feeding and dosing. The cages are then


Page 53 of 76

moved systematically as a group through all locations within the rack during the course of the study. The racks themselves are systematically moved through all locations in the room. This assures uniform exposure of the test animals to the full range of environmental variations within the room. The moves are often performed with the same frequency as other animal room procedures, such as cage changing. The moves should be documented by recording the date of the move. Laboratories may use diagrams of racks and rooms to document the movements and specific location of racks and cages.

2.11 Exposure of animals to a test or control substance

There are two major concerns during this phase of the study. Firstly, known quantities of defined substances should be administered to specifically identified test animals over a given period of time. Secondly, test animals must not be exposed to uncontrolled stress that may affect their response to the test substance. Both of these concerns can be met by appropriate care and use of laboratory animals.

The administration of substances should be carried out in accordance with the SOP and/or the study plan requirements to assure proper treatment of the test animals. A laboratory should have SOPs to cover fully every aspect of receipt, testing, preparation, distribution and use of test and control substances. Laboratory procedures should ensure that properly defined and known quantities of correctly labelled test and control substances are delivered to the laboratory personnel for administering to the test animals. Personnel should have their SOPs and study plan readily available to assure proper administration of the materials. The study plan should specify the route of administration and the dosage levels. However, there

should also be SOPs to cover the technical aspects of administering the substances, as well as specific procedures to prevent confusion of animals or test and control substances during administration.

Examples of SOPs for dosing animals might include instructions on how to insert a stomach tube for administration of materials by oral gavage or how to administer substances intravenously. Examples of SOPs designed to prevent confusion would include procedures that address the order in which the animals are to be dosed, how many animals can be removed for dosing at one time, and procedures for verifying animal and test substance identification. The SOP or study plan directions for the administration of test substances should provide directions on how to document the administration of the test and reference substances. This is crucial to the subsequent validation of study findings. Such documentation usually consists of a dosing record kept by means of a standard form. Documentation should include the following information: the name, lot number and expiration date of the test and reference substances, as applicable; the exact quantity of substance administered; the specific identity of the individual animal receiving it; the date, time and duration, as applicable, of administration; and the names of the individuals administering the dose. The individuals involved should sign or initial the record. During test and reference substance administration, the fact that each animal was dosed and the specific weight or volume of dose that each animal received must be recorded. If the test substances must be prepared or manipulated by technicians, this should also be documented. It is equally important to document positively that control animals were treated according to study plan requirements.

2.12 Control of laboratory environment


Page 54 of 76

The second major concern during the dosing phase is to prevent the exposure of test animals to uncontrolled stress. This is important throughout the in-life portion of the study. Uncontrolled stress can be caused by numerous factors: these include inadequately controlled animal room environment; disease; contaminants in feed, bedding and water; exposure to cleaning agents, pesticides and uncontrolled test substances; poor husbandry practices; improper handling; and inadequate facilities.

To prevent interference from environmental factors, the laboratory must have animal facilities that will provide a controlled environment appropriate for the species housed. This generally means that temperature, humidity, lighting, ventilation, noise and personnel activity in the animal rooms should be monitored and/or controlled. The monitoring and control activities should be recorded.

Laboratories housing rodents should have the capability to control temperature and humidity. There should be SOPs to define acceptable ranges for environmental parameters and to describe actions

to be taken if acceptable ranges are exceeded. Some laboratories continuously monitor and record the temperature and humidity (by means of hygrothermographs). The recording charts from such devices should be regularly checked and retained as study records. The charts should be initialled and dated by the individual checking them and there should be explanations for any reading that is abnormal. Such readings should be brought to the attention of the study director so that their impact on the study can be assessed.

Although they are desirable, hygrothermographs or other elaborate temperature and humidity recording devices are not absolutely essential. Many laboratories monitor these parameters with nonautomatic instruments on a twice daily basis to coincide with other activities that are being performed. All that is required is an accurate thermometer and a hygrometer or sling psychrometer. If these are used there should be SOPs for their use and the collected records should be reviewed and retained as raw data. If temperature is to be observed only once a day, it is desirable to have a thermometer that will show the minimum and maximum temperatures reached over the 24-h period. It is also good practice to have any temperature monitoring device periodically calibrated against an official standard to assure accuracy. This also applies to thermocouples or other electronic sensors used to monitor temperature and humidity.

Other environmental factors that affect animals include lighting, noise and ventilation. Lighting is often controlled by the use of timers to cycle lights on and off on a regular schedule. For small animals, it is common practice to provide equal periods of light and dark over a 24-h period (i.e. 12 h on, 12 h off). Lighting cycles should be monitored periodically to assure that they meet study plan requirements. Such monitoring should be recorded. This sometimes presents a problem and may be overlooked when the light cycles occur after normal working hours. To overcome this problem, some laboratories have SOPs that direct the security or building maintenance personnel to monitor and document the light cycles after working hours. This is an acceptable practice, provided accurate documentation is maintained. It is also good practice to have animal room doors identified with the name and telephone number of the study director or other responsible individual so that they may be contacted should a problem with the animal room be detected after working hours.

Noise in animal facilities is generally controlled by facility design and proper placement of animals. In most laboratories, noise usually comes from three sources: animals, equipment and personnel.


Page 55 of 76

To control animal noise, noisy animals such as primates and dogs should be housed separately from other animals. Their housing should have doors and windows designed to reduce noise transmission between rooms and provide for arrangement of animals to accommodate their social behaviour. For example, animals housed so that they can see each other are often quieter. Limiting the entrance of unnecessary personnel into these areas will greatly reduce noise levels.

The most stressful noise probably originates from equipment and personnel. For example, cage washing operations often generate sharp irregular and/or continuous noise. Noise from these sources can be controlled by locating these areas out of hearing range of animal housing areas. Employees slamming doors, moving racks and cages, and playing radios can also create uncontrolled stress in the animals.

Ventilation of animal rooms should always be controlled and monitored. Proper ventilation with clear, filtered air is necessary to maintain acceptable temperature and humidity levels and to remove offensive odours or other airborne contaminants. SOPs for animal care and housing should specify acceptable air exchange rates for animal rooms. These should be monitored and documented periodically.

Another factor that may produce uncontrolled stress in test animals is disease. In cases where animals become diseased during a study, the disease may be treated. However, such treatment must be fully authorized and documented as discussed in section 2.6, and the study director must determine that treatment will not interfere with the purpose or conduct of the study.

Contaminants in feed, water and bedding can adversely affect the animals. To preclude this, feed and water used for animals should be analysed periodically to ensure that contaminants known to be capable of interfering with the study, and reasonably expected to be present in such feed and water, are not present at levels above those specified in the study plan. Documentation of such analysis should be maintained as raw data. The study director should identify any such contaminants in the study plan. Likewise, bedding should be free of any interfering contaminants or naturally occurring constituents that could interfere with the test. For example, pine and cedar shavings may contain naturally occurring aromatic hydrocarbons that can affect the liver metabolism of laboratory animals.

Other potential contaminants may be introduced into the animals' environment in the form of cleaning agents, pesticides and test or control substances from different studies. There should be SOPs covering the identification, use and monitoring of these agents. For example, the facility should have an SOP detailing the cleaning agents acceptable for use in animal facilities or on equipment coming into contact with animals. This would include detergents and sanitizers for cleaning floors, walls, ceilings and counter tops in animal rooms, as well as these same agents used to clean cages, pans, water bottles, racks and feed mixers. The SOP for cleaning must also include procedures for documenting that cleaning was performed and that the agents used were in accordance with the SOP. A similar SOP should exist for any pest control materials that are used. The SOP should identify which chemical pesticides are approved for use, the method of application and the specific locations where they can be used. The SOP should include the provision that responsible laboratory personnel should accompany any contract pest control personnel when they apply

pesticides to assure that they are applied in accordance with the SOP. This should be documented in writing. Many laboratories forbid the use of any pesticides and instead rely on mechanical traps and barriers. The use of these should also be described in the SOP.


Page 56 of 76

Other sources of uncontrolled stress to animals include laboratory personnel and poor husbandry practices. There should be an SOP regarding the admittance of personnel to the animal facilities; this should identify which employees are permitted in the animal rooms and under what circumstances. It should also describe any special health or safety precautions necessary to protect employees and animals. For example, the SOP should describe any protective clothing to be worn or decontamination procedures to be followed by personnel entering or leaving animal areas. Many laboratories have procedures designed to control disease and environmental contamination of the animal colony by prohibiting movement of employees between different animal facilities or laboratories. SOPs should also exclude from direct contact with animals an employee with an illness that may adversely affect animal health. Such employees should be reassigned until their health recovers.

Poor husbandry practices can be avoided by the use of adequate SOPs covering housing, feeding, handling and care of animals. This should include procedures to assure that animal cages, racks and accessory equipment are cleaned and sanitized at appropriate intervals. SOPs should also specify minimum acceptable cage sizes and room capacities to prevent animals being overcrowded. Every activity performed in the animal room during a study should be documented. To do this, some laboratories keep room activity logs. These records are maintained in the animal rooms during a study. Each time anyone enters the room they must sign the log documenting who they are, when they entered the room, what activity they were performing, and when they finished and left the room. This is a good practice for controlling and monitoring the level of activity in an animal room. Excessive activity can increase stress in the animals.

These animal care and use procedures are not all inclusive but they do cover the major factors. The procedures apply not only during the exposure of the animals to the test substances, but also to every phase of studies involving the maintenance and use of animals in laboratories.

2.13 Evaluation of in-life animal responses to test and control substances

This phase of animal use covers the period from the first administration of the test substances until the animals are submitted for postmortem evaluation. It is during this phase that crucial data are obtained by observing the response of test animals to exposure of test and reference substances.

In a typical chronic rodent study, animals are observed as specified in the study plan and/or SOPs. Examples of observations to be made include the following: general physical examination; daily observations for mortality and toxicological or pharmacological effects, body weight and feed consumption measurements; palpation for tissue masses; and special procedures such as ophthalmoscopy. The study plan must specify the frequency of these observations. Also, each of these procedures should be covered by the study plan or an SOP; SOPs often complement the study plan by providing specific guidance. For example, the study plan may simply state that animals are to be observed for toxicological effects. The SOP may define such effects as lethargy, hyperactivity or convulsions.

SOPs can standardize observations by prescribing the use of standard terminology and methods. For example, in palpating animals for tissue masses, the SOP should require that the location and size of masses be reported. It should define the limits of specific


Page 57 of 76

location description, such as dorsal front right, to distinguish masses in this area from adjoining areas. The SOP should also require that masses reported in a previous observation be positively accounted for during each subsequent observation session from the time they appear until the time they regress or are observed at postmortem observation. SOPs should also cover how and where observations will be recorded.

Proper identification of animals is important because all observations made must be related to specific animals. There should be SOPs that assure verification of the accuracy of animal identification during dosing, observation and transfer of animals. All documentation should clearly identify who made an observation, when it was made and to which animal it pertained.

An important consideration is the training of personnel. It is crucial to have employees who are trained and experienced in working with animals to make in-life observations. All personnel should have documentation of their training and experience. Complete familiarity with a species and its characteristics is essential to detect the often subtle clinical changes that may be indicative of toxic effects of the test substances.

2.14 Removal of animals from a study

Animals may be removed from a study because they die while the study is in progress, because they become sick or because they are sacrificed at the times specified in the study plan. In these cases, it is important that proper identification of the animals and related specimens is maintained and that the animals are submitted promptly for postmortem evaluation to preclude the loss of data due to autolytic changes in tissue. This is accomplished by requiring multiple daily checks for mortality and morbidity and specifying after-hours procedures for storage of dead animals.

Documentation should be maintained for unscheduled removal of a dead or moribund animal and include, at a minimum, the identity of the animal, the date and time of removal, the reason for removal, and the identity of the individual who removed the animal. For moribund animals, the reason for removal should include specific observations of animal behaviour and physical condition. The study records should clearly indicate whether the animal was sacrificed or found dead. Animals that are killed accidentally during the study should be fully reported.

The same general requirements, with respect to the required documentation, apply to scheduled deaths. An important requirement in the scheduled sacrifice of animals is that documentation should be maintained to demonstrate the method of sacrifice used. This may be specified in the study plan or SOP, but should include a method that will not interfere with postmortem evaluation of animal tissues and specimens.

Postmortem evaluation includes gross necropsy, histological preparation of tissues and organs, and histopathological evaluation. A major concern is integrity of animal identification. There should be procedures covering the identification of animals and their respective tissues and specimens from the time they are submitted for postmortem evaluation until they are sent to storage.

There should always be positive identification attached to or accompanying each animal. Many laboratories have a form that accompanies the animal to necropsy, identifying it by number, sex, dose group and study. For animals that are individually numbered with


Page 58 of 76

ear tags, tattoos or other permanent identification, SOPs generally require the necropsy personnel to compare the animal received against the form to confirm proper submission. Animals are then necropsied and tissues, organs and specimens are collected and processed as directed by the study plan. Each of the items collected should be identified in a manner that relates it back to the animal it came from. This is often accomplished by placing unique identifying information on containers used in the storage or processing of these tissues, organs or specimens. A unique sequential accession number can be assigned to containers that relate to the animal, or the unique animal identification number may be utilized. Whatever method is used, it must be traceable back to a specific animal in a specific test. The procedures used must be fully described in the SOP. Not only must tissues be identified, but all documentation for the receipt, preparation and evaluation of tissues must be identified to relate them to a specific animal from a specific study. The records of gross necropsy findings, microscopic findings and in-life observations must account for all gross observations. For example, if an animal technician reports four tissue masses during the last in-life observation period of an animal prior to submission for necropsy, the necropsy records must confirm the presence or absence of each of the masses reported.

To achieve this, the last in-life observations must be available to the individual making necropsy observations. Each mass accounted for at necropsy should be uniquely reported and identified in the gross necropsy findings for the animals. Trimming and tissue processing records must also account for masses collected at necropsy for processing. Finally, necropsy findings should be provided to the pathologist making microscopic evaluation of the tissues. Records of this evaluation, likewise, must account for all tissues, masses and lesions reported. To facilitate tissue accountability for animals with multiple masses, number or letter designations should be assigned to each mass at necropsy. The method used to assure identification and accountability of animals and tissues during this phase of the study must be fully described in the SOP.

2.15 Transfer of animal tissues and specimens to archives

Safety studies generally produce large amounts of data and specimens. To accommodate their storage and retrieval, a laboratory should have space designated as an archive for all raw data and specimens from completed studies. This area should have access limited to authorized personnel only. Although most laboratories have a centrally located archive to support the whole testing facility, some have a number of archives. For example, some support departments, such as histology and chemistry, may maintain space for storage of data and specimens they generated or analysed. This is acceptable, provided access to these materials is limited to authorized personnel and the data and specimens are properly identified and indexed as to their location. The best situation, however, is to have archive facilities available to provide central storage of all data and specimens. These should be under the control of a designated individual.

After evaluation of animal tissues and specimens, the final phase is the transfer of these materials to the archives. At this point, the gross remains of animals are placed in sealed containers while the tissues are contained in blocks and slides. There should be an SOP to assure that all these items are properly identified, accounted for, inventoried and placed in the archives. It is the study director's responsibility to assure that all specimens are transferred to the archives at the conclusion of the study.


Page 59 of 76

3. QUALITY MANAGEMENT APPLIED TO HUMAN AND ENVIRONMENTAL MONITORING STUDIES

3.1 Introduction

The manufacture and use of chemicals can lead to the deliberate or unintentional exposure of natural ecosystems to potentially hazardous substances. In this case, humans may be affected by contact with the chemicals through the consumption of food and drinking-water, and air inhalation. The natural environment can change its structures due to effects on the complex processes governing the functions of ecosystems.

In recent years, the awareness of risks of adverse health effects due to exposure to various environmental factors has increased substantially. In many countries major health problems and nuisances are still related to particulate matter, sulfur dioxide and pesticides. In the more developed industrialized countries the interest has shifted to effects such as cancer, allergy and disturbed function of the central nervous system, caused by exposure to pollutants such as hydrocarbons, nitrogen oxides and toxic metals. In the future, interest is likely to focus on environmental health effects, e.g., effects on the nervous and immune systems, that are more difficult to detect and often appear after long periods of exposure to relatively low doses of specific environmental factors or to combinations of different factors.

One aim for continuous health surveillance is to follow the development of morbidity and mortality patterns within various population groups. This requires access to extensive descriptive data. In order to relate observed health effects to certain environmental factors, reliable exposure data are needed. It is important that observed changes in the health effect pattern are validated on a local level. Many observed changes have been shown to be caused by artefacts.

The function of an ecosystem is determined by the physical and chemical processes, as well as by the relationships and interactions of the living organisms within the system. With the overall aim of the safe manufacture and use of chemicals, several objectives are covered by monitoring studies in natural environments. These can be described, for instance, as the monitoring of the levels of chemicals in various compartments within ecosystems, i.e. a descriptive study of situations and trends. Furthermore, programmes monitoring the fate and effects of chemical substances in the field are carried out in order to compare the results with existing information from similar studies carried out in the laboratory, e.g., the investigation of exposure-response relationships for risk assessment. In addition, monitoring of contamination levels in a selected environment is

performed in order to evaluate compliance with specific environmental quality criteria or targets, for instance, from a regulatory point of view.

Consequently, in environmental monitoring studies of natural ecosystems, it is predominantly the concentrations of chemicals, transformation processes and products which are investigated, as well as accumulation and ecological effects in wildlife, and the abundance and distribution of species. For this purpose, environmental monitoring studies are carried out in different compartments of various ecosystems, e.g., sediments and soils, air and water, and terrestrial and aquatic biota.

The incorporation of quality management approaches in


Page 60 of 76

environmental monitoring studies should ensure that the resulting data are reliable and reconstructible. Quality management should cover all phases of an environmental monitoring study, i.e. the planning of the study, selection and preparation of sampling equipment, sampling procedures, analyses, measurements and observations, and the reporting.

Reliable exposure data are essential for the establishment of dose-response relationships for toxic effects of environmental pollutants or other chemicals in human subjects, in epidemiological studies and for the assessment of risks of adverse health effects upon contact with such substances. Traditionally, pollution monitoring has been concerned primarily with the movement of pollutants through the environment and the concentrations in various environmental media such as air, food and water. Such monitoring provides little information on the amounts of pollutants actually coming in contact with people.

Direct measurements of human exposure levels may involve determination of the chemical under study in air, food and water, preferably through personal monitoring, or determination of the chemical or its metabolites in tissues or body fluids (biological monitoring). Ideally, the concentration of a pollutant, or its metabolites, in an indicator medium will give information on the degree of exposure, the dose at the critical organ and the risk of adverse health effects. Some information concerning the type and degree of exposure may also be obtained from determinations of pollutant concentrations in micro-environments combined with studies of human activity patterns.

The World Health Organization (WHO) and United Nations Environment Programme (UNEP) have been involved in human exposure monitoring since 1977, when global studies on the biological monitoring of lead and cadmium (Braux et al., 1979; Vahter, 1982; Friberg & Vahter, 1983; Bruaux & Svartengren, 1985; Vahter & Slorach, 1989) and organochlorine compounds (Slorach & Vaz, 1983) were initiated. On the basis of this work the WHO health-related programme, which involves monitoring of pollutants in air, water and

food, has included a new component, the Human Exposure Assessment Locations (HEAL) programme. Initially, the HEAL programme has focused on methods for exposure monitoring, including methods for quality assurance. General principles and procedures for the development of quality assurance in relation to exposure monitoring have been prepared (UNEP/WHO, 1984; WHO, 1986b). A comprehensive document on quality assurance in biological monitoring of metals has been published (Friberg, 1988).

3.2 Procedural requirements

A crucial point in exposure monitoring is to ensure correct sampling, e.g., that the collected air particles, food, blood and other samples really cover a representative period and that the correct sampling procedures have been used. As in other types of studies, it is important that the field personnel and the laboratory staff are properly trained. In many exposure monitoring activities the subjects under study collect the samples themselves, e.g., duplicate diets. In this case it is necessary to train the subjects properly, and to have staff available for assistance or advice during the entire sampling period. A study plan and detailed SOPs for the sampling procedures should be prepared and discussed with all the people concerned. It is also important to motivate the subjects and to give them adequate information about the aim and the design of the project before starting collecting the samples.


Page 61 of 76

The SOPs should cover all aspects of the monitoring exercise, particularly the sampling procedure, use of equipment and materials, transport and processing of samples, and analytical and observation methods. For example, a description of the equipment to be used for field sampling should take into account that outdoor application requires robust constructions, simple handling and easy-to-read instructions. Careful transportation is necessary to maintain the functioning of equipment. Furthermore, calibration procedures should be established which appropriately consider that the performance of many instruments, not particularly designed for outdoor use, may alter under field conditions; the laboratory calibrations may not be valid. The SOP should consequently define how the functions of the instruments for field measurements are to be tested.

The correct, unambiguous and non-erodible labelling of samples and specimens is particularly essential, since the samples taken in the field are usually transported and analysed or measured at other locations and at a later date. Thus the conditions under which the samples have to be transported and stored must be established in an SOP. The correct recovery of a chemical from an environmental sample, for instance, is very much dependent on its chemical and biological stability in the medium. Considerable care has, therefore, to be taken to ensure that the content and often the structure of a sample remain unchanged until it is analysed, or until observations or measurements have been carried out.

In the study plan, the personnel responsible for different parts of the study should be clearly identified, since the field work may be carried out by different institutions to those that carry out the laboratory analyses and measurements. Therefore, the study plan is the most important document for providing information to all participants on all aspects of the study, including sampling dates, amounts and character of samples.

Deviations from the study plan, particularly concerning changes in the selected sampling stations, sampled populations and sampling tissues, necessitate a detailed amendment to the study plan. This is of great importance, since in many monitoring studies sampling occurs on a regular basis, and the data obtained may be worthless if the preset sampling scheme is not followed. Therefore, changes need authorization of the study director. Acceptable levels of deviations in respect of time or space of sampling should also be stated.

If a change in a given parameter or the impact of a certain process has to be monitored over time or space, a control site or population often has to be selected. The selection of the control must ensure that conditions are similar to those of the site under investigation but that the influencing factor is lacking. In contrast, background sites (or populations) that are not affected by human activities (Black, 1988) can be treated as "blanks" in environmental monitoring programmes.

3.3 Selection of sampling strategies and study design

The study plan for a monitoring study must state specifically the selected sampling area, sampling station or population to be sampled, medium to be sampled, frequency and time of sampling, numbers of samples and the parameters for evaluation. For instance, when the contamination level in an aquatic ecosystem is monitored over a long period, samples from the water column, sediments and living organisms are commonly taken on a regular basis. The number of samples is selected according to the size and character of the investigation zone. The volume of the sampled medium is chosen according to the expected concentration of the chemical and the sensitivity of the


Page 62 of 76

analytical method. The wildlife species to be sampled should be representative for the biota and known to accumulate the substance of concern sufficiently.

Different objectives of environmental monitoring programmes require various kinds of sampling techniques. In general, it has to be decided if random or non-random sampling should be applied to achieve the objectives.

For example, if the fate and effect of a contaminant distributed by a diffuse source has to be monitored, a random sampling technique is appropriate. On the other hand, if the dilution of an industrial effluent by natural surface waters is monitored, the sampling stations

need to be selected according to various factors, e.g. water flow, while sampling times may be randomly chosen.

In cases where exposure data for a group of individuals are used for estimating exposure in the population, the selection of individuals for monitoring is a critical step. The sample of individuals has to be selected so that it will provide valid inferences for the target population. Thus, the selected individuals must be representative of the population under study. Quality assurance related to selection of study groups and individuals is described in the guidance provided by WHO on sample selection and data analysis for HEAL studies (WHO, 1992).

Three major random sampling techniques exist; these are commonly described as "stratified random sampling", "simple random sampling", and "systematic sampling" (Cochran, 1963; Kelley, 1976; Sokal & Rohlf, 1981). All three techniques have a common purpose, namely that the samples are representative for the population or compartment to be sampled.

In simple random sampling the selection process is totally, unconditionally random. The disadvantage of this method lies in the possibility that the samples may unintentionally be clumped together, while other parts of the population or compartment may not be sampled at all. This may be particularly unfavourable when samples show a large variation.

Distribution problems can be solved by dividing the population or compartment into either equal segments, where the investigator selects the interval systematically and in which segment a sample is to be taken, or into segments that are unequal in size or number ("strata"), and where at least one sample is taken in each segment. The first situation (systematic sampling) is unfavourable in cases where, during repeated sampling, the functions to be monitored vary, perhaps periodically. In the second case (stratified random sampling), some information on functions that are monitored must be available before the study starts and has to be used in designing the strata. Within each stratum the samples are selected randomly. The sampling size can be adjusted for each segment of the population or compartment.

Thus, the quality and value of the data obtained by the monitoring exercise is governed to a great extent by the selected sampling technique. For example, if the effects of the so-called acid rain on forest ecosystems should be monitored, simple random sampling might not lead to valid results, since the effects might vary considerably between, for instance, deciduous and coniferous forest systems.

All these aspects demonstrate the extent to which a well- designed study influences the quality of the results.


Page 63 of 76

3.4 Sampling procedures and documentation

The parameters to be measured or observed must be specified by SOPs and/or the study plan. Where and by which means the samples are taken and how measurements and observations are carried out should be described. For instance, if concentrations of a substance are monitored in natural aquatic ecosystems, it is necessary to establish the volume of water to be sampled, from which depth the samples must be taken, and which device should be used. For blood samples the time of sampling can be of great importance.

For collecting human samples, new syringes and containers should preferably be used, but for environmental samples this may not always be feasible. In any event, clean sampling devices and containers should always be used. In order to avoid contamination of samples, the necessary cleaning procedures for sampling devices and containers must be specified. During sample collection the sampling devices can contribute to chemical contamination due to the use of improper or unstable material as well as improper cleaning procedures. The SOPs should state clearly the material to be used for sampling and storage of samples, as well as the cleaning procedures. The cross-contamination of biological material can also occur, e.g., a net sample with planktonic organisms, where organisms from a previous sample are introduced into another because the net was not properly rinsed. In this example, if the species composition is monitored, biased results can be expected. Thorough rinsing would avoid this. In the case of chemical contamination, cleaning procedures such as acid washing may be useful.

Containers and chemicals may be potential sources of contamination, for instance with trace metals. Special cleaning procedures are needed to minimize this problem.

3.5 Handling of samples

The samples obtained during a study are normally shipped from the field into the laboratory where they are to be evaluated. Often there is a considerable delay between sampling and arrival at the laboratory where the samples and specimens are to be stored.

For many pollutants there is a great risk of introducing errors during sampling, sample handling and chemical analysis. The risk of contamination of the sample during sampling and sample handling is particularly great for samples with low concentrations of pollutants ubiquitous in the environment or present in materials and tools coming into contact with the sample. For example, a blood sample of 1 ml normally contains as little as 0.1-0.5 ng cadmium, and it is obvious that considerable measures have to be taken in order to avoid contamination. Tobacco smoke often contains cadmium at levels that may seriously contaminate the blood samples. Therefore, all sample

handling must be carried out in rooms where smoking is prohibited, and preferably by non-smokers.

Concentration changes during storage may be due to precipitation or evaporation of the analyte or to evaporation of the solvent. Adsorption of the analyte to the container may also occur. Changes in the sample matrix, e.g., clotting of blood, may change the concentration of the chemical to be measured. Often the samples have to undergo a series of preparation steps before the determination of the concentration of a pollutant is carried out. Mistakes in the dilution, weight determination and calculations may appear. Chemicals added to the samples may be contaminated with the substance under


Page 64 of 76

study.

The use of blanks is the most common analytical tool for controlling contamination. Blanks can be used in the laboratory in the form of instrument blanks, calibration blanks and reagent blanks. In the field, matched-matrix blanks (determination of contamination during sample collection, handling, storage and transport) are used to simulate the sample matrix and are carried through all processes. Blanks of the media used in sampling (like filters, nets, traps and containers) and of the sampling devices (by collecting the rinsing media) should also be used.

Appropriate provision must be made and measures taken in order to keep the samples under the conditions required to maintain the character of the samples. The stability of the chemical to be analysed, in terms of, for instance, physical, chemical or biological degradation, must be known. If there are special storage conditions required, adherence to these conditions must be controlled and documented.

One means of controlling the stability of the samples for chemical analysis is the use of spiked samples. These samples contain the same matrix as the environmental sample but without the respective analyte, which is then added in a known quantity. The spike samples are then handled and stored in the same way and under the same conditions as the environmental samples and are concomitantly analysed. It should be noted that the integrity of volatile fractions in samples is particularly difficult to maintain.

The stability of biological material is typically ensured by the use of preservation materials or processes, but proper and appropriate preservation methods must be used so that very fragile structures, such as single cells in plankton samples, are not damaged or destroyed. This is necessary to ensure that measurements or observations of the samples are representative.

3.6 Analytical performance evaluation

The main objective of the analytical performance evaluation is to assess the accuracy of data. This evaluation should be published along with the data so that users can assess the quality. The following techniques are available for analytical performance evaluation:

* external quality control programmes;

* comparison with results using an independent technique;

* comparison with results of a reference laboratory;

* analysis of commercial standard reference materials;

* analysis of spiked samples prepared at the laboratory.

Ideally, the analytical performance evaluation should be coordinated by an external quality assurance coordinating centre or by the quality assurance unit of the laboratory. The coordinators provide the laboratory with external quality control (EQC) samples, the concentrations of the substance being unknown to the analyst. The EQC samples should be analysed together with the collected samples, and the results should be evaluated by the quality assurance personnel.

If it is not possible to have the analytical performance


Page 65 of 76

evaluated by an external quality assurance personnel, duplicate samples should be analysed using other analytical techniques, preferably at a reference laboratory. If other techniques are not available, duplicate samples should be analysed at a reference laboratory.

Since the certified concentrations of standard reference materials are published, and this is known to the analyst, analysis of such samples alone is often not enough for independent evaluation of the accuracy of the resulting data. Correct results for the internal quality control (IQC) samples do not always guarantee accurate results for the EQC samples. Also, standard reference materials are often only available at one or two concentrations. The quality control samples should cover the range of concentrations likely to be found in the monitoring samples. Good analytical performance at one concentration is no guarantee of good performance at other concentrations.

The analytical performance evaluation should be carried out at the time of the analysis of the monitoring samples. Reference to previous participation in interlaboratory comparison programmes cannot be used for evaluating the accuracy of the data produced.

3.7 The regression method

Unfortunately it is not possible to measure of the accuracy of data produced, but the limits of uncertainty can be estimated. Principles for analytical quality assessment in human exposure monitoring, giving limits for the uncertainty, have been recommended by WHO (1986b). The method is based on the quality control programmes developed in the UNEP Biological Monitoring of Lead and Cadmium (Vahter, 1982; Friberg & Vahter, 1983; Vahter & Slorach, 1990). Basically, it involves the analysis of sets of quality control samples and evaluation of the results using linear regression analysis. In the WHO/UNEP Human Exposure Assessment Locations (HEAL) monitoring programme for lead and cadmium, a quality control set consisted of 3-6 external EQC samples, the metal concentrations of which were not known to the laboratories, as well as 1-2 IQC samples, the metal concentrations of which were given. The IQC samples are used for the analyst's own control of the analytical conditions, while the EQC samples are used for the analytical performance evaluation. One or more sets of quality control samples were analysed together with the monitoring samples, and the results were evaluated by a coordinating centre.

The regression method, i.e. the evaluation of the regression line of reported versus "true" values for a set of quality control samples analysed together with the monitoring samples, is a useful method of guarding against systematic errors in the whole range of concentrations likely to occur (Vahter, 1982; UNEP/WHO, 1984; Friberg, 1988). The regression line represents the average analytical performance.

In order to obtain limits for the uncertainty of the data produced, the maximum allowable deviation (MAD) of the empirical regression line from the ideal line y = x is defined. The MAD criteria have to be decided separately for each pollutant and for each medium. In the UNEP/WHO HEAL project on lead and cadmium, the MAD was generally set to ± (5-10% ± sigma), where sigma was the estimated error of the method, based on several quality control runs.

Since the regression line, based on the results of a set of quality control samples, has an operating error, the decision on acceptance or rejection of the regression line must be based on


Page 66 of 76

statistical criteria, i.e. the probability of making right or wrong decisions. A laboratory's results may be erroneously rejected when in fact the laboratory performance is satisfactory, or erroneously accepted when the performance is bad. Table 1 illustrates the different decisions that can be made on the basis of the results of the quality control analyses, and the associated probabilities (UNEP/WHO, 1984).

Table 1. Decision-making on the basis of quality control resultsa

True condition Decision

Acceptance Rejection

Methodology is correct decision wrong decision satisfactory (1-alpha) Type I error (alpha)

Methodology is not wrong decision correct decision satisfactory Type II error (ß) Power (1-ß)

a From: UNEP/WHO (1984)

In the HEAL project, a total power of 90% was employed, which means that the probability of accepting an unsatisfactory performance (the true regression line falling outside the MAD interval) was not more than 10%. For acceptance of data the empirical regression lines had to fall not only inside the MAD interval, but also inside an acceptance interval.

Fig. 1 shows an example of a regression line of the results of six quality control samples, the MAD interval (solid lines) and the acceptance interval (broken lines). The distance between the MAD lines and the acceptance lines is 1.645 times the operating error, sigmað, calculated according to the formula:

sigma2ð = sigma2y/x (1 + d2 ) n (n-1).sigma2x

where

n = number of observations

d = difference between x value and x mean

sigmax = standard deviation of x values

sigmay/x = error of method or residual deviation (estimated from previous analyses)


Page 67 of 76

It is obvious from the formula that the acceptance interval (AI) lines will get closer to the MAD lines with increasing numbers of data points (quality control samples). Also, a smaller error of method will decrease the difference between the MAD lines and the AI lines.

The random error of method may be calculated from the results of each quality control set. However, with quality control sets consisting of only 4-6 samples, the empirical error of method may be largely influenced by one or two occasional gross errors. Therefore, it is good practice to estimate the error of method based on previous experience. It is nevertheless important to calculate the empirical error of method, since it may serve as a supplementary guidance in the evaluation of the analytical performance. When the current random error of method deviates too greatly from the estimated one, this is an indicator of bad performance, and the evaluation should not be based on the estimated error of method.

For many types of chemical measurements, the error variance tends to vary with the true concentration. Variance-stabilizing transformations, e.g., logarithmic ( z = ln x ) or square-root ( z = x ) transformation, of the data may make the error variance independent of the true concentration (Starks, 1989). In the HEAL nitrogen dioxide project, where many quality control samples were


Page 68 of 76

used, the quality control results were considered satisfactory if the regression line, the 90% confidence interval and all data points were inside the MAD interval (Matsushita & Tanabe, 1991).

The regression method may give valuable information concerning the type of error. For example, a regression line parallel to the ideal line ( y = x ) indicates an absolute error caused by, for instance, a false blank value. A slope deviating from 1.0 indicates a constant relative error due to, for instance, incorrect standards or errors in the concentrations of the standards.

3.8 Practical application of the regression method

The regression method for analytical performance evaluation was developed for use in a WHO study on the assessment of human exposure to lead and cadmium through biological monitoring. Since then, the method has been used in a number of studies, involving both metals (Lind et al., 1987, 1988a,b; Zheng & Ji, 1987; Vahter & Slorach, 1990) and nitrogen dioxide (Matsushita & Tanabe, 1991).

3.9 Other analytical performance evaluation programmes

There are several other methods for external analytical performance assessment (ISO, 1986b; Horwitz, 1988). Some examples are given below. Most of these methods are descriptive and do not give acceptability limits for possible errors.

The WHO European Regional Study on Health Effects of Exposure to Cadmium, coordinated by the Coronal Laboratory for Occupational and Environmental Health, was specially developed for countries participating in the United Nations Development Programme. A quality control programme was developed for the determination of lead and cadmium in blood, as well as cadmium, ß2-microglobulin and retinol binding protein in urine (Herber, 1990). In the metal programme, 30-40 laboratories each analysed six samples of human blood and six samples of human urine. The samples contained cadmium and lead nitrate at levels up to about 20 µg/litre for cadmium and up to 800 µg/litre for lead.

Regarding the proteins in urine, there were only six or seven participating laboratories, and another, more simple, evaluation procedure was used. Urine from patients with kidney disease was diluted with urine with normal physiological protein concentrations.

The acceptance criteria were ± 20% of the median for albumin and ± 40% of the median for ß2-microglobulin and retinol binding protein.

The Guildford Trace Element Quality Assessment Scheme, coordinated by Robens Institute of Industrial and Environmental Health and Safety and St. Luke's Hospital, Guildford, United Kingdom, is an external quality assessment scheme for trace elements in human biological fluids (Taylor et al., 1985). More than 100 laboratories from over 15 countries participate in the scheme. The programme includes several combinations of analytes and biological samples. Every month during a six-month cycle, three specimens of each matrix are sent to the participants. Monthly reports include consensus mean, standard deviation, relative standard deviation, a histogram of distribution and a tabulation of the results. At the end of the six-month cycle, the 18 results for each sample-matrix analyte are summarized and the analytical performance is assessed based on the proximity to the consensus mean, the difference between results analysed on two occasions and the recovery of added analyte. A "performance score" is calculated for each individual laboratory. Targets or markers of satisfactory performance have been established


Page 69 of 76

based on what is necessary for clinical purposes and what can be achieved with available analytical techniques.

The Guildford external quality assessment (EQA) programme for aluminium in serum has been in operation since 1981 (Taylor, 1988). Samples of horse serum, supplemented with known amounts of aluminium, are prepared for a series of 6-monthly cycles. During a cycle, nine different specimens are distributed in duplicate, three samples each month. The results are evaluated as described above. The programme has shown poor performance in large numbers of laboratories and excellent performance in a few laboratories. In an attempt to explain this, an evaluation of instrumentation and methodologies was carried out. It showed that the quality of the results was not influenced by, for example, specific features of equipment or instrumentation but rather by good, careful analysts, who were able to ensure that everything was properly set up and that contamination was avoided.

The National External Quality Assessment Scheme (NEQAS), coordinated by the Wolfson Research Laboratory in Birmingham, is another external quality assessment programme in the United Kingdom (Bullock & Wilde, 1985). Samples are distributed to the participating laboratories for analysis, and based on the results obtained, a variance index (VI) is calculated. The VI is the difference between the result obtained and a trimmed mean, divided by a chosen relative standard deviation for the analyte and expressed as a percentage. A Mean Running Variance Index Score (MRVIS) and a Mean Running Bias Index Score (MRBIS) are also calculated. It is believed that a MRVIS below 33 should be the target for the participants, and that any MRVIS over 66 should stimulate investigation of the laboratory's method.

3.10 Analytical performance criteria

The limits of the uncertainty in the produced data should be based on the accuracy requirements for the monitoring data and what can be achieved with available analytical techniques. The accuracy and precision of routine and reference methods should be evaluated in order to determine the feasibility of the proposed criteria.

Often the criteria for the analytical performance are determined by the sensitivity and accuracy of the analytical method. In the WHO/UNEP monitoring project on cadmium and lead in blood, the limits for the MAD lines for lead in blood were set to

y = x ± (0.1 x + 20). This means that if the average concentration of lead in blood in a group of people was found to be, for example, 100 µg/litre, the criteria guaranteed with a probability of 90% that the true average concentration was somewhere between 70 and 130 µg/litre. If the average blood lead concentration found was 50 µg/litre, the criteria guaranteed that the true average was between 25 and 75 µg/litre. With such a great "uncertainty" in the monitoring data, it is of course difficult to detect differences in lead exposure between various groups in the general population.

The experience from the quality control activities in the HEAL pilot project on lead and cadmium shows that, for lead concentrations in blood (µg/litre), experienced laboratories can meet the MAD criteria y = x ± (0.05 x + 10), which guarantees that an obtained mean blood lead concentration of 50 µg/litre lies with 90% probability between 37.5 and 62.5 µg/litre. For concentrations of cadmium in blood, well-experienced and well-equipped laboratories met the MAD criteria y = x ± (0.05 x + 0.2), which guarantees that an obtained mean blood cadmium concentration of, for example, 0.5 µg/litre lies with 90% probability between 0.28 and 0.72 µg/litre.

3.11 Quality control samples


Page 70 of 76

The analytical procedures and performance may vary considerably between various types of samples. Good analytical performance for a pollutant in one type of medium is no guarantee of good performance with other media. Thus, it is important for quality control samples to have a matrix similar to that of the monitoring samples.

As already mentioned above, the quality control sample should cover the range of concentrations likely to be found in the monitoring samples. It must be emphasized that good analytical performance at one concentration is no guarantee for good performance at other concentrations.

Various types of reference samples are commercially available (Muntau et al., 1983; Belliardo & Wagstaffe, 1988; Klich & Caliste, 1988; Okamoto, 1988; Parr et. al., 1988; Rasberry, 1988). However, many of the commercially available reference materials are certified

for a limited number of substances, and usually only for one or two different concentrations. Commercially available reference materials are suitable for internal quality control purposes but cannot, as a rule, be used for published data.

If possible, specimens of the samples should finally be archived in order to prove again the validity of an environmental monitoring study, if it should be necessary. This may be appropriate where the samples are still integral after evaluation.

Biological tissue is typically contained in slides or blocks, while specimens are stored in containers with appropriate fixation. Aliquots from the various media for chemical analysis may be stored after appropriate preservation, carrying a label with an expiry date based on stability analysis. All written documentation including the raw data is also transferred to the archive at the conclusion of the study.

REFERENCES

AOAC (1984) Quality assurance principles for analytical laboratories. Washington, DC, Association of Official Analytical Chemists, 224 pp.

Belliardo JJ & Wagstaff PJ (1988) BCR reference materials for food and agricultural analysis: An overview. Fresenius Z Anal Chem, 332(6): 533-538.

Black SC (1988) Defining control sites and blank sample needs. In: Keith LH ed. Principles in environmental samples. Washington, DC, American Chemical Society, pp 109-118.

Bruaux P & Svartengren M ed. (1985) Global Environmental Monitoring System (GEMS). Assessment of human exposure to lead: Comparison between Belgium, Malta, Mexico and Sweden. Stockholm, National Swedish Institute of Environmental Medicine and Department of Environmental Hygiene, Karolinska Institute, and Brussels, Institute of Hygiene and Epidemiology, 57 pp (Prepared for United Nations Environment Programme and World Health Organization).

Bruaux P, Claeys-Thoreau F, Ducoffre G, & Lafontaine A (1979) Exposition saturnine de la population bruxelloise. Choix des groupes de population. Choix des indicateurs. Premiers résultats. Arch Belg Méd Soc, 37: 488-501.

Bullock DG & Wilde CE (1985) External quality assessment of urinary pregnancy oestrogen assay: further experience in the United Kingdom.


Page 71 of 76

Ann Clin Biochem, 22: 273-282.

Burck K (1979) The role of quality assurance in good laboratory practices. Clin Toxicol, 15: 627-640.

Cochran WG (1963) Sampling techniques. New York, John Wiley & Sons, 13 pp.

FDA (1987a) Good laboratory practice regulations. Fed Reg, 52(172): 33768 (21 CFR Part 58) (Docket No. 83N-0142).

FDA (1987b) Part 58 - Good laboratory practice for non-clinical laboratory studies. Code Fed Regul, 21(Ch1): 228-242.

Friberg L (1988) Quality assurance. In: Clarkson TW, Friberg L, Nordberg GF, & Sager PR ed. Biological monitoring of toxic metals. New York, Plenum Press, pp 103-126.

Friberg L & Vahter M (1983) Assessment of exposure to lead and cadmium through biological monitoring. Results of a UNEP/WHO global study. Environ Res, 30: 95-128.

Herber RFM (1990) The World Health Organization study on health effects of exposure to cadmium. Toxicol Environ Chem, 27: 1-10.

Herber RFM & Schaller KH (1986) Analytical variability of biological parameters of exposure and early effects. In: Notten WRF, Herber RFM, Hunter WJ, Monster AC, & Zielhuis RL ed. Health surveillance of individual workers exposed to chemical agents. Berlin, Springer Verlag, pp 102-109.

Horwitz W (1988) Protocol for the design, conduct and interpretation of collaborative studies: IUPAC Workshop on the Harmonization of Collaborative Analytical Studies, Geneva, Switzerland, 4-5 May 1987. Pure Appl Chem, 60: 855-864.

Horwitz W (1989) Harmonization of methodology: international perspective. In: Cowgill UM & Williams LR ed. Aquatic toxicology and hazard assessment. Philadelphia, American Society for Testing and Materials, pp 5-10 (Special Technical Publication No. 1027).

ISO (1986a) International Standard 8402. Quality - Vocabulary. Geneva, International Organization for Standardization, 15 pp.

ISO (1986b) International Standard 5725: Precision of test methods - Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests: 2nd edition. Geneva, International Organization for Standardization, 49 pp.

ISO (1987a) International Standard 9000. Quality management and quality assurance standards: guidelines for selection and use. Geneva, International Organization for Standardization, 6 pp.

ISO (1987b) International Standard 9004. Quality management and quality system elements - Guidelines. Geneva, International Organization for Standardization, 19 pp.

Kelley JC (1976) Sampling the sea In: Cushing DH & Walsh JJ ed. The ecology of the seas. Oxford, Blackwell Scientific Publications, pp 361-387.

Klich H & Caliste JP (1988) COMAR - database for certified reference materials. Fresenius Z Anal Chem, 332(6): 552-555.


Page 72 of 76

Lepore PD (1979) FDA's good laboratory practice regulations. Pharm Technol, 3: 71-74.

Lind B, Elinder C-G, Friberg L, Nilsson B, Svartengren M, & Vahter M (1987) Quality control in the analysis of lead and cadmium in blood. Fresenius Z Anal Chem, 326: 647-655.

Lind B, Vahter M, Rahnster B, & Bjors U (1988a) Quality control samples for the determination of lead and cadmium in blood, feces, air filters and dust. Fresenius Z Anal Chem, 332(6): 741-743.

Lind B, Bigras L, Cernichiari E, Clarkson TW, Friberg L, Hellman M, Kennedy P, Kirkbride J, Kjellstrom T, & Ohlin B (1988b) Quality control of analyses of mercury in hair. Fresenius Z Anal Chem, 332(6): 620-622.

Matsushita H & Tanabe K (1991) Earthwatch - Global Environmental Monitoring System (GEMS). Exposure monitoring of nitrogen dioxide. An international pilot study within the WHO/UNEP Human Exposure Assessment Location (HEAL) Programme. Nairobi, United Nations Environment Programme, 109 pp.

Muntau H, Schramel P, Brätter P, & Knapp G (1983) Trace element investigations on some new biological test materials. In: Brätter P & Schramel P ed. Trace element - Analytical chemistry in medicine and biology. Berlin, New York, Walter de Gruyter & Co., vol 2, pp 819-831.

OECD (1982) Good laboratory practice in the testing of chemicals. Paris, Organisation for Economic Co-operation and Development, 62 pp.

Okamoto K (1988) Biological reference materials from the National Institute of Environmental Studies (Japan). Fresenius Z Anal Chem, 332(6): 524-527.

Parr RM, Schelenz R, & Ballestra S (1988) IAEA biological reference materials. Fresenius Z Anal Chem, 332(6): 518-523.

Rasberry SD (1988) NBS activities in biological reference materials. Fresenius Z Anal Chem, 332(6): 528-532.

Slorach SA & Vaz R ed. (1983) Global Environmental Monitoring System (GEMS). Assessment of human exposure to selected organochlorine compounds through biological monitoring. Uppsala, Swedish National Food Administration, 134 pp (Prepared for United Nations Environment Programme and the World Health Organization).

Smith F, Kulkarni S, Myers LE, & Messner MJ (1988) Evaluating and presenting quality assurance sampling data. In: Keith LH ed. Principles in environmental samples. Washington, DC, American Chemical Society, pp 157-168.

Sokal RR & Rohlf FJ (1981) Biometry: The principles and practice of statistics in biological research, 2nd ed. New York, Oxford, W.H. Freeman, 859 pp.

Starks TN (1989) Global Environmental Monitoring System (GEMS). Statistical guidelines for quality assurance in human exposure assessment location studies. Geneva, World Health Organization, 48 pp (Document PEP/89.9).

Taylor A (1988) Reference materials for measurement of aluminium in biological samples. Fresenius Z Anal Chem, 332(6): 616-619.

Taylor JK (1987) Quality assurance of chemical measurements. Chelsea,


Page 73 of 76

Michigan, Lewis Publishers, 328 pp.

Taylor JK (1988a) Defining the accuracy, precision and confidence limits of sample data. In: Keith LH ed. Principles in environmental samples. Washington, DC, American Chemical Society, pp 101-108.

Taylor JK (1988b) The role of statistics in quality assurance. Fresenius Z Anal Chem, 332(6): 722-725.

Taylor A, Starkey BJ, & Walker AW (1985) Determination of aluminium in serum: findings of an external quality assessment scheme. Ann Clin Biochem, 22: 351-358.

UNEP/WHO (1984) Principles and procedures for quality assurance in environmental pollution exposure monitoring. Geneva, World Health Organization, 31 pp (EFP/HEAL/84.4).

Vahter M (1982) Global Environmental Monitoring System (GEMS). Assessment of human exposure to lead and cadmium through biological monitoring. Stockholm, National Swedish Institute of Environmental Medicine and Department of Environmental Hygiene, Karolinska Institute, 136 pp (Prepared for United Nations Environment Programme and World Health Organization).

Vahter M & Slorach S ed. (1989) Global Environmental Monitoring System (GEMS). Exposure monitoring of lead and cadmium: An international pilot study within the UNEP/WHO Human Exposure Assessment Location (HEAL) Programme. Nairobi, United Nations Environment Programme, 82 pp.

Vahter M & Slorach S ed. (1990) Global Environmental Monitoring System (GEMS). Exposure monitoring of lead and cadmium: An international pilot study within the WHO/UNEP Human Exposure Assessment Location (HEAL) Programme. Nairobi, United Nations Environment Programme, 82 pp.

WHO (1986a) Environmental Health Criteria 57: Principles of toxicokinetic studies. Geneva, World Health Organization, pp 20-24.

WHO (1986b) UNEP/WHO Global Environmental Monitoring System (GEMS). Human Exposure Assessment Location (HEAL) Project: Guidelines for integrated air, water, food and biological exposure monitoring. Geneva, World Health Organization, 389 pp (Document PEP/86.6).

WHO (1992) Earthwatch - Global Environmental Monitoring System (GEMS). Guidance on survey design for Human Exposure Assessment Locations (HEAL) Studies. Geneva, World Health Organization (Document WHO/PEP/92.6).

Youden WJ (1967) The role of statistics in regulatory work. J Assoc Off Anal Chem, 50: 1007-1013.

Youden WJ & Steiner EH (1975) Statistical manual of the Association of Official Analytical Chemists. Washington, DC, Association of Official Analytical Chemists, 88 pp.

Zheng XQ & Ji RD (1987) Assessment of lead contamination of the general population through blood lead levels. Environ Monit Assess, 9: 169-177.

APPENDIX I

GLOSSARY


Page 74 of 76

Batch: A specific quantity or lot of a test or reference substance produced during a defined cycle of manufacture, in such a way that it could be expected to be of a uniform character and should be designated as such.

Carrier (vehicle): Any agent that serves as a vehicle used to mix, disperse, or solubilize the test or reference substance to facilitate the administration to the test system.

Calibration: Assigning values to the output of an instrument or other device. Calibration can apply to instruments, glassware or any other measuring device.

Calibration verification (calibration check): Determining if the output of an instrument or device is within some established tolerance.

Code number: A unique number assigned to each packaged unit of the test substance.

Final report: A comprehensive report that includes all information relative to the evaluation of the results of a specific nonclinical laboratory study.

Laboratory technician: A laboratory worker who performs an operation covered by an SOP, under the supervision of a professional scientist.

Master schedule sheet: A document that contains essential information on completed and in-progress studies in the laboratory, i.e. a list of studies, initiation and completion dates, test systems, route of administration and name of study director.

Quality assurance programme (QAP): An internal control system of inspections and study audits designed to ascertain that a study is in compliance with defined guidance principles. It assures laboratory management that facilities, equipment, personnel, methods, practices, records and controls conform with these principles.

Quality assurance unit (QAU): Any person or organizational element, designated by testing facility management to perform inspections of laboratory operations and study audits relating to quality assurance of studies.

Quality control system: A system used to assess product quality whenever the quality attributes of the final product can be expressed as discretely measured parameters.

Raw data: All original laboratory records and documentation, or verified copies thereof, which are the result of the original observations and activities in a study.

Sample: Any quantity of the test or reference substance. Specimen: Any material derived from a test system for examination,

analysis or storage.

Sponsor: A person or entity who commissions and/or supports a study. A testing facility can also be a sponsor if it both initiates and actually conducts the study.

Standardization: Determining the response of an instrument to known quantities of a test agent according to a specific method; accurately determining the concentration of a solution.


Page 75 of 76

Standardization verification (standardization check): Determining if the response of an instrument to a particular standard is within some established tolerance (e.g., verifying that a previously prepared standard curve (or other procedure) is still valid).

Standard operating procedure (SOP): A written procedure that describes how to perform certain routine laboratory tests or activities that are normally not specified in detail in study plans or test guidelines.

Study: An experiment or set of experiments in which a test substance is examined to obtain data on its properties and/or its safety with respect to human health and the environment.

Study audit: A comparison of the raw data and associated records with the interim or final report in order to determine whether the raw data was accurately reported, and whether testing was carried out in accordance with the study plan and SOPs to obtain additional information not provided in the report, and to establish whether practices were employed in the development of data that would impair their validity.

Study director: The individual responsible for the overall conduct of the study.

Study plan: A protocol which defines the entire scope of the study. Test facility (testing facility): The persons, premises and

operational unit(s) that are necessary for conducting the study.

Test facility management: Consists of the executive level of management charged with the ultimate responsibility of the test facility and studies.

Test substance: A chemical substance or a mixture that is under investigation.

Test system: Any human, other animal, plant, microbial, as well as other cellular, subcellular, chemical or physical system or combination of these, that is exposed to a test, control or reference substance.

Validation: Establishing documented evidence that provides a high degree of assurance that the intended use of such things as a procedure, test system, test substance or control substance is accomplished.

Validation procedure(s): A written procedure stating how validation will be conducted. Validation procedures should exist for all elements of the test system and should specify the procedures and tests that will be conducted and the data to be collected. Examples include the validation procedure used for animal supply vendors, facility equipment, test substances control substances, and SOPs. See Also:

Toxicological Abbreviations


Page 76 of 76

quality management for chemical safety

Documents