Environmental Health Criteria 72 PRINCIPLES of STUDIES ON

Environmental Health Criteria 72

PRINCIPLES OF STUDIES ON DISEASES OF SUSPECTED CHEMICAL ETIOLOGY AND THEIR PREVENTION

Please note that the layout and pagination of this web version are not identical with the printed version. Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 72

PRINCIPLES OF STUDIES ON DISEASES OF SUSPECTED CHEMICAL ETIOLOGY AND THEIR PREVENTION

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, 1987

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.

ISBN 92 4 154272 1

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

Page 1 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

to the text, plans for new editions, and reprints and translations already available.

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

PRINCIPLES OF STUDIES ON DISEASES OF SUSPECTED CHEMICAL ETIOLOGY AND THEIR PREVENTION

INTRODUCTION

1. SUMMARY

2. DEFINITION OF THE PROBLEM

3. GENERAL APPROACHES IN THE INVESTIGATION OF DISEASES OF SUSPECTED CHEMICAL ETIOLOGY

4. DISEASE APPROACH: RECOGNITION AND DESCRIPTION OF A HEALTH PROBLEM

4.1. General background 4.2. Recognition of the index cases 4.3. Screening of populations for suspected cases 4.4. Case definition 4.5. Case identification 4.6. Importance of pathological studies 4.7. Reporting systems and case records

5. EPIDEMIOLOGICAL APPROACHES: SEARCH FOR DETERMINANTS

5.1. General background 5.2. Collection of information and criteria for obtaining specimens 5.3. In-depth study of cases 5.4. Evaluation of epidemiological data

6. TOXICOLOGICAL APPROACHES: STUDIES ON CHEMICALS POSSIBLY INVOLVED

6.1. Experimental animal studies 6.2. Analysis for toxic substances 6.3. Toxic chemical information data systems

Page 2 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

7. PROBLEMS IN ESTABLISHING CHEMICAL ETIOLOGY

7.1. Causation in human disease 7.2. Consideration of a chemical etiology 7.3. Temporal relationships 7.4. Biological plausibility 7.5. Dose-response and dose-effect relationships 7.6. Effects of intervention 7.7. Confirmation of a causal relationship

8. STEPS TO BE TAKEN FOLLOWING THE RECOGNITION OF THE CHEMICAL ETIOLOGY OF AN OUTBREAK

8.1. Preventive action and control 8.2. Surveillance and monitoring system 8.3. Health education

9. THE NEED FOR INTERNATIONAL COOPERATION

9.1. Cooperation and collaboration among countries 9.2. Global and regional activities 9.3. Final recommendations

REFERENCES

APPENDIX I. LIST OF BACKGROUND PAPERS

APPENDIX II. CONCEPTUAL FRAMEWORK USED IN SEVERAL STUDIES LEADING TO IDENTIFICATION OF THE CHEMICAL ETIOLOGY OF OUTBREAKS OF DISEASES

APPENDIX III. SUMMARY REPORT ON THE IPCS/WPRO/PRC MEETING ON KASHIN-BECK DISEASE

WHO TASK GROUP ON PRINCIPLES OF STUDIES ON DISEASES OF SUSPECTED CHEMICAL ETIOLOGY AND THEIR PREVENTION

Members

a,c Professor L. Amin-Zaki, Former Professor of Paediatrics (Baghdad), Abu Dhabi, United Arab Emirates

a Professor E.A. Bababunmi, Laboratory of Biomembrane Research, Department of Biochemistry, University of Ibadan, College of Medicine, Ibadan, Nigeria

a Dr R.V. Bhat, National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India

a,b,d Dr J. Borgoño, University of Chile, Department of International Affairs, Ministry of Health, Santiago, Chile (Vice-Chairman)

a Dr Y. Egashira, National Institute of Health, Hatano Research Institute, Food and Drug Research Centre, Hatanoshi, Kanagawa-ken, Japan

a,b,d Dr R.A. Goyer, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA

a,c,d Dr P. Grandjean, Institute of Community Health, Department of Environmental Medicine, Odense

Page 3 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

University, Odense, Denmark

a Dr V.N. Ivanov, Chita Medical Institute, Chita, USSR

a Dr V.V. Ivanov, Department of Pathophysiology, Krasnoyarsk Medical Institute, Krasnoyarsk, USSR

a,b,e Dr R.D. Kimbrough, Center for Environmental Health, Centers for Disease Control, Atlanta, Georgia, USA

a,d Professor P. Krogh, Department of Microbiology, Royal Dental College, Copenhagen, Denmark (Rapporteur)

a,c Dr O.A. Levander, Vitamin and Mineral Nutrition Laboratory, Human Nutrition Research Center, US Department of Agriculture, Beltsville, Maryland, USA

a,c Dr K.R. Mahaffey, Division of Standards Development and Technology Transfer, National Institute for Occupational Safety and Health, Cincinnati, Ohio, USA

a,d Dr G. Martin-Bouyer, Ministère de la Santé, Direction Générale de la Santé, Paris, France (Rapporteur)

a Professor V.A. Nasonova, Institute of Rheumatology, Academy of Medical Sciences of the USSR, Moscow, USSR

a,d Professor H.D. Tandon, New Delhi, India (Temporary Adviser) (Chairman)

a Professor Yan Jianbo, Haerbin Medical University, Haerbin, People's Republic of China

a,b Professor H. Yanagawa, Department of Public Health, Jichi Medical School, Minamikawachi, Tochigi-ken, Japan

a Professor Yang Guangqi, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China (Vice-Chairman)

a Professor Yu Weihan, Haerbin Medical University, Haerbin, People's Republic of China

Observers

a Dr Chen Junshi, Department of Nutrition and Food Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Chen Xiaoshu, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Chen Qing, Department of Environmental Health, School of Public Health, Beijing Medical University, Beijing, People's Republic of China

a Dr Dai Yin, National Institute of Food Safety Control and Inspection, China National Centre for Preventive Medicine, Beijing, People's Republic of China

Page 4 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

a Dr He Xingzhou, Department of Environmental Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Li Guangshen, Institute of Endemic Disease, Norman Bethune University of Medical Sciences, Changchun, Jilin province, People's Republic of China

a Professor Li Yurui, Division of Pneumoconiosis, Department of Labour Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Liu Yuying, Division of Industrial Toxicology, Department of Labour Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Lu Boqin, Division of Industrial Toxicology, Department of Labour Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Ma Tai, Tianjin Medical College, Tianjin, People's Republic of China

a Professor Niu Shiru, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Qin Yuhui, Institute for Environmental Health Monitoring, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Su Zhi, Department of Health and Epidemiological Control, Ministry of Public Health, Beijing, People's Republic of China

a Professor Tan Jianan, Institute of Geography, Chinese Academy of Sciences, Beijing, People's Republic of China

a Professor Wang Huizhou, Department of Nutrition and Food Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Wang Juning, Division of Environmental Toxicology, Department of Environmental Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Xu Genlin, Office of Research Management, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Xu Guanglu, Research Laboratory of Keshan Disease, Xian Medical University, Xian, People's Republic of China

Page 5 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

a Dr Yo Peipei, Division of Pneumoconiosis, Department of Labour Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

a Dr Zhen Xiwen, Institute of Epidemiology and Microbiology, China National Centre for Preventive Medicine, Beijing, People's Republic of China

Secretariat

e Dr T. Kjellström, Prevention of Environmental Pollution, World Health Organization, Geneva, Switzerland

a Dr M. Mercier, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland

a Dr M. Mitrofanov, Division of Noncommunicable Diseases, World Health Organization, Geneva, Switzerland

a,d Dr J. Parizek, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary)

d Dr A. Prost, Division of Environmental Health, World Health Organization, Geneva, Switzerland

------a Attended the Task Group meeting in Beijing, 28 October - 2 November 1985. b Chairman of a subgroup of the Beijing Task Group meeting. c Rapporteur of a subgroup of the Beijing Task Group meeting. d Attended the editorial meeting in Geneva, 28 April - 2 May 1986. e Available for discussions at the Geneva editorial meeting on a part-time basis.

NOTE TO READERS OF THE CRITERIA DOCUMENTS

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

* * *

INTRODUCTION

The prevention and control of locally endemic diseases is one of the essential components of primary health care. The IPCS Environmental Health Criteria documents dealing with mycotoxins

Page 6 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

(WHO, 1979b), aquatic biotoxins (WHO, 1984b), and pyrrolizidine alkaloids (WHO, in press), describe situations in which the prevention and control of outbreaks of locally-occurring diseases were made possible by the successful elucidation of the chemical etiology of the diseases. The strategies used in this disease- oriented approach, including the sequence and character of the questions addressed, differ from those used in predictive toxicology, where the starting point is not a disease but a known, defined chemical, evaluated for possible health effects.

When developing the methodology component of the International Programme on Chemical Safety, it was evident that there was a need for a document that would summarize the principles of studies aimed at the elucidation of the role of chemicals in the etiology of diseases, benefiting from the experience of experts who have successfully carried out such studies. The disease-oriented approach in relation to chemical exposures and malignant and occupational diseases has been dealt with in other programmes of the World Health Organization (WHO), including the International Agency for Research on Cancer (IARC), and will not be discussed specifically in this document. The document focuses on the principles of studies dealing with endemic non-malignant diseases and outbreaks of diseases of unknown origin and the possible role of exposure to chemicals in their causation.

Situations in which populations are permanently resident in an area and mainly dependent on locally produced food, offer conditions favourable for the occurrence of locally endemic diseases and also for the study of these diseases. These conditions are often found in developing countries and much can be learned concerning methods of studying the problem of the chemical etiology of diseases, from studies carried out in such regions. Thus, it was of great advantage that the Ministry of Public Health of the People's Republic of China agreed that the Institute of Preventive Medicine and its Institute of Health, Beijing, would host the IPCS Task Group meeting on the principles and criteria for establishing the chemical etiology of diseases of uncertain causality as a basis for their prevention, together with a joint conference on Kashin-Beck disease, a highly disabling disease of permanent character affecting a very large group of people in endemic areas in China.

The resulting draft document was finalized at an editorial meeting held in Geneva from 28 April to 2 May 1986 under the guidance of PROFESSOR H.D. TANDON (Chairman of the Task Group). Background papers, prepared by the participants at the request of the Secretariat, for the IPCS Task Group meeting in Beijing, are listed in Appendix I. The Chairman of the Task Group, responding to a request of the Secretariat, prepared another paper summarizing the conceptual framework that had been used in several studies in

the past and had led to the successful identification of the chemical etiology of certain diseases and their prevention and control. This paper, which was used as a guide for the Task Group during discussions in plenary and subgroups and was further developed at the editorial meeting, is presented in Appendix II. The summary report of the Beijing meeting, reviewing the experience on Kashin-Beck disease, is presented in Appendix III.

The help of the host Institutions in Beijing, the organizers of the meetings, and all participants, including those who finalized the document at the editorial meeting in Geneva, is gratefully acknowledged. It is hoped that their life-long experience and enthusiastic involvement throughout the preparation of this

Page 7 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

publication will make it useful to all concerned with the prevention and control of diseases of possible chemical etiology.

The United Kingdom Department of Health and Social Security generously supported the costs of printing.

1. SUMMARY

It is suspected that some endemic diseases or sudden outbreaks of unknown diseases in several regions of the world might be related to exposure of affected populations to one or more natural or man-made chemical substances.

This publication is based on experience from studies and investigations in which the chemical origin of several unknown or unusual pathological conditions has been successfully identified. The basic principles of these studies are summarized and a step- by-step approach is described starting with the recognition of the disease pattern among the affected people, evaluation of the magnitude of the problem (number of people affected, geographical impact area, etc.), and collection of clues pointing to, and eventual identification of, its chemical etiology. Such an approach is called the disease approach. The search for etiology begins after the recognition and description of the health problem being investigated.

The second section of the paper deals with the epidemiological approach. The search for specific determinants is described, including methods that should be used, and how data should be analysed. This is followed by a description of the toxicological studies necessary for testing hypotheses on possible exposure to various substances, the hypotheses having been generated through the combined evaluation of clinical and epidemiological data. Problems, including bias, in establishing the causal relationship between one or several suspected agents and the observed disease patterns are also considered.

A further section deals with steps that, in the opinion of the Task Group, should be taken when the chemical etiology of an outbreak of a disease has been recognized. The publication ends with a section on the need for international cooperation in the field of recognition of the chemical etiology of certain diseases.

A sequential list of activities that has been followed in several successful studies and can be used as a checklist, when a similar approach needs to be developed, is included as Appendix II.

The meeting at which this publication was prepared was held in the People's Republic of China together with another international meeting organized jointly by the IPCS, the WHO Regional Office for the Western Pacific, and the health authorities of the People's Republic of China. The second meeting dealt with the present status of research on Kashin-Beck disease, and osteoarthrosis deformation, which is endemic in certain parts of Asia and, in certain regions of the People's Republic of China, has affected more than one million inhabitants, mainly children. This disease is given special attention, because it affects a very large segment of the population in a developing country and because of growing evidence that deficiency of a certain essential nutrient combined with exposure to a specific chemical may be the factor responsible for this peculiar disease and its specific endemicity.

2. DEFINITION OF THE PROBLEM

Page 8 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

The prevention of disease constitutes a major component in world-wide efforts to achieve "Health for all by the year 2000". Preventive measures must be directed towards the control or eradication of specific etiological factors that cause human diseases. Thus, a prerequisite for successful prevention is adequate knowledge concerning disease causation.

While several diseases are known to be heritable and therefore to have a genetic etiology, other diseases are known to be caused by specific environmental factors, as in the case of intoxications, infections, or nutritional deficiencies. However, at present, the etiology of most human diseases is not known or is only partially known, and methods for primary prevention based on recognition of etiology can only be considered for a limited number of diseases. The search for etiological factors will therefore achieve increasing importance as a basis for future preventive measures. In this regard, diseases caused by chemical factors are of particular interest, and their effects on human health have not been sufficiently investigated, even though exposure to naturally- occurring chemicals has always been a part of human life. With further industrialization as an essential step in development, people are likely to be exposed to more and increasingly diverse chemicals, even though careful steps may be taken to monitor and control their proper use and the safe disposal of industrial wastes. New chemicals are developed for a variety of purposes, both industrial and non-industrial, and the lack of preventive measures, or insufficient application of such measures, may result in toxic effects in populations exposed to them. Furthermore, exposure to chemicals or combinations of chemicals under various conditions of human life may result in toxic effects, not previously recognized. Chemicals in the form of fertilizers and pesticides are particularly essential in modern agriculture, to meet the increasing needs for food, and exposure of the general population to the residues of these chemicals is increasing. In most parts of the world, the agricultural community is often not sufficiently informed about the proper use of chemicals and the associated hazards, and dangerous exposures are likely. The episode of alkylmercury poisoning (WHO, 1977) is an example of large-scale casualties caused by such accidents.

Exposure to chemicals may cause human disease in several ways. First, a certain disease may result directly from exposure to a specific chemical compound, for example, Minamata disease, which is caused by exposure to methylmercury (WHO, 1977). Second, exposure to a chemical may be only one of several factors contributing to the development of a disease, and, thus, be part of a multicausal relationship. Itai-itai disease is an example of a disease with a complex etiology, caused by several factors in combination with cadmium toxicity (Tsuchiya, 1978). Chemical exposure may also aggravate a pre-existing disease. For example, air pollution with nitrogen oxides will provoke air-way symptoms in patients with respiratory diseases (WHO, 1979a). Thus, exposure to chemicals may constitute a leading factor in the development of a range of human diseases.

An important determinant of health is the balance between the status of the milieu intérieur and that of the milieu extérieur. Excess or deficiency of naturally-occurring chemicals in the natural environment or the presence of extraneous chemicals may alter this balance by making the body tissues more vulnerable. Fluorosis is known to be endemic in several parts of the world because of excess fluorides in the drinking-water; similarly, there are large areas where goitre is endemic, because of iodine deficiency in the environment. Nutritional factors play an

Page 9 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

important role in both exposure and susceptibility to various chemical agents.

The practising physician may not be familiar with the ever increasing diversity of chemicals that can be hazardous for man, nor with the initial symptoms and signs of toxicity. This is one of the reasons why the establishment of a cause and effect relationship between disease and exposure to chemical agents is less straightforward than between disease and infections. Episodes of chemical poisoning in the form of isolated cases, groups of cases, or outbreaks, which present a pattern of disease that does not fit in with the symptom complexes of known diseases, particularly in developing countries, are likely to be first considered as of infectious origin or to be the result of malnutrition (Anon., 1984). The Afghan outbreak of veno-occlusive disease was considered to be a water-borne infection, until the chemical etiology was discovered through recognition of a plant growing among the staple food crop of wheat. The chemicals responsible for the disease were the pyrrolizidine alkaloids in the seeds of the plant, which were mixed with the grain (Tandon & Tandon, 1975).

Generally speaking, there are no sets of clinical characteristics of diseases that, alone, could lead the investigator to suspect chemical etiology and exclude other causes. It is only when the total context of the disease pattern and the circumstances of its appearance is considered that such an etiology might be suspected, though some clues may emerge, even from the initial epidemiological characteristics.

The epidemiological characteristics of diseases of unknown etiology may vary in the following ways:

(a) A sudden outbreak of a disease that is considered "new" is often attributed to an unknown infectious agent. The search for the etiology should take into account factors of a chemical nature. An evaluation of the epidemiological pattern of the disease might provide useful orientations that can be fruitfully pursued. For example, the outbreak of liver disease in India, which was later confirmed to be due to aflatoxin exposure (Tandon et al., 1977, 1978a), was initially considered to be one of infectious hepatitis, because the dominant features of the disease were jaundice and other systemic symptoms that suggested hepatocellular failure. The initial epidemiological characteristics of the disease indicated

that transmission of the virus was highly unlikely to be a causal factor, and this prompted the search for another environmental factor.

(b) Endemic diseases continuously occur and recur in defined geographical areas. Although such diseases constitute a chronic health problem that could be studied by conventional epidemiological methods, many endemic diseases have an environmental etiology of unknown nature. For example, according to the recent estimates, Kashin- Beck disease has affected about 2 million individuals in certain endemic regions of China, but the specific chemical etiology has so far escaped discovery (Appendix III).

When the exposure is not sufficient to result in the clustering of cases, the disease may remain unnoticed or

Page 10 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

unknown.

(c) Some commonly occurring diseases may include exposure to chemicals as a risk factor. However, the role of widespread, long-term, low-level complex exposure in the multifactorial etiology of such diseases is more difficult to establish and this problem is not addressed in this publication.

In searching for an etiology, it is important to recognize the source of the chemical. Diseases that have been identified as chemically induced, have been associated with various chemicals with the following origins:

(a) Naturally-occurring inorganic chemicals

There are many naturally-occurring inorganic chemicals that have been found to be toxic for man, e.g., mercury, lead, and cadmium. Although these chemicals are neither created nor destroyed by man, geochemical conditions, natural processes, such as accumulation in certain biota, and industrial activities may result in their widespread but uneven distribution, or in the formation of new compounds that may be more or less toxic than the naturally-occurring forms.

Diseases can also be caused by excessive exposure to inorganic chemicals that may be beneficial or even be a metabolic requirement at low levels. A relationship has been established between skeletal fluorosis and the long-term elevated intake of fluoride (WHO, 1984a).

(b) Chemicals of plant origin

Several episodes of disease have been reported resulting from the accidental contamination of food crops by certain weeds and their seeds. Thus, contamination of staple food crops with pyrrolizidine alkaloid-containing seeds has caused large-scale outbreaks of veno-occlusive disease in Afghanistan (Tandon &

Tandon, 1975; Mohabbat et al., 1976) and the USSR (Dubrovinski, 1952). Furthermore, veno-occlusive disease may also result from the use of pyrrolizidine alkaloid-containing herbs as traditional home remedies, as seen in the West Indies (Stuart & Bras, 1955) and elsewhere (Huxtable, 1980; Ridker et al., 1985).

Staple food sometimes contains toxic substances that are removed during food processing or do not represent a hazard for human health under normal circumstances. However, as seen in Mozambique, modifications in plant constituents induced by environmental changes, together with changes in methods of food preparation, and patterns of consumption, can lead to acute or subacute poisoning. An outbreak of spastic paraparesis in Mozambique was shown to be related to cyanide intoxication caused by cassava. During a severe drought, the cyanide content of the cassava increased naturally. Moreover, famine forced most families to turn to bitter cassava, which has a higher cyanide content, and to eat it after drying it for a few days only, whereas several weeks are required for proper detoxication (Mozambique Ministry of Health, 1984).

Exposure to toxins from fungi (mycotoxins) takes place mainly

Page 11 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

through the ingestion of contaminated foods, though airborne exposure to fungal spores containing mycotoxins is also possible (WHO, 1979b). Contamination of foods of plant origin takes place generally in the post-harvest period through invasion by moulds. It is difficult to detect by conventional food inspection practices because of the microscopic appearance of the fungi. An example of mycotoxin-associated disease is the toxic effects caused by aflatoxins in food grains (WHO, 1979b). Exposure to algal toxins is generally caused through the ingestion of fish and shellfish that have been feeding on toxic microscopic algae (plankton) and thereby retaining part of the algal toxins, without actually showing any organoleptic changes. Examples of such diseases are paralytic shellfish poisoning caused by saxitoxin and derivatives, and diarrhoeic shellfish poisoning caused by okadaic acid and derivatives (WHO, 1984b). Diseases induced by bacterial toxins are caused by inadequate food processing, during which the toxin- producing bacteria may infect foods and produce toxins, just prior to consumption. For example, staphylococcal food poisoning is caused by enterotoxins from Staphylococcus aureus, and botulism, by neurotoxins from Clostridium botulinum growing in processed meat, fish, and vegetables.

(d) Man-made chemicals

A number of acute outbreaks and incidents of illness have been found to be caused by exposure to pesticides, fertilizers, germicides, or industrial chemicals. Some of these episodes have been explained as outbreaks of acute poisoning caused by the accidental contamination of food, for example by parathion (Diggory et al., 1977), or of items of household and personal use, such as

baby powder, by hexachlorophene (Martin-Bouyer et al., 1982), and laundry rinses, by pentachlorophenol (Robson et al., 1969). Although only short-term exposure may have occurred, health effects may be permanent, or recovery may be very slow as in the Yusho episode in Japan where the deceased people had ingested a mixture of polychlorinated biphenyls, dibenzofurans, and quarterphenyls (Kuratsune et al., 1969) or in Turkey where liver disease and porphyria cutanea tarda resulted from contamination of grain with hexachlorobenzene.

3. GENERAL APPROACHES IN THE INVESTIGATION OF DISEASES OF SUSPECTED CHEMICAL ETIOLOGY

The nature and magnitude of the problem, as well as the facilities available for investigating diseases possibly caused by chemicals, vary from country to country for a number of reasons including the extent to which the health services have been developed, and whether stray cases or outbreaks of toxic disease are being studied. Countries with well-developed infrastructures in the health services usually have rigid control systems with regard to food quality, sanitation, and water supplies, and good investigative facilities. Reasonably good health-care facilities are available, even in outlying areas. If outbreaks of chemically- induced diseases occur, they are usually the result of a short-term exposure to a toxic chemical, affecting a limited number of cases, which are likely to be noticed as soon as they appear. Thus, expeditious steps can be taken for their investigation or control. Investigation and control of outbreaks in countries with inadequately developed health services need a different approach, though the principles may be the same.

In this publication, a strategy is discussed, which might be used in the investigation of episodic or endemic diseases of

Page 12 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

suspected chemical etiology. Instead of asking what are the effects of a specific chemical, the question can be reversed to ask which chemicals might cause a particular health effect. It may be difficult to distinguish a chemically-induced from an infectious disease and to identify the etiological chemical agent. Clues are often elusive and inextricably mixed with day-to-day occurrences or the "not-so-unusual" disease phenomena affecting a population. The search for an etiology often necessitates sequential planning for a progressive build up of clues.

The identification of the chemical causes of a disease involves a group of interrelated disciplines, in particular pathology, epidemiology, and laboratory medicine and toxicology. The approach may be a straightforward process when a large group of people is exposed to a chemical with known toxicity, but this may not always be the case. The search for an etiology will depend on scientifically clear evidence of linkages between exposure to a chemical, the biological effects, and subjective clinical symptoms.

Appendix II includes a description of a conceptual framework of studies on outbreaks (episodes), which resulted in the successful identification of their chemical etiology and, through this, their control and prevention. However, it should be said at the outset that it is not a blueprint for action, but an example that could be suitably modified as the situation demands. It was designed to investigate the causes of diseases in which chemicals in the environment were the principal, rather than a minor, contributory factor.

Appendix III includes a summary report on the present state of research concerning the etiology of a wide-spread endemic disease in which exposure to certain environmental chemicals is considered an important etiological factor.

In the following sections, the approaches that should be considered simultaneously, when the chemical etiology of a disease is being investigated, are discussed at some length.

4. DISEASE APPROACH: RECOGNITION AND DESCRIPTION OF A HEALTH PROBLEM

4.1. General Background

To investigate the etiology of outbreaks of diseases caused by chemicals, the "disease approach" is the first step in the methodological framework. Epidemiological and toxicological approaches are then developed as analytical tools to generate valid etiological hypotheses and to test them at each stage of the investigation. The steps or phases recommended are intended to be progressive and the decision to terminate or modify the process might be considered at any phase, if appropriate.

In order to investigate outbreaks of disease, or the endemicity of a disease, it is important to be familiar with the general disease patterns in a specific geographical area. The incidence and types of diseases may vary, to some extent, in different countries and may change with socioeconomic conditions and industrial activity. However, awareness of the epidemiological pattern in a given population will make it possible to recognize changes in disease patterns, the occurrence of new diseases, the sudden outbreak of epidemics of infectious diseases, outbreaks of poisoning, or the presence of endemic diseases in localized geographical areas. The recognition of such diseases is usually based on observations of health personnel that the frequency of a common disease has increased, that a hitherto unknown constellation

Page 13 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

of clinical manifestations has been encountered, or that the age- specific incidence of a disease is shifting. When such observations are reported, they must be critically evaluated to determine whether these observations are truly abnormal. For example, it is possible that a physician with a special interest in certain diseases may simply recognize them more frequently, or that diseases of different etiological factors may be erroneously grouped together.

The methodology of epidemiology as described in a recent publication (WHO, 1983) outlines choices of study design, methods for the assessment of exposure, and signs and symptoms for the ascertainment of health effects. However, the precise etiology of the condition under study cannot be established from the resulting association alone. In addition, there may be lack of information on exposure, evidence of combined toxic exposures, or, perhaps most important of all, lack of proof or evidence supporting hypotheses linking exposure to specific environmental factors with the disease. Several disciplines must be involved in providing precise identification of etiological factors.

Disease investigation begins with awareness of an apparent and unusual number or cluster of cases with a clinically characterized disease entity (index cases as discussed below). Therefore, the initial investigations must almost always involve some level of epidemiological investigation. An unusual clinical phenomenon in a population may:

(a) be characterized by pathognomonic signs that do not occur in a disease of known etiology;

(b) be associated with combination of non-specific clinical features, signs, symptoms, and laboratory and other data that do not fit into a known disease category;

(d) constitute an endemic disease of unknown etiology.

After ascertaining that there is an unusual clinical phenomenon in the population, the search for a chemical etiology and subsequent action can proceed in the following phases:

(a) descriptive phase, in which the disorder is characterized by clinical, pathological, and epidemiological features;

(b) hypothesis-generation phase, in which a hypothesis is assumed concerning the possible etiological factors, on the basis of clues obtained from the initial studies; further supportive clues are searched for;

(c) hypothesis-testing phase, in which the clues obtained are tested by the identification of biological markers of exposure or injury, detailed pathological studies on diseased tissues in man and affected animals, and toxicological studies on animals;

(d) follow-up evaluation, in which follow-up action is taken, if the causative agent is successfully identified.

These steps are outlined in the Appendix II.

4.2. Recognition of Index Cases

Page 14 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

Index cases refer to the first reported case or cluster of cases of the disease under study. It may happen that the first reported case is not the first one to occur, and that the true index case has remained unnoticed. A retrospective analysis of all available information should always attempt to trace the disorder backwards to the true index case. In the rural areas of some developing countries, these cases may be identified by paramedical personnel or practitioners of traditional medicine. In urban areas, they may initially come to the attention of the emergency medical services, or, in the industrial sphere, be identified by occupational medicine specialists. In order to reduce, or possibly exclude, distractors from among the cases included in the affected category, it is essential to define the characteristics of such index cases. Typically, the index cases exhibit severe forms of the disease with overt clinical manifestations. At this stage, the whole range of signs and symptoms associated with the disease may not be recognized, and index cases should later be thoroughly evaluated and a general case definition developed.

Once it is presumed that a normally occurring illness is suddenly occurring at a higher frequency (or has reached epidemic proportions), or that there is an outbreak of a disease that has not been observed previously, it must be established whether a true outbreak exists. For instance, it is possible that certain conditions in the general population may be overlooked, until special interest develops or appropriate diagnostic methods are introduced, with the result that suddenly such conditions are reported more frequently. This increase merely represents a reporting artifact and not a true increase. Furthermore, a variety of diseases may be thought to represent the same condition and the increase in this condition is again merely a reporting artifact.

After having defined the diagnostic criteria on the basis of identified index cases, a search should be carried out to identify all suspect cases. The principles of epidemiological studies are described in detail in a previous publication (WHO, 1983). Disease characterization can be carried out by a sequential approach. Initial screening of the population aimed at identifying all cases suspected of being involved in the outbreak is followed by phases in which the disease becomes progressively more clearly defined as more specific clinical and laboratory criteria are applied. As an example, in the investigation of the aflatoxin outbreak in India (Tandon et al., 1977), all cases of jaundice associated with hepatomegaly and other systemic symptoms were included in the primary screening. When liver biopsies were taken, features characteristic of acute infectious hepatitis were seen in occasional cases, and these were excluded at the stage of case definition, described in section 4.4.

4.3. Screening of Populations for Suspected Cases

After the index cases have been evaluated, a decision may be made to screen the affected community. This decision is not automatic and must be based on an assessment, by appropriately trained medical and public health personnel, that an outbreak of the disease has occurred and may be more widely distributed than appears from the index cases. The aim of screening the population is to estimate the total incidence or prevalence of a set of signs and symptoms considered to suggest a disease related to the suspected etiology. Such screening in developing countries is frequently carried out by paramedical personnel. Accordingly, the tests need to be easy to use, e.g., standard questionnaires based on information obtained from the index cases. An example would

Page 15 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

include simple tests of motor coordination to evaluate nervous system changes, without the use of specialized medical equipment. Personal history should include age, sex, occupation, life-style variables, and place and duration of residence. In addition, past medical history should include relevant data on previous and current diseases, pregnancy, and lactation.

The number of persons to be included in the initial screening depends on the specific circumstances of the outbreak. Clearly, the key limiting factors are economic resources and available personnel. The efficacy of the screening will be improved by

eliciting the full cooperation of the persons potentially affected by the disease. The benefits for the population of participating in the primary screening should be emphasized. Support from the affected person's family and, for example, the village or tribal heads, is helpful in conducting primary screening. In more urbanized areas, the support of community groups or factory workers' organizations could be beneficial.

4.4. Case Definition

At this stage of the investigation, identification of different stages of the disease becomes possible. Cases range from those with the most severe symptoms to those with only subclinical effects. The case definition should not be so restrictive that only the "tip of the iceberg" is identified. It should be specific enough to separate the disease of concern from diseases of other origin. Ideally, one sign or symptom would be pathognomonic of the disease under investigation. However, this is rarely the case. Generally, a number of signs and symptoms and the pattern of their appearance are used to identify the disease. Individually, these signs and symptoms would not be either sensitive or specific. However, the clustering and relative severity of these signs and symptoms greatly increase their use in identifying cases of the disease.

Procedures used in case definition are generally much more reliable and complex, and involve laboratory support and techniques not applicable in the initial screening. This part of the study requires more highly trained personnel than the initial screening. The criteria are based on the most severe cases at the initial stages and must therefore be considered provisional, since they will not include mild disorders and subclinical effects. The case definition should include:

(a) Identification of the target organ or organs and the characteristic effects. It is critical to recognize that organ systems have limited types of response and that these are rarely pathognomonic for a disease caused by a particular chemical.

(b) Identification of specific groups that appear to be particularly vulnerable, e.g., infants, or persons living within a certain radius of a chemical industry.

A description of the clinical appearance of the disease should include the signs and symptoms, clinical course of the disease, and pathological findings. Pathological study of diseased human or animal tissues, early in the outbreak, might provide useful etiological clues. If autopsies are not available, biopsies from patients or autopsies of affected domestic animals, as mentioned in section 4, should be carried out. More detailed information should be gathered concerning the extent of the problem, i.e., precise

Page 16 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

figures on morbidity and mortality rates and time sequence.

Analysis of the demographic data obtained during screening will indicate whether or not there is a subpopulation at high risk, e.g., residents in a given area, an occupational group, young children, or persons with pre-existing problems. The spectrum of clinical severity may differ among groups of individuals affected by toxic chemicals to a greater extent than that among patients with an infectious disease. The wide variation in involvement may be due to differences in the levels and lengths of exposure and differences in host susceptibility, because of differences in age, nutritional status, etc., which should be specified in detail during the process of identification.

At this phase in the study, it is desirable to review toxicological information on the potential health effects of the toxic chemical suspected of playing a role in the etiology of the disease (section 6). Comparison may identify clinical or biochemical effects that have not been previously investigated and may provide a basis for confirming the relationship between the toxic agent, the case definition, and observed effects. Finally, toxicological studies may provide predictive information regarding prognosis, including the progression or reversibility of the health effects observed.

Because the background prevalence of signs and symptoms in the general population is not known, it is appropriate at this stage to survey all persons who appear to be affected by the disease. The inclusion of a comparison group is essential, in order to identify a tentatively discriminating factor.

4.5. Case Identification

The definition of the clinical entity, based on rigid quality control, high specificity and sensitivity, results in the identification of:

(a) early symptoms that may not have been recognized during the initial screening;

(b) associated clinical pathological/radiological features of the disease;

(d) complications beyond the acute phase, and sequelae.

In this phase of the analysis, the population is screened again to identify borderline, subclinical, or latent cases using established diagnostic criteria and specific biological markers. This may also give a more precise idea about the magnitude of the outbreak.

4.6. Importance of Pathological Studies

The pathological study of biopsy or autopsy specimens may provide the crucial clue(s) in the identification of the etiology of a disease. The proper conduct of pathological studies may contribute to the formulation of hypotheses regarding etiology, and influence the selection of subsequent studies. Morphological changes in the principal target organ may prove to be pathognomonic of the etiological agent. In the investigation of an outbreak of a disease in India, the presence of syncitial giant cells in human

Page 17 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

liver tissue and recognition of the fact that an identical lesion had been previously observed in the liver of rhesus monkeys exposed to aflatoxins, suggested the etiological role of aflatoxins in this human disease outbreak (Tandon et al., 1977, 1978a). The morphological changes observed may also help to exclude infectious disease as a cause of the illness.

For various reasons, there may be difficulties in obtaining human autopsy material. In such an event, biopsy of the diseased organ or examination of autopsy material from field animals suffering from a disease identical to that in man may prove useful, as again happened in the aflatoxicosis outbreak (Tandon et al., 1978a). The biopsy could be carried out as part of a diagnostic or therapeutic procedure for the benefit of the patient, for example a liver or a kidney biopsy could be carried out following paracentesis, or the bony tissue, resected as a part of corrective surgery, could be examined. Histological appearance may identify the specific target tissue and the affected cell type or could decisively indicate infection or some other specific disease. Descriptions should be in the internationally recognized standard medical nomenclature.

The usefulness of pathology specimens may be enhanced by providing advice and materials for proper tissue fixation, and storage instructions. Parallel sampling for chemical analysis should always be considered and such specimens should be stored by freezing and without fixation.

4.7. Reporting Systems and Case Records

In most countries, reporting systems exist for recording infectious diseases. A similar approach would be useful for the reporting of outbreaks and epidemics of diseases of unknown origin.

Delays in notifying central health authorities may occur for many reasons. If different patients are treated by different physicians, there may be a time lag before a disease outbreak is recognized. The delays should be avoided, as far as possible, as prompt and concise reporting is necessary for establishing the etiology of the disease and controlling the outbreaks. Biological specimens should be secured, especially from the index cases, and the time interval between onset of illness and biological sampling should be noted. Even initial studies might provide useful clues. Records should be kept of the index cases, if further evaluation or periodic reexamination of index cases is thought to be warranted,

e.g., for the development of a case registry and long-term, follow-up studies. Such a registry would allow the investigators to study the natural history (or progression) of the disease. Unfortunately, a number of index cases may be lost to follow-up, when it is not initially recognized that an outbreak is occurring. On the other hand, development of registries is not always desirable or cost productive. If long-term follow-up of affected persons is deemed desirable, certain possible constraints should be noted.

The following problems are generally faced in the development of a case registry:

(a) high costs and lack of financial support;

(b) cases of chemical toxicity are generally not reportable;

Page 18 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

personnel, capable of maintaining a registry;

(d) mobility of populations;

(e) lack of cooperation of health personnel;

(f) inconsistency of disease nomenclature.

The problems encountered in recognizing and recording diseases of chemical etiology are illustrated by the historical evidence on lead poisoning (Grandjean, 1975; Mahaffey, 1985). The failure to recognize lead exposure as a cause of the disease was due to: (a) failure to appreciate the importance of individual susceptibility; (b) unsuspected sources of exposure; (c) non-specific pathological changes; and (d) classification into several ill-defined clinical syndromes. With the occurrence of ubiquitous lead pollution, there may be other insidious health effects caused by past or long-term exposure, which may be difficult to identify or recognize.

5. EPIDEMIOLOGICAL APPROACHES: SEARCH FOR DETERMINANTSa

5.1. General Background

One of the important steps, when searching for the etiology of diseases, is to establish and validate associations between the occurrence of the disease and exposure to one or more common factors that could be considered as determinantsa. The classical epidemiological approach is to generate a hypothesis from preliminary information collected through descriptive study and then test it in an analytical epidemiological study of appropriate design, as described in detail by WHO (1983). Although this sequence is logical, the exploration of the chemical etiology of a disease may not necessarily follow this sequence, particularly when an acute outbreak of disease occurs. In such situations, rapid recognition and resolution of the problem is of the utmost importance. Although a series of steps is outlined below, in practice, they will often have to be carried out almost simultaneously.

The early recognition of an outbreak of, or unusual increase in, disease depends on careful observation of cases and a reliable reporting network. Expeditious investigation of the outbreak/occurrence of cases cannot be overemphasized. The investigator should obtain as much information as possible, such as the geographical extent of the outbreak, the population at risk, the number of cases, distribution of cases in the population, clinical manifestations, time of onset of the outbreak, and whether similar outbreaks have occurred in the past. This preliminary information will enable the investigator to develop the tentative hypotheses necessary to decide on the technical support systems needed. Additional advice can be obtained from the pertinent scientific literature and from international agencies and other institutions in relation to the nature of samples that should be collected, and the names and addresses of specialized laboratories for the analysis/identification of toxic materials and biological fluids/tissues from diseased animals and patients.

5.2. Collection of Information and Criteria for Obtaining Specimens

The availability of properly trained and briefed personnel is an essential element in the conduct of a study. The investigating team will have to obtain detailed data on the disease characteristics and must include adequately trained clinical staff. The help and cooperation of international agencies and other

Page 19 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

departments within the country, or in other countries, may be

------a A determinant is any factor, whether event, characteristic, or other definable entity, that brings about change in a health condition, or other defined characteristic (Last, ed., 1983). The same source recognizes predisposing factors (e.g., age, sex, occupation), enabling factors (e.g., climate, income), precipitating factors (e.g., exposure to noxious agents), and reinforcing factors (e.g., repeated exposures) in causation of disease.

needed, but the investigating team should comprise local investigators, as a cultural and language identity with the populations being investigated is essential. When possible, the laboratory worker who will analyse the samples should participate, otherwise this team should receive adequate briefing from the responsible laboratory regarding the type and amount of samples to be collected, method of storage, transport, etc. In particular, a statistician should be consulted regarding the size of population to be studied, when dealing with epidemiological studies of chronic diseases. In the absence of ideally trained personnel in the investigating team, efforts should be made to brief and train available people appropriately.

A wide range of specific information should be collected in order to identify, as far as possible, all the relevant factors common to the identified cases. Examples of such factors are given in Table 1. Another possibility could be the use of the classification proposed by Last (1983) with an identification of the predisposing, enabling, precipitating, and reinforcing factors. However, situations will differ, and some of the items listed may not apply in certain situations.

Table 1. Factors to be considered during an epidemiological investigation of an unknown disease ------(a) Factors relating to the physical environment

Geographical location, including altitude, nature of terrain, distance to rivers, lakes, swamps Climatic conditions Seasonal occurrence of symptoms

(b) Man-made environmental factors

Diet Food preparation, cooking, and eating habits; storage practices Water supply, sanitation Agricultural practices, including the use of fertilizers and pesticides Industrial environment, including mining

Age distribution Sex distribution Family aggregation of cases and family structure Social and cultural factors: ethnic groups, religion, education, etc. Occupation Socio-economic status, including income level Mobility of the population, migration

Page 20 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

Duration of residence in the area ------Note: Recent changes concerning any of these factors should be carefully documented.

Background information on general health statistics should be collected through government/municipal/county/hospital/primary health centres, together with the following items: morbidity, mortality, case fatality rates, and the trends in these indicators. Special attention should be paid to the possible occurrence in the past, or in nearby areas, of similar outbreaks, or even sporadic cases, constituting the unusual clinical entity.

Information on the environment must include recent changes in the environment if any, such as the destruction of forests, building of dams, opening of new industries, discharge of effluents, new range of products, or change in manufacturing processes. The agro-ecological factors include the introduction of new varieties of food grain, mechanization of agriculture, use of agrochemicals, storage practices of harvested food, changes in the cultivation pattern, etc.

A co-existence between a disease in human beings and a similar disease in animal species provided important clues in some of the earlier studies of diseases later identified as being caused by exposure to chemicals in the environment. Examples of these studies are those on Minamata disease (WHO, 1977), veno-occlusive disease (Tandon & Tandon, 1975; Tandon et al., 1976), and the aflatoxin outbreak in India (Tandon et al., 1977, 1978a). Information from farmers or veterinary officials on the outbreak or occurrence of related diseases in farm and domestic animals or other fauna and flora may yield valuable data.

Various additional sources can be used in the collection of relevant information. The most useful information would generally come from the members of the affected community themselves. Further sources of information include community elders, social workers, and health personnel, including primary health and paramedical workers, general practitioners, and practitioners of traditional medicine. Attention must also be paid to additional factors. Thus, a family member or a member of the affected community may have very specific useful information such as practices in food storage or the special use of pesticides. Some data could be collected through formal questionnaires for the village (unit) level, family level, or individual level. Other methods could include less-structured personal interviews. Some useful information may already be recorded elsewhere, for instance demographic characteristics.

An intensive or extensive sampling strategy should be designed, depending on a balanced trade-off between what would be desirable and what is feasible. The specimens to be sampled should include biological samples, as well as samples of food, air, water, and other commodities. In the absence of precise knowledge of the possible etiological factor and particular source, a large number of samples covering a wide range should be collected, the sample size being adequate for a reasonable range of analyses. The range of sampling to be carried out should be decided on after generating a tentative hypothesis on the possible etiological factors, based on previously collected information. However, even if a precise

hypothesis has not been defined, potentially useful samples must be obtained as early as possible. The samples should preferably be representative of the index cases, and sampling could be restricted

Page 21 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

to the families or individuals with the most typical case history. The samples should be obtained from both affected and non-affected areas.

For practical purposes, there are limitations to the number of samples that can be collected and processed. For this reason, some judgement must be made as to what would represent the most useful samples. Such decisions are influenced by the time that has elapsed between the onset of the illness and the beginning of the investigation, the pertinence of the samples, and the possible routes of exposure. For instance, if a food commodity is suspected in acute outbreaks, the food that was consumed immediately prior to onset of illness would represent the most valuable samples. Collection of the cooked food actually consumed is emphasized. Samples can also be collected from the food grains and the cooking medium from both local and commercial stores. Identifying pertinent exposure to environmental sources may be more difficult in the case of chronic illnesses, as such exposures may have occurred some time in the past or may have varied over time.

There is a danger that samples might be contaminated, destroyed, or damaged during storage or transit. Biological samples should be properly preserved in formalin or in an insulated container with ice or dry ice. Agricultural commodities should be adequately dried before transporting. These requirements must be agreed on with the analytical laboratory.

5.3. In-Depth Study of Cases

The result of the initial investigation is the identification of one or several factors that are common to all identified cases of the disease or seem to be related to its distribution in the community. An in-depth study of the outbreak/occurrence of cases requires that population-based studies, aimed at identifying the determinants of the disease, should be complemented with case- control studies. In the case-control design, cases of the disease are matched to a similar number of controls (people with no symptoms of the disease), in order to test the strength of the association between the exposure to the suspected etiological conditions and the occurrence of the disease. The relative risk associated with exposure can be calculated on the basis of such studies (section 5.4).

Once the situation has been evaluated in the field, a special subgroup of the diseased population can be selected, unless only a small group of people have been affected, and studied in depth as a supplement to more formal epidemiologically designed investigations. Such studies include detailed background questionnaires, medical examination, and relevant laboratory tests. The type of information to be collected will depend on the situation. It is important to be as concise as possible and not to obtain a great deal of extraneous information, as this will only impede analysis of the data (see also sections 5.2 and 6).

The selection of cases for detailed investigation will depend greatly on the specific factors involved in the particular outbreak. The following factors deserve consideration. The most typical cases should be included. If more than one member of a family is affected and a common factor among families is sought, then it may suffice to examine only one member per family. However, if the family is to be characterized, all members of the family should be examined. It might be useful to confine the examination to children or other specific segments of the population. In other situations, a common factor might be sought

Page 22 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

in individuals belonging to diverse subpopulations. Cases that suddenly develop in another geographical area may be useful in identifying a common factor. Alternatively, random sampling can be used to obtain representative samples for the epidemiological studies.

Having dealt with the more readily available information in an acute outbreak of disease, additional studies should then be contemplated, such as more detailed morphological examination of affected target organs, and chemical analysis of biological fluids and tissue samples. In addition to the fixation of tissues for morphological studies, selected tissues and biological fluids should be frozen for possible chemical analysis, only after obtaining initial clues suggesting a possible toxic agent. For the successful identification of an etiological agent, it is important that the potential source of the toxic agent is identified and the exposure established through analysis of the incriminating material, such as food, or of body tissues and fluids. The samples of such tissues and fluids, as well as of the incriminating material have to be properly collected and preserved following the advice of the analytical laboratory. In the absence of specific information to the contrary, freezing at the lowest possible temperature is the best method of preservation. Individual samples should be identified and stored separately, and pooling or mixing of samples should be avoided. However, discretion in using this approach is advised, since extensive analysis is laborious and will rapidly deplete the supply of available samples.

5.4. Evaluation of Epidemiological Data

The data collected must be analysed for the existence of dose- response and dose-effect relationships and for the occurrence of specific differences between affected and comparable unaffected individuals. Although results obtained should preferably be representative for the population examined, it may not be possible to meet this criterion very rigidly in studies of acute outbreaks. Appropriate reservations as to the general validity of such results must then be made. In some outbreaks of acute poisoning, the illness experienced and the development of the epidemic may be so specific that it may not be feasible or necessary to apply a statistical approach. A statistically significant result may only indicate a "spurious" association occurring by chance, and a non- significant association could be due to an insufficient number of observations, lack of precision in the measurements performed, and other factors.

The information obtained must be carefully scrutinized for the presence of bias. For example, bias in selection may be of importance if only the most severely affected patients are examined and differ in some important aspects from the group of patients as a whole. Ascertainment bias may occur if a local informant is particularly aware of, and reports, cases. Reporting bias in information may occur if prior information about the disease in the news media influences answers to questionnaires. Errors may occur in many situations, if samples are mislabelled or if artifacts are introduced. Confounding is likely to pose a problem, especially in studies of multifactorial diseases.

Epidemiological data can be analysed using the case-control approach and the follow-up approach (WHO, 1983). In both approaches, control groups need to be selected. In the first approach, the controls are people without signs and symptoms of the disease; in the other approach, the controls are those who have not been exposed to the chemical suspected of causing the disease.

Page 23 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

In the study of acute outbreaks of unknown etiology, it is usually not feasible to select proper control groups, as the healthy people in the affected area may also be exposed, but to a lesser extent, and usually the causative agent has not been identified at this stage. The usual procedure is to select as a comparison group, people who do not present signs and symptoms characteristic of the disease, or who appear not to have been exposed to the suspected agent. In fact, these comparison groups are never fully comparable to the group under study and it should be emphasized that the absence of valid comparison groups may severely hamper the value of the study. Although, during outbreaks, it may not be possible to identify valid comparison groups, tentative conclusions must be drawn on the basis of a critical assessment of all available data by the observer. On the other hand, when dealing with endemic disease, it should be possible to select proper comparison groups controlled for specific suspected determinants on a prospective basis.

6. TOXICOLOGICAL APPROACHES: STUDIES ON CHEMICALS POSSIBLY INVOLVED

6.1. Experimental Animal Studies

Animal studies are being used to complement investigations of outbreaks of disease and of endemic diseases of suspected chemical etiology. The principal aim of these studies is to assist in the identification of the toxic substance and also to clarify the mechanism of action of the substance, after it is identified, and to develop an animal model for further study of the disease.

When a possible route of entry of the toxic agent is suspected, the material can be administered to animals by this route to see whether or not an illness is produced. The animal model developed should reproduce the pathological features of the disease closely similar to those in human beings, and attention should be given to the target organ and types of cells involved (e.g., neurons, chondrocytes, etc.), the presence of storage materials in tissue or inclusion bodies, and the types of cellular reaction in the injured tissues. It may not be possible to reproduce the exact clinical disease in animals, because the species chosen for study may simply not react to the toxic agent in exactly the same way as a human being. Alternative species may be more or less sensitive to the offending agent, or the target organs may vary in different species. Therefore, toxicological testing should preferably be done using a species in which a disease similar to that encountered in human beings has been observed in the field. At times, it may not be possible to develop an animal model by simple exposure to the suspect chemical, since the disorder may be multifactorial, and simultaneous exposure to other environmental factors may be necessary to develop a syndrome comparable to the human disease. Such factors could include: concurrent infectious disease, nutritional deficiencies, and mixed chemical exposures. For instance, in encephalitis associated with the oral intake of bismuth salts, it was impossible to reproduce the disease in any animal species, in spite of the fact that the role of this compound in human disease had been established (Martin-Bouyer, 1981).

As mentioned, human beings may be more or less susceptible to the chemical than animals. Nevertheless, the dose-response relationship is an essential consideration in the design of an experimental study. The study should be designed to determine the minimal toxic dose, whether the toxic chemical may be accumulated, and if there is a time lag between the exposure and the effect. For instance, in the methylmercury outbreak in Iraq, loaves of

Page 24 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

bread containing the toxic substance were fed to domestic animals, but time was not allowed for the toxic effects to become manifest (latency period), resulting in the false interpretation that the bread was not toxic (Bakir et al., 1973; WHO, 1977). Also, if the material fed to animals is a foodstuff, it must be ascertained that a caloric or nutritional imbalance or deficiency is not produced, resulting in disease symptoms not related to the toxic material.

Animal studies can provide information on the development of characteristic pathological changes produced in the affected organs by the chemical suspected of being involved in the etiology of the disease. They can also help to identify factors influencing susceptibility to this compound. When the possibility cannot be excluded that the etiology concerns exposure to a complex mixture, or is otherwise multifactorial, it would be misleading to base the evaluation only on the results of tests for the effects of exposure to a single compound.

Studies on biochemical effects should be designed and interpreted with cautious consideration of other measures of toxicity. Studies of the metabolism and toxicokinetics of a putative chemical agent in animal models can provide information, important for planning and for the interpretation of results of screening and of analysis of biological specimens for toxic substances and their metabolites. Such information includes identification of metabolites, determination of excretory rates, and elimination half-times of the chemical agent and its metabolites. Principles of toxicological investigations and toxicokinetic studies are dealt with in detail in previous EHC documents.

6.2. Analysis for Toxic Substances

Analytical chemistry is important in investigations of endemic disease or of acute and chronic poisoning episodes in order to identify and quantify the toxic chemical within the environment and in affected persons. It is desirable to quantify the offending agent in tissue samples to identify and, if possible, quantify, new exposure and to determine a dose-response relationship. However, analytical methods with appropriate sensitivity and precision may not be accessible within the area of disease outbreak. Furthermore, some chemicals are rapidly metabolized and excreted making their detection in body fluids and tissue samples difficult, or even impossible. Thus, the time between exposure and the collection of human specimens may be crucial, not only for the detection of the responsible chemical(s) and metabolites, but also for estimating the dose associated with the observed effects.

Analysis for microbial toxins represents a special problem. Intoxications caused by microbial toxins are mainly foodborne, but respiratory exposure has also been known in the case of fungal and algal toxins. Microbiological investigation of suspected foods can be an important step in the identification of the chemical involved, indicating the chemical method of analysis to be applied for unequivocal identification of the causative agent. Thus, if Staphylococcus aureus is the predominant member of the microflora in foods that have been associated with an acute gastrointestinal disorder, then the food should be analysed for Staphylococcus enterotoxins. Identification of Aspergillus flavus growing in foods (cereals, oil seeds), associated with acute hepatitis, may suggest analysis for aflatoxin B1, B2, G1, and G2 (WHO, 1979b). Routine methods are not available for the chemical analysis of some microbial toxins. Identification and quantification can then be

Page 25 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

achieved through bioassays, though the analytical power is drastically reduced compared with chemical methods. Thus, exposure conditions during outbreaks of paralytic shellfish poisoning (PSP) are estimated on the basis of a standardized mouse assay (WHO, 1984b; AOAC, 1980). However, chemical procedures for the determination of paralytic shellfish poisoning components, such as high-pressure liquid chromatography, are available and may soon replace a bioassay.

6.3. Toxic Chemical Information Data Systems

Most data systems are based on studies exploring toxic effects produced by exposure to known, specific chemicals, plants or their extracts, natural toxins, etc. In other words, these systems are developed under circumstances in which the agent is known. When a disease is the starting point of investigation, the signs and symptoms are known, but the chemical is unknown or only suspected. A number of existing data systems listing the toxic effects of chemicals are computerized. These systems can be used by professional information specialists to list all chemicals reported to produce a specific effect, e.g., neurotoxic effects. However, the process of establishing diagnostic criteria involves knowledge of the relative specificity of various signs and symptoms as well as of the order of their appearance. Because of this, relevant information from such existing systems can be obtained through the interaction of a professional information specialist with a specialist of a suitable medical background.

A source would be useful, providing information on specific requirements for the collection of biological specimens, the types of specimens to be collected, the amount of sample needed for chemical analysis, means of preservation of the specimens, as well as a directory of laboratories performing specialized analyses.

7. PROBLEMS IN ESTABLISHING CHEMICAL ETIOLOGY

7.1. Causation in Human Disease

Human disease occurs as a result of a series of events, beginning with some initial insult or insults, which then develop further. The final manifestation of disease may be the result of various promoting, modulating, or interfering factors related to a number of factors including genetic make-up, nutrition, life style, and environment. In some situations, a specific disease simply seems to develop as a direct result of a particular factor, i.e., a monocausal relationship. However, frequently, more than one factor may be involved in the evolution of the disease. Also, several conditions or characteristics may be associated with the occurrence of the disease, without necessarily being causally related. Thus, in the search for the chemical etiology of a human disease, specific causal factors that constitute necessary (though rarely sufficient) conditions for the development of the disease need to be identified. When searching for these causal factors, case definitions and diagnostic criteria must adequately distinguish the particular disease under investigation from similar conditions with different etiologies. However, the disease should not be separated into too many subentities as this blurs the impression of a common causation. Individual disease entities frequently have a heterogeneous etiology, as in the case of end-stage kidney disease or organic brain dysfunction. Some diseases are called "idiopathic" or "essential", their causes being unknown, and eponyms and pathological features are frequently the basis for names of diseases or their classification. The same chemically- induced liver disease has even been given different names in

Page 26 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

independent outbreaks, viz., "hepatitis with ascites" (Dubrovinski, 1952) and "veno-occlusive liver disease" (Tandon & Tandon, 1975). It may be difficult to determine whether an outbreak of a disease is new or, if similar outbreaks have occurred in the past, whether the etiology has, perhaps, already been elucidated.

In the investigation of disease outbreaks or endemic disease, the analysis must first of all focus on the clinical and epidemiological features of the disease. Pathology, toxicology, and other related disciplines provide further information. Detailed analysis of the clinical manifestations and morphological changes in organs may at times help to identify etiological agents (Parish et al., 1979; Cooper & Kimbrough, 1980). On the other hand, some chemicals that cause acute poisoning and death may show few, if any, morphological changes in organs or only very non- specific changes (Diggory et al., 1977). Several compounds produce morphological changes that can be considered characteristic, even if not necessarily specific for one compound, e.g., status spongiosus of the white matter of the nervous system, which is observed after exposure to hexachlorophene or triasultane (Kimbrough, 1976). In the same way, some biochemical alterations can be considered characteristic, such as the inhibition of cholinesterase by organophosphorus compounds (Wills, 1972), or the inhibition of uroporphyrinogen decarboxylase in the liver by hexachlorobenzene, resulting in acquired porphyria cutanea tarda

(Cam & Nigogosyan, 1963; Taljaard et al., 1971; Felsher et al., 1982). However, the circumstances of the events leading to the illness need to be investigated in detail, since the nosographic specificity may not allow proper judgement without supporting information.

For many chronic diseases, the situation is more difficult. A chronic disorder could result from an acute insult, such as the permanent paralysis caused by triorthocresyl phosphate (Morgan & Penovich, 1978), or the onset may be insidious resulting from a cumulative effect following repeated insults, such as the development of emphysema after years of smoking. Finally, a disease may have a composite etiology, such as glucose-6-phosphate dehydrogenase-associated anaemia, which is caused by a combination of genetic abnormality and an environmental chemical.

An individual disease is recognized by its characteristic combination of symptoms and signs. Each component may not necessarily be pathognomonic, but the association between otherwise non-specific symptoms and signs may constitute a typical appearance. For example, Thomas Addison, in the 19th century, first described pernicious anaemia by separating it from general anaemia, since it occurred without any discoverable cause, such as loss of blood or malignant disease. In the case of pernicious anaemia, the absence of other signs present in previously established forms of anaemia, was a surprising observation. Subsequently, the discovery of vitamin B12 and the mechanism of its absorption in the gut was discovered. Research on several other diseases has followed a similar line.

In approaching the etiology of a "new" disease, a step-wise series of considerations may be useful, starting from more general criteria and concluding with a detailed evaluation of the strength of the accumulated evidence. The initial steps are particularly useful, when considering strategies to elucidate an outbreak of a new disease, while the final evaluation of the data is similar to criteria generally used when considering causal links.

Page 27 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

If a disease occurs in a particular environmental setting, this should be characterized with regard to a set of factors, discussed in section 5.2 and summarized in Table 1. Genetic disease should be considered. The following considerations may be useful with regard to inherited diseases: (a) occurrence of the disease in definite proportions among persons related by descent; (b) failure of the disease to appear in unrelated lines (e.g., spouses, in- laws); (c) characteristic age of onset and course, in the absence of known precipitating factors; (d) greater concordance of incidence in monozygotic than in dizygotic twins. Furthermore, a genetic component may be indicated by studies of genetic isolates or races, more detailed examination of clustering within families, relationship to blood types, and occurrence of characteristic chromosomal anomalies (Murphy, 1972). Many diseases have a genetic component that may determine individual susceptibility (Kimbrough, 1984). Also, some forms of inherited or inborn disease are not expressed, unless an individual is exposed to environmental

chemicals or drugs, e.g., with atypical plasma-cholinesterase (Baker et al., 1977) or deficient glucose-6-phosphate dehydrogenase (G6PD).

In the case of environmentally-related disease, individuals who move into the environmental setting run a risk of developing the disease, while individuals who leave will normally not suffer further deterioration and may even recover.

If an acute illness of unknown etiology is encountered, both infectious and chemical causes should be considered. Principal investigations into the causes of the disease should be governed by the most likely etiology. However, other possible causes (infectious versus chemical, versus multi-factorial) should not be ignored. Koch's postulates, which were developed to elucidate microbial infections, rarely if ever apply to chemical-induced disease.

7.2. Consideration of a Chemical Etiology

The distinction between chemical and infectious etiology is of special importance when considering the possible strategies for prevention. In this regard, two main sets of data should be considered, i.e., clinical symptoms and epidemiological characteristics. In addition, the results of laboratory investigations and experimental animal studies may further elucidate the question, if supplementary evidence is needed.

In considering clinical patterns, sudden deaths early in the outbreak after a prodromal period of a few hours would suggest a chemical etiology. Non-pathognomonic individual signs and symptoms taken alone are of little value in distinguishing between poisoning and infection. Thus, although it is not a common feature of diseases caused by chemicals, fever may be seen with compounds that uncouple oxidative phosphorylation, or otherwise impair temperature regulation. Examples of such compounds are: pentachlorophenol (Robson et al., 1969), dicoumarol (Toolis et al., 1981), hexachlorophene (Martin-Bouyer et al., 1982), and aflatoxin (Ngindu et al., 1982). Sudden enlargement of lymph nodes, increased peripheral white cell counts, persistent diarrhoea after "cessation of exposure", and early acute inflammatory exudates are usually not part of the clinical picture of a chemical poisoning. On the other hand, in metal fume fever, both fever and increased white cell counts are seen (Parke, 1982). Thus, the total clinical picture should be considered. Moreover, chemicals may modulate the function of the immune system or otherwise increase individual

Page 28 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

susceptibility to infection. In this case, infection would be seen as pre-disposed by chemical exposure.

Consideration of the following epidemiological features may be helpful when investigating the etiology of a disease not previously encountered:

(a) case-to-case transmission of the disease (presence or absence of secondary cases);

(b) common source of exposure;

(d) geographical distribution of cases within well-defined areas; and

(e) distribution within particular age groups, sex, socioeconomic strata, and other demographic subgroups.

Early in an outbreak, it is often difficult to identify features characteristic of diseases caused by certain chemicals (e.g., hexachlorophene) or infectious agents (e.g., Legionnaire's disease). The occurrence of secondary cases within a family, days to weeks later, might suggest an infectious agent. If such secondary cases occur within several families at regular intervals, an infectious agent with an incubation period is suggested. However, no secondary cases were observed in, for example, Legionnaire's disease. Therefore, it may not be possible, early in an outbreak, to determine whether a disease is infectious by nature or caused by a chemical, and both leads will have to be followed up. As the epidemic progresses, this distinction is likely to become easier.

7.3. Temporal Relationships

The temporal relationship between the disease and associated variables must be known in order to establish a causal relationship. Thus, the time sequence is an important criterion in evaluating causality and should be considered early in the process.

Sometimes, specific events can be associated with the onset of illness, such as the change of a production process in a chemical factory, or the use of a new chemical in a commercial product, as in the case investigated by Mallov (1976) of methyl-isobutyl-ketone which was replaced by methyl- N-butyl ketone. These and similar leads should be carefully investigated. Though a time-relationship should be sought, the temporal association between cause and effect may not be very obvious and may follow an unpredictable pattern.

Exposure to the chemical factor must occur or begin prior to the development of disease. In the case of acute toxicity, the adverse effects follow as an obvious consequence of the chemical exposure. However, this sequence may frequently not be apparent. This is the case when exposures are long-term, irregular, or mixed. Some chemicals are rapidly metabolized and excreted, thus disappearing from the body shortly after exposure. In other cases, the chemical is accumulated in the body, and may remain in body depots for decades, perhaps resulting in disease much later than the initial exposure. This sequence would depend on dose and on individual susceptibility. Also, when the effect is non-specific, develops insidiously or is delayed, the time of onset of the disease may not be well defined. In addition, the expression of disease may be determined by a factor independent of the chemical

Page 29 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

exposure.

A chemical may cause an acute illness from which the patient appears to recover only to develop a permanent injury after a delay of several days to weeks, as in the case of neuropathy after triorthocresyl phosphate intoxication (Morgan & Penovich, 1978) or the development of cataracts following ingestion of dinitrophenol (Horner et al., 1985). Continuous internal exposure to, e.g., persistent chemicals or asbestos fibres, may ultimately lead to disease. Certain diseases, such as cancer or lung fibrosis, may continue to progress, even after the exposure has long ceased.

7.4. Biological Plausibility

While specific chemical etiology may be suggested by the clinical and epidemiological features, additional leads can be obtained from detailed examination of autopsy samples and clinical laboratory data including results of chemical analysis for toxic substances (section 6.2). Once a chemical or a family of related chemicals is suspected in an environmentally-induced disease, the criterion of biological plausibility in establishing the role of the suspect agents as etiological factors in the disease has to be considered. This criterion is important when investigating causal relationships. However, in many cases, the toxicokinetics and/or biochemical mechanism of toxic action of a particular chemical are not known with certainty. In such cases, it is more difficult to use biological plausibility as a criterion to determine the association between a given disease and a particular chemical agent. Nevertheless, as toxicological investigations advance, the linkage of various metabolic effects with specific chemicals becomes possible and should be pursued. When reviewing the toxic effects of chemicals, the extent to which the affected population could have had access to the substances under consideration should be determined. Furthermore, if a suspect chemical compound is known to have, for instance, haemolytic effects, and the disease in question does not involve haemolysis, then this compound can effectively be ruled out as a causative factor in the disease. On the other hand, if the suspect agent is known to inhibit or induce certain metabolic pathways or enzymatic reactions and these biochemical activities are depressed or activated in the disease concerned, then this can be taken as supportive evidence for a role of the chemical in the etiology of the disease.

When the effects of exposure to a chemical, found to be associated with the disease in question, do not fulfil the criterion of biological plausibility, the possibility should be explored of contamination of the chemical with a more toxic substance. The purity of manufactured chemicals may vary and they can occasionally contain trace amounts of highly toxic contaminants. Furthermore, exposures to such mixtures may result in additive, synergistic, potentiating, or antagonistic effects. If only exposure to the technical grade or less than pure compound is identified, it may be difficult to explain the illness observed, as in the case of Yusho disease, where the clinical manifestations greatly exceeded the severity expected from polychlorinated biphenyl exposure alone, but would be explained by the presence of trace amounts of polychlorinated dibenzofurans (Masuda & Yoshimura, 1984). In such situations, it may therefore be useful to evaluate the production process of the chemical involved and to determine the extent to which toxic impurities are present (Baker et al., 1978).

Toxic impurities may also develop on storage at high environmental temperatures, or because of exposure to sunlight.

Page 30 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

Furthermore, human beings may be exposed simultaneously to more than one chemical, resulting in possible interactions.

From the examples presented above, it should be clear that both pathophysiological and biochemical alterations can be used to establish whether or not the suspect agent satisfies the criterion of biological plausibility. If the criterion cannot be met at an early point in the investigations, a re-evaluation should be made, when more information has been obtained.

7.5. Dose-Response and Dose-Effect Relationships

If possible, the search for, and confirmation of, chemical etiologies for specific diseases should include studies on relationships between the incidence and severity of the particular disease and the intensity and duration of exposure to the factor or factors suspected of causing the disease. However, a simple relationship between "dose" and disease outcome may not necessarily be apparent.

In outbreaks of acute poisoning, the severity of the clinical manifestations will generally depend on the dose of the chemical. For instance, food may be contaminated, but the level of contamination and individual intake may not be uniform. Some people ingesting much of the food item may receive a high dose and others a much lower dose. Therefore, some of the exposed may develop severe disease, while others may only develop a few of the symptoms and signs related to the disease, or none at all. For some diseases, a quantal relationship exists where the same type of exposure will result either in a certain type of disease or in no obvious ill effects. Different segments of the population may vary in their susceptibility to toxic effects. Depending on the dose, the effects may be acute, following one-time inhalation, ingestion, or dermal exposure to a toxic quantity of a substance. However, at very low doses, a single dose may not result in any illness, particularly for chemicals that are cumulative. Thus, the integrated exposure over a long period, as reflected by the build- up of the chemical in the body, should be taken into account in dose-effect, dose-response considerations. Such long-term exposure levels should be compared to general background levels, and, if possible, properly selected control areas should be examined as a reference.

From results in experimental toxicology, it is known that many chemicals affect cellular and subcellular systems before overt disease occurs. Some such effects can be quantified using non- invasive techniques or by analysing a blood samples. The clinical significance of many of these tests is not clear at the moment, and

they need to be validated further. On the other hand, some of these tests, for example the determination of red blood cell- cholinesterase and, within certain risk groups, determination of urinary-beta-microglobulin excretion, have become useful markers of chemical exposure, and/or effects, and have been used for dose- effect and dose-response evaluations.

7.6. Effects of Intervention

There are two types of intervention. Primary intervention involves efforts to prevent continued exposure to the offending environmental factor, while secondary intervention prevents or ameliorates the damage caused by the exposure. Distinguishing between these two categories may not always be straightforward, particularly when the pathogenesis of the disease is not known.

Page 31 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

However, if adequate prevention can be achieved by secondary intervention, the search for specific chemical etiology becomes less urgent.

As soon as a chemical etiology has been suggested, primary intervention should be considered to prevent further occurrence of new cases or progression of the disease. Through this approach, further proof of a causal relationship can frequently be obtained. The results of a preliminary intervention will frequently give rise to subsequent, more specific interventions that may further support the causal relationship. Efforts in this area should preferably include a control group, and the effects of the intervention must be separated from the natural cycles of the disease and the effects of other environmental factors. For example, outbreaks of disease of chemical origin may be self- limiting. The question of whether intervention occurred when the number of cases was still increasing or whether they were already decreasing at this time may be crucial. Examples of intervention include the removal of contaminated food items from circulation and the warning of the population against hazards, such as not to consume grain treated with fungicide. Every effort should always be made to identify and conduct intervention measures, in order to augment disease prevention.

7.7. Confirmation of a Causal Relationship

After an association between a particular chemical and the disease has been identified, the validity of the association should be confirmed. The causal relationship could be supported, for instance, through the detection of the toxic chemical or its metabolites in tissues or biological fluids, or through laboratory studies on appropriate experimental species, though, as discussed in section 6, an animal model may not be readily identified. Information on adverse effects in wild or domestic animals exposed to the chemical factor may be useful in this regard. For example, for confirmation of the involvement of aflatoxins in human aflatoxic hepatitis, the contaminated corn grains consumed by affected individuals were fed to ducklings, which then developed bile-duct proliferation in the liver, which was typical of aflatoxicosis (Krishnamachari et al., 1975).

Another means of confirmation is to relate the type, levels, and extent of exposure to the chemical with previous or subsequent disease outbreaks in human beings, if any. For example, the clinical symptoms, levels of aflatoxins in the staple food, and conditions of exposure during the outbreak of aflatoxic hepatitis in Kenya were similar to those reported earlier from India. Similarly, conditions related to veno-occlusive disease in India associated with pyrrolizidine alkaloid contamination of food grains were similar to those that caused the disease in Afghanistan. Furthermore, the cause of endemically-occurring osteosclerosis was discovered when a similar disease, skeletal fluorosis, was described in workers exposed to excess levels of fluoride.

Ideally, when considering the association between the suspected causal factor and the resulting disease, the overall evidence accumulated should be critically reviewed, according to the following established criteria discussed in detail by Susser (1973) and Lilienfeld & Lilienfeld (1980):

(a) consistency of association, i.e., the repeated observation of the connection between chemical exposure and effects, under different circumstances;

Page 32 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

(b) strength of association, i.e., more intense exposure leads to more frequent or more severe effects;

The criteria of time sequence and of coherence (biological plausibility) and also of dose-response relationship were dealt with in previous sections (7.3, 7.4, and 7.5, respectively). However, all these criteria may sometimes be difficult to apply in practice. Outbreaks of poisoning may be isolated events, and idiosyncratic reactions or individual susceptibility may blur the relationship to dosage levels. With chronic disease, where chemical exposure constitutes only one of the risk factors, the evaluation of the significance of a particular chemical exposure is more difficult.

8. STEPS TO BE TAKEN FOLLOWING THE RECOGNITION OF THE CHEMICAL ETIOLOGY OF AN OUTBREAK

Appropriate preventive measures must be implemented as soon as is possible and feasible after the nature of the chemical agent, the pathway of exposure, and the source have been identified.

Having determined the magnitude of the hazard in terms of the size of population affected and the geographical areas involved, the following public health measures should be undertaken in addition to the obvious measures for the treatment and rehabilitation of patients.

8.1. Preventive Action and Control

Strategies have to be planned for: (a) immediate steps to control the outbreak; and (b) prevention of such outbreaks in the future. Many of these steps have multisectorial implications and need coordinated and integrated approaches for their success. Immediate preventive measures depend on the type of chemical involved and the pathway of exposure. If the contamination is through food, steps have to be taken to seize and destroy the damaged or contaminated food grain or food material, to prevent the community from consuming such food, and alternative sources of food must be made available. Wherever possible, the commodity should be retrieved by cleaning or detoxifying under supervision. After identifying the chemical(s) involved and their source, it may be possible to prevent the entry of the chemical(s) into the food chain. For example, recognition that pyrrolizidine alkaloids contaminated food through the seeds of certain plants made it possible to eradicate veno-occlusive disease by introducing simple preventive measures, and removing the incriminating seeds from the grain (Tandon & Tandon, 1975).

8.2. Surveillance and Monitoring System

A surveillance system should be established or strengthened in all cases of chemically-induced disease, and should be suitably designed according to the nature of the exposure. If the chemical is identified and can be removed from the environment, apart from monitoring for its recurrence in the environment, a long-term follow-up, especially of the persons affected by the disease, may be necessary. This is especially important if, according to known experimental or human data, some health effects could manifest after a period of time. This is of particular relevance for carcinogenic effects. Thus, the creation of long-term machinery is

Page 33 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

necessary for follow-up surveillance, including periodic physical examination of the affected cases and monitoring for the appearance of the earliest signs of the disease.

If the chemical has not been precisely identified, or if it cannot be removed from the human environment, the following surveillance measures are recommended:

(a) to ensure the reliability of disease-specific morbidity and mortality data;

(b) to monitor the incidence, i.e., the occurrence of new cases;

(d) to monitor the evolution of disease symptoms and complications to initiate rehabilitation programmes, when appropriate;

(e) to monitor specific environmental hazards, if identified.

A formal registry of cases is a valuable tool.

8.3. Health Education

With the identification of the chemical etiology of a disease and the initiation of preventive steps, the need to disseminate the information to the professionals involved and to educate the public in appreciating the nature of the hazard, and ways and means of preventing and minimizing them, cannot be over-emphasized. For this purpose, the role of the mass media is particularly important. To improve the preceptiveness of the medical and paramedical personnel for such information, the medical/nursing/paramedical curricula should include adequate sections on the role of chemicals in contributing to disease.

9. THE NEED FOR INTERNATIONAL COOPERATION

9.1. Cooperation and Collaboration among Countries

Technical cooperation among countries is extremely important because:

(a) The same diseases of suspected chemical etiology may be found in several countries in different parts of the world or in countries belonging to the same geographical area, where the populations are exposed to the same environmental hazards. Thus, there is a need for the sharing of knowledge by countries with experience of the endemic disease and for cooperative efforts in the control of common public health problems. The relevant discussion on Kashin-Beck disease can be found in Appendix III.

(b) There is a need for joint efforts in activities such as the training of personnel, exchange of experts and specialists, the use of sophisticated laboratory tests for clinical diagnosis, and pathological and toxicological studies.

(c) It is essential that clinical, epidemiological, and public health information and experiences should be shared to develop surveillance and monitoring activities for common,

Page 34 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

and newly emerging, problems.

(d) Joint ventures in the field of research are necessary concerning problems of common interest. This can be facilitated by the establishment of "Sister Institutions" dealing with the same problem in different countries.

(e) Cooperation among countries makes it possible to carry out more extensive investigative programmes on common health problems, which would exceed resources available in one country, and to develop and implement preventive measures of benefit to all of them.

(f) Mutual technical and economic assistance is needed in case of disasters to deal with, and solve, emergency situations.

All these aspects should be considered in the context of the social and economic development of the member countries, taking into account the need for a multisectorial approach in the prevention and control of diseases of chemical etiology.

9.2. Global and Regional Activities

The disease-oriented approach, which has been used when establishing the role of certain chemicals in the etiology of outbreaks of locally endemic diseases, has provided a basis for the prevention and control of these diseases; thus, it must be recognized as an important component of chemical safety. There is a need to strengthen methodological cooperation at both the global

and regional levels, and the methodological component of the IPCS has a well-defined role in this respect. It is also necessary to develop, through the IPCS, regional and global registries of endemic diseases with suspected chemical etiologies, to facilitate the harmonization of activities aimed at improved understanding of their causation, and at the prevention and control of these diseases.

Thus, at both global and regional levels, there is a need to:

(a) Increase awareness of the potential of chemicals to induce disease and of the need for international cooperation to achieve the goal of chemical safety, including the prevention and control of diseases of chemical etiology;

(b) Strengthen national capabilities in environmental epidemiology, toxicology, and related fields;

(d) Develop guidelines on environmental epidemiology;

(e) Identify relevant institutions at the country level to promote training and research in the field;

(f) Apply health criteria in establishing national standards, and in promoting the national regulations according to the needs of the different countries and specific epidemiological situations;

(g) Promote the evaluation of existing control programmes;

(h) Develop new, and improve existing, indicators to be used in the epidemiological investigation of outbreaks of

Page 35 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

disease and chronic endemic diseases, and for the evaluation of control measures;

(i) Strengthen health monitoring and surveillance systems at the country level;

(j) Strengthen the infrastructure of health services to implement activities for the control of environmental hazards, and to create or improve the surveillance system, the laboratory capabilities, and the research institutions;

(k) Help member countries to establish a set of priorities for chemical hazards control action within the framework of health for all by the year 2000 and its main strategy, primary health care.

9.3. Final Recommendations

In summary, the following three main recommendations should be implemented through the global, regional, and country levels:

- to promote and stimulate the collaboration and cooperation among member countries towards joint efforts for the better knowledge and implementation of control measures in common problems involving chemical safety;

- to accelerate and increase the training of specialized personnel in the different regions and member countries;

- to promote and stimulate the collection and sharing of clinical, epidemiological, and biological information concerning outbreaks of disease and chronic endemic diseases that are chemically-related.

REFERENCES

ANON. (1984) Pyrrolizidine alkaloids: editorial. Lancet, 1: 201-202.

AOAC (1980) Official methods of analysis of the Association of Official Analytical Chemists, 14th ed., Washington DC, Association of Official Analytical Chemists, pp. 344-345.

BAKER, E.L., SMREK, A., KIMBROUGH, R.D., HUDGINS, M., LANDRIGAN, P.J., & LIDDLE, J.A. (1977) Hereditary cholinesterase deficiency: A report of a family with two rare genotypes. Clin. Genet., 12: 134-138.

BAKER, E.L., ZACK, M., MILES, J.W., ALDERMAN, L., MCWILSON, W., DOBBIN, R., MILLER, S., & TEETERS, W.R. (1978) Epidemic malathion poisoning in Pakistan malaria workers. Lancet, 1(8054): 31-34.

BAKIR, F., DAMLUJII, S.F., AMIN-ZAKI, L., MURTADHA, M., KHALIDI, A., AL-RAWI, N.Y., TIKRITI, S., DHAHIR, H.I., CLARKSON, T.W., SMITH, J.C., & DOHERTY, R.A. (1973) Methylmercury poisoning in Iraq. Science, 181: 230-241.

BRAS, G., JELLIFFE, D.B., & STUART, K.L. (1954) Veno- occlusive disease of liver with nonportal type of cirrhosis, occurring in Jamaica. Arch. Pathol., 57: 285-300.

CAM, C. & NIGOGOSYAN, G. (1963) Acquired toxic porphyria cutanea tarda due to HCB. J. Am. Med. Assoc., 180: 88-91.

Page 36 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

COOPER, W.W. & KIMBROUGH, R.D. (1980) Acute dimethylnitros- amine poisoning outbreak. A case report. J. forensic Sci., 25(4): 874-882.

DIGGORY, H.J.P., LANDRIGAN, P.J., LATIMER, K.P., KIMBROUGH, R.D., LIDDLE, J.A., CLINE, R.E., & SMREK, A.L. (1977) Fatal parathion poisoning caused by contamination of commercial flour. Am. J. Epidemiol., 106: 145-153.

DUBROVINSKI, S.B. (1952) [The etiology of toxic hepatitis with ascites.] In: Millenkov, S.M. & Kizhaikin, Y., ed. [Symposium on Toxic Hepatitis With Ascites,] Tashkent, V.M. Molotov Medical Institute (in Russian).

FELSHER, B.F., CARPIO, N.M., ENGLEKING, D.W., & NUNN, A.T. (1982) Decreased hepatic uroporphyrinogen decarboxylase activity in porphyria cutanea tarda. New Engl. J. Med., 306: 766-769.

GRANDJEAN, P. (1975) Lead in Danes: historical and toxicological studies. Environ. Qual. Saf. Suppl., 2: 6-75.

HORNER, W.D., JONES, R.B., & BOARDMAN, W.W. (1985) Cataracts following the use of dinitrophenol: preliminary report of 3 cases. J. Am. Med. Assoc., 105: 108-110.

HUXTABLE, R.J. (1980) Herbal teas and toxins: novel aspects of pyrroliziding poisoning in the US. Perspect. Biol. Med., 24: 1-14.

KIMBROUGH, R.D. (1976) Hexachlorophene toxicity and use as an antibacterial agent. Essays Toxicol., 7:: 99-120.

KIMBROUGH, R.D. (1984) Relationship between dose and health effects. Clin. lab. Med., 4: 507-519.

KRISHNAMACHARI, K.A.V.R., BHAT, R.V., NAGARAJAN, V., & TILAK, T.B.G. (1975) Hepatitis due to aflatoxicosis. Lancet, 1: 1061.

KURATSUNE, M., MORIKAWA, Y., HIROHATA, T., ET AL. (1969) An epidemiological study on "Yusho" or chlorobiphenyls poisoning. Fukuoko Acta med., 60: 513-532.

LAST, J.M., ed. (1983) A dictionary of epidemiology, New York, Oxford University Press.

LILIENFELD, A.M. & LILIENFELD, D.E. (1980) Foundations of epidemiology, 2nd ed., New York, Oxford University Press.

MAHAFFEY, K.R. (1985) Dietary and environmental lead: human health effects, Amsterdam, Elsevier Scientific Press.

MALLOV, J.S. (1976) MBK neuropathy among spray painters. J. Am. Med. Assoc., 235(14): 1455-1457.

MARTIN-BOUYER, G. (1981) Epidemiological study of encephalopathies following bismuth administration. Clin. Toxicol., 19(11): 1277-1283.

MARTIN-BOUYER, G., LEBRETON, R., TOGA, M., STOLLEY, P.D., & LOCKHART, J. (1982) Outbreak of accidental hexachlorophene poisoning in France. Lancet, 1: 91-95.

Page 37 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

MASUDA, Y. & YOSHIMURA, H. (1984) Polychlorinated biphenyls and dibenzofurans in patients with Yusho and their toxicological significance a review. Am. J. ind. Med., 5: 31-44.

MOHABBAT, O., SRIVASTAVA, R.N., YOUNOS, M.S., SEDIQ, G.G., MERZAD, A.A., & ARAM, G.N. (1976) An outbreak of hepatic veno-occlusive disease in Afghanistan. Lancet, 2: 269-271.

MORGAN, J.P. & PENOVICH, P. (1978) Jamaica ginger paralysis. Arch. Neurol., 35: 530-532.

MOZAMBIQUE MINISTRY OF HEALTH (1984) Mantakassa: an epidemic of spastic parapesis associated with chronic cyanide intoxication in a cassava staple area of Mozambique. Bull. World Health Organ., 62: 477-494.

MURPHY, E.A. (1972) The application of genetics to epidemiology. In: Stewart G., ed. Trends in epidemiology, Springfield, Illinois, Thomas, pp. 102-138.

NGINDU, A. (1982) Outbreak of acute hepatitis caused by aflatoxin poisoning in Kenya. Lancet, 2: 1346-1348.

PARISH, G.G., GLASS, R., & KIMBROUGH, R. (1979) Acute arsine poisoning in two workers cleaning a clogged drain. Arch. environ. Health, 34: 224-227.

PARKE, W.R. (1982) Occupational lung disorders, 2nd ed., London, Butterworth.

RIDKER, P.M., OHKUMA, S., MCDERMOTT, W.V., TREY, C., & HUXTABLE, R.J. (1985) Hepatic veno-occlusive disease associated with consumption of pyrrolizidine alkaloid containing dietary supplements. Gastroenterology, 88: 1050-1054.

ROBSON, A.M., KISSANE, J.M., ELVICK, N.H., & PUNDAVELA, L. (1969) Pentachlorophenol poisoning in a nursery for newborn infants. I. Clinical features and treatment. Pediatr. Pharmacol. Ther., 75: 309-316.

STUART, K.L. & BRAS, G. (1955) Clinical observations on veno-occlusive disease of the liver in Jamaican adults. Br. med. J., 2: 348-352.

SUSSER, M. (1973) Causal thinking in the health sciences; concepts and strategies of epidemiology, New York, Oxford University Press, pp. 140-162.

TALJAARD, J.J.F., SHANLEY, B.C., & JOUBERT, S.M. (1971) Decreased uroporphyrinogen decarboxylase activity in experimental symptomatic porphyria. Life Sci., 10: 887-893.

TANDON, H.D. & TANDON, B.N. (1975) Epidemic of liver disease in Gulran District, Herat Province, Afghanistan, Alexandria, WHO Regional Office for the Eastern Mediterranean (Unpublished assignment report No. EM/AFG/OCD/001/RB).

TANDON, R.K., SINGH, D.S., ARORA, R.R., LAL, R., & TANDON, B.N. (1975) Epidemic dropsy in New Delhi. Am. J. clin. Nutr., 28: 883-887.

TANDON, B.N., TANDON, H.D., TANDON, R.K., NARENDRANATHAN, M.,

Page 38 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

& JOSHI, Y.K. (1976) Epidemic of veno-occlusive disease in central India. Lancet, 2: 271-272.

TANDON, B.N., KRISHNAMURTHY, L., KOSHY, A., TANDON, H.D., RAMALINGASWAMI, V., BHANDARI, J.R., MATHNI, M.M., & MATHUR, P.D. (1977) Study of an epidemic of jaundice in North-West India, presumably due to toxic hepatitis. Gastroenterology, 72: 488-494.

TANDON, H.D., TANDON, B.N., & RAMALINGASWAMI, V. (1978a) An epidemic of toxic hepatitis in India of possible mycotoxic etiology. Am. J. Gastroenterol., 70: 607-613.

TANDON, H.D., TANDON, B.N., & MATTOCKS, R.R. (1978b) An epidemic of veno-occlusive disease of the liver in Afghanistan. Am. J. Gastroenterol., 72: 607-613.

TOOLIS, F., ROBSON, H.R., JULIAN, A., & CRITCHLEY, J.H. (1981) Warfarin poisoning in patients with prosthetic heart valves. Br. med. J., 283: 581-582.

TSUCHIYA (1978) Cadmium studies in Japan: a review, Amsterdam, Elsevier Biomedical Press, 376 pp.

WHO (1977) EHC 1: Mercury, Geneva, World Health Organization, 121 pp.

WHO (1979a) EHC 8: Sulfur oxides and suspended particulate matter, Geneva, World Health Organization, 93 pp.

WHO (1979b) EHC 11: Mycotoxins, Geneva, World Health Organization, 127 pp.

WHO (1983) EHC 27: Guidelines on studies in environmental epidemiology, Geneva, World Health Organization, 349 pp.

WHO (1984a) EHC 36: Fluorine and fluorides, Geneva, World Health Organization, 136 pp.

WHO (1984b) EHC 37: Aquatic (marine and freshwater) biotoxins, Geneva, World Health Organization, 72 pp.

WHO (in press) EHC: Pyrrolizidine alkaloids, Geneva, World Health Organization.

WILLS, J.H. (1972) The measurement and significance of changes in the cholinesterase activities of erythrocytes and plasma in man and animals. CRC crit. Rev. Toxicol., 1: 152-202.

APPENDIX I. LIST OF BACKGROUND PAPERS

AMIN-ZAKI, Professor L. A tale of two alkylmercury poisoning epidemics in Iraq

BABABUNMI, Professor E.A. (a) Possible involvement of Cassava in certain tropical endemic diseases (b) A biochemical approach for establishing the role of cyanide in the etiology of tropical ataxic neuropathy

BHAT, Dr R.V. Endemic disease outbreaks in India due to chemical toxins

BORGONO, Dr J.

Page 39 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

The problem of chemical etiology of certain human diseases

EGASHIRA, Dr Y. An outline of history of research on Kashin-Beck disease in Japan

GOYER, Dr R.A. Identification of diseases caused by chemicals in the environment

GRANDJEAN, Dr P. Constraints in establishing the etiology of environmentally- induced disease

IVANOV, Dr V.V. Biochemical criteria of environmentally-induced diseases

KIMBROUGH, Dr R.D. Investigation of acute outbreaks of human illness caused by chemicals

KROGH, Professor P. Strategies employed in the elucidation of causal associations in human diseases caused by fungal and algal toxins

LEVANDER, Dr O.A. Theories of nutritional deficiency in the etiology of endemic diseases

MAHAFFEY, Dr K.R. Techniques for investigating the etiology of disease

MARTIN-BOUYER, Dr G. Thoughts on three epidemiological enquiries into collective accidents due to a poison

SHIBATA, Professor S. (a) Nephritogenic glycopeptide, nephritogenoside (b) Vasculitis

TANDON, Professor H.D. On investigating the chemical etiology of endemic diseases and episodes or outbreaks of chemical poisoning with special emphasis on developing countries

YANAGAWA, Professor H. & SHIGEMATSU, Professor I. The epidemiological approach to the chemical etiology of specific diseases: Itai-itai disease, Minamata disease, and Yusho (PCB poisoning)

APPENDIX II. CONCEPTUAL FRAMEWORK USED IN SEVERAL STUDIES LEADING TO IDENTIFICATION OF THE CHEMICAL ETIOLOGY OF OUTBREAKS OF DISEASE

INTRODUCTION

This framework, originally presented at the Task Group meeting held in Beijing from 28 October to 8 November 1985, is based on experience during studies on the aflatoxin-induced outbreak in India (Tandon et al., 1977; WHO, 1979b) and the outbreaks of veno- occlusive disease in Afghanistan (Tandon & Tandon, 1975; Tandon et al., 1978b) and India (Tandon et al., 1976). It was amended at the editorial meeting held in Geneva from 28 April to 2 May 1986, to include as examples, studies in which a similar approach had been used when establishing the chemical etiology of disease outbreaks

Page 40 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

related to hexachlorophene (Martin-Bouyer et al., 1982) and dicoumarine (Martin-Bouyer, 1983).

CONCEPTUAL FRAMEWORK

To investigate the etiology of a disease, follow a "disease- oriented" approach as opposed to the classical toxicological "substance-oriented" approach. Recognition of the occurrence of an unusual disease is the initial requirement.

In order to be considered unusual, the disease entity may:

(a) be characterized by pathognomonic signs that do not occur in a known disease;

(b) include non-specific clinical features, signs, and symptoms and clinical laboratory and other data that do not fit into a known disease category;

(d) be present as an endemic disease of unknown etiology.

The search for an etiology can be carried out in the following three phases.

1. DESCRIPTIVE PHASE

After ascertaining that the disease is "unusual", the general features of the disease, the circumstances under which it occurs, and the size and nature of the population, or type of patients involved, can be studied. The following steps are suggested:

1.1 Development of a Case Definition

For conducting an epidemiological study and to ensure the uniformity of collected data, guidelines for screening the population have to be developed. Criteria are needed for two levels of screening, initial or mass screening and a later screening that requires diagnostic criteria to confirm the cases.

1.1.1 Criteria for recognizing index cases during mass screening

Criteria for the identification of index cases consist of major clinical signs and symptoms, for example:

(a) acute jaundice in the aflatoxin outbreak;

(b) rapidly developing ascites in the outbreak caused by pyrrolizidine alkaloids;

1.1.2 Criteria for confirming cases suspected in mass screening

These may include a combination of: symptoms, signs, laboratory data, X-rays, etc, and pathological features.

The criteria selected in the studies used as examples in this

Page 41 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

paper were:

(a) characteristic morphological features observed in the liver biopsies in cases of toxicosis due to aflatoxins and pyrrolizidine alkaloids;

(b) characteristic morphological lesion in the central nervous system and presence of chemical in blood and bones at autopsy, in cases of hexachlorophene poisoning; and

1.2 Descriptive Pattern of the Disease

This may include a study of the clinical features of the disease, the dominant signs and symptoms, the clinical course of the disease and, in case of an outbreak, an approximate idea of the

size of the population affected, the mortality rates, etc. Visits to the field or site of occurrence by the investigating team, which should include adequately trained clinical staff, are essential and cannot be over-emphasized. Study of the habitat, and the family style of living, cooking, and eating, may provide crucial clues. The investigating team should mainly consist of persons with the same cultural background as the affected population, or at least persons familiar with it. In addition, the following information can be obtained:

(a) Cluster analysis of cases, either according to factors related to the physical environment, e.g., geographical, seasonal, or to factors related to affected subjects, e.g., age, sex, family structure, socio-economic status, occupation.

(b) Retrospective study of health statistics of the area, obtained through government, municipal, or county data, or from hospitals and other health-care facilities in the region.

(d) Study of the original source of food and of food storage practices;

(e) Identification of locally endemic diseases, if any.

In the case of a disease outbreak, reports providing some of the above information and often important clues to the etiology of the disease, especially if a similar disease has been known to occur in the past, may be obtained from local general practitioners, primary health/paramedical workers, health officials, social workers, and school teachers.

1.3 Study of Locally Prevalent Animal Diseases

Unusual happenings, if any, should be noted in the fauna, farm animals, and domestic animals.

2. HYPOTHESIS GENERATION PHASE

Having collected the descriptive data on the disease and the

Page 42 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

above information, there may be possible clues suggesting, at least, the nature of the likely etiological factor(s). While generating a hypothesis, attention should be focused on the most typical cases. The following steps are suggested:

2.1 Examination of Diseased Tissue

Examination of even one or two tissue specimens, especially the main diseased organ, may provide crucial clues to the etiology of the disease, particularly in the light of the descriptive pattern, in the initial phases of the study. If available, the diseased tissue of both animals and patients should be examined.

For example, in the study on the aflatoxin-induced outbreak, the finding of syncitial giant cells in human liver tissue, identical to those found in rhesus monkeys administered aflatoxin, suggested mycotoxin etiology.

2.2 Possible Clues in the Environment

The following factors should be investigated:

(a) Unusual happenings in the environment, e.g., drought, floods, heavier than normal rainfall, etc

(b) Opening of new industries, manufacture of new chemicals or products using new chemicals, change in the manufacturing process

(d) Introduction of new seed/agricultural practices, involving the use of a new chemical

(e) Disposal of industrial waste

(f) House-keeping practices in the local industry

(g) Change in food habits

(h) Possible contamination of staple food/cooking medium

(i) The use of a new household chemical.

2.3 Consideration of Possible Routes of Exposure

Exposure usually occurs orally or via inhalation. However, the more unusual dermal route of exposure should also be considered.

3. HYPOTHESIS-TESTING PHASE

On the basis of the above information, one or more etiological factors may be suspected and a hypothesis generated, which then has to be tested as described below:

3.1 Exploration of Reports of Occurrence of Similar Episodes or Diseases Elsewhere in the World, Using Available Data Base Systems

3.2 Evolution of a Strategy for Further Study, Taking Into Account the Specific Characteristics of the Suspected Agent

This step includes:

(a) Identification of specific biological markers of exposure

Page 43 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

and of toxicity;

(b) Listing of laboratory procedures/analyses to be carried out:

(i) in the field; (ii) in local institutions; (iii) at specialized laboratories;

(d) Decisions on type and expertise of specialized personnel required to conduct field study, besides essential clinical and pathology-trained staff;

(e) Listing of toxicological studies to be carried out on laboratory animals.

3.3 Collection of Additional Pathological Samples

Detailed autopsy/biopsy studies can be made on animals and patients. A biopsy could be carried out as part of a diagnostic or therapeutic procedure, for example, carrying out a liver biopsy/kidney biopsy following paracentesis.

3.4 Collection and Storage of Biological and Environmental Samples for Further Analysis

4. FOLLOW-UP EVALUATION

At the end of the entire process, after testing the validity of the hypothesis and after establishing the causal relationship between a chemical agent and the identified disease cases, the following steps are necessary:

(a) Dissemination of knowledge concerning signs and symptoms among health practitioners, in order to identify all cases of the disease and to get an exact evaluation of the magnitude of the outbreak/episode.

(b) Search for even milder signs and symptoms among subpopulations that have been exposed to the agent. This may result in the identification of borderline cases, which will improve the accuracy of the evaluation.

(d) Finally, identify the most relevant measures to either remove the causal agent or minimize the exposure of the population at risk.

APPENDIX III. SUMMARY REPORT ON THE IPCS/WPRO/PRC MEETING ON KASHIN-BECK DISEASE

Background

Kashin-Beck disease was first described by Kashin and later by Beck & Beck in the Urov River area of eastern Siberia in the last century. Further research and practical measures in the USSR resulted in the successful control of the endemic disease in this area.

Page 44 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

In China, this disease, which has probably existed in the endemic regions for centuries, was first studied during the first half of this century. However, it is only during the last decades that this health problem, prevalent in 14 out of 29 provinces, municipalities, and autonomous regions of mainland China, has received appropriate attention. The basic pathological change in this disease, commencing mainly in children, is the degeneration and necrosis of articular cartilage and the growth plate, which can result in permanent disability. Present estimates indicate that about 2 million people are affected by this disease and that more than 30 million people living in the endemic areas of China are at risk of acquiring it. The disease seems to be linked with the consumption of food produced in the endemic areas and is possibly related to the presence of certain specific chemicals in food and/or water. The People's Republic of China, expressing interest in the WHO/ILO/UNEP International Programme on Chemical Safety (IPCS), underlined the importance of this disease and, for this reason, a joint meeting was convened by the Ministry of Public Health of the People's Republic of China, the International Programme on Chemical Safety, and the Western Pacific Regional Office of the World Health Organization to review the status of present knowledge on this disease and its possible etiology.

Scope of the Meeting

The meeting was held at the Institute of Health of the China National Centre for Preventive Medicine, in Beijing, from 28 October to 1 November 1985. After the opening by Professor Chen Chunming, Director the China National Centre for Preventive Medicine, the meeting was addressed by Dr Guo Ziheng, Vice-Minister of the Ministry of Public Health of the People's Republic of China, who explained the importance of the meeting for the promotion of studies on the chemical etiology of specific diseases. The Vice- Minister stressed the importance of the fact that the meeting on Kashin-Beck disease would be followed by a second IPCS meeting on criteria for establishing the chemical etiology of specific diseases as a basis for their prevention, which would be hosted by the same Institute, during the following week. Dr E. Goon (WHO Representative, Beijing) and Dr J. Parizek (IPCS/WHO) addressed the meeting on behalf of WHO. Professor Chen Chunming was elected Chairman, Professor V.A. Nasonova and Dr Niu Shiru, Vice-Chairmen, and Dr Chen Junshi and Dr O.A. Levander, Rapporteurs. The background papers for the meeting are listed in Appendix I.

The papers on Kashin-Beck disease presented at the meeting by Chinese scientists reviewed:

(a) the epidemiology, geographical distribution, and clinical features;

(b) pathological and biochemical features; and

In addition, a paper expressing the present views of a leading scientist from the region in the USSR where Kashin-Beck disease is endemic was presented, at the request of the organizers of the meeting from the People's Republic of China.

During the last day, related papers were presented on Keshan disease and of endemic human selenosis in China.

Summary of the results presented at the meeting: the current situation concerning Kashin-Beck disease

Page 45 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

1. Main epidemiological characteristics

(a) Geographical distribution

The disease mainly occurs in the mountainous and hilly areas of temperate forest and forest-steppe zones and is very rarely observed in the plains. The climate in endemic areas usually includes a long period of frost and big differences between the daily minimum and maximum temperatures. Affected villages are distributed in small foci within the endemic area. The prevalence in neighbouring villages can vary significantly and can change with time. New patients can be found in villages where patients have not been reported before. In rare cases, the prevalence in certain, severely affected villages can decrease "spontaneously" so that, after about 10 years, no new cases occur.

(b) Age of patients

The disease mainly develops in 5- to 13-year-old children, and very few new cases are seen among adolescents and adults. In heavily affected areas, some new cases might be only 2 - 3 years of age, whereas, in lightly affected areas, some new cases may not occur until 10 years of age.

Within endemic areas, patients mainly occur in farming families. Children of farm (government-owned) employees are also vulnerable). Only very few cases occur in "professional" families. However, patients may occur in professional families, if they consume a large amount of the staple grains (corn, highland barley, wheat) produced in endemic areas. The incidence of new and severe cases of the disease may decrease after changing the staple grains.

In contrast with other endemic diseases, such as Keshan disease, it is important to note that, in some sites where Kashin- Beck disease is endemic, in recent years, the number of recognized cases is increasing. The etiology of the disease remains obscure, but has been linked with certain chemical constituents of food and possibly water. In China, Kashin-Beck disease mainly occurs in low-selenium areas, but selenium deficiency alone is not sufficient to explain the disease. The possible role of fungal (Fusarium oxysporum) or bacterial toxins was considered in some of the papers presented at the meeting.

2. Clinical signs and symptoms

In the clinical development of the disease, weakness is followed by joint stiffness. Limitation of flexion of the index, middle, and ring fingers towards the palm can be detected followed by limitation of elbow joint movement and joint enlargement and deformity. Signs are similar to those of primary osteoarthrosis. In China, the disease has been clinically classified as follows:

- Early stage: flexion of distal joint part of fingers, bow-like fingers, and pain in knee and ankle joints;

- First degree: enlargement and crepitus of small joints;

- Second degree: short fingers, enlargement and dysfunction of medium-sized joints;

- Third degree: enlargement and dysfunction of large joints

Page 46 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

(stunted growth).

"Muscular dystrophy" can be seen during the course of the disease.

X-ray examination reveals:

(a) the calcification line becoming blurred, thin, interrupted, and disappearing as is characteristic for chondronecrosis;

(b) defects of the metaphyseal plate, the end of distal bone, and carpal and metacarpal bones following chondronecrosis;

(c) deformity of the epiphysis, synostosis of the epiphyseal plate resulting from necrosis of the whole layer of the epiphyseal plate; and

(d) enlargement of joints and stubby fingers, the effects of secondary osteoarthrosis.

Anatomical pathological examination reveals that lesions mainly involve hyaline cartilage. The epiphyseal cartilage, articular cartilage, and epiphyseal growth plate are the most affected sites. Changes are "dystrophic" in nature. The most important pathological feature is multiple localized chondronecrosis in the deep portion of cartilage tissue. Chondronecrosis of the growth

plate may result in disturbance of endochondral ossification and may even induce the early closure of the epiphyseal growth plate. The growth of long bones ceases and causes short fingers and toes, short limbs, and even stunted growth. The chondronecrosis in articular cartilage may induce scar formation and result in bony enlargement, osteophyte formation, and disfiguration of the joints affected ("endemic osteoarthrosis deformans"). Disturbances of endochondral ossification of the growth plate and advanced secondary osteoarthrosis are the two cardinal manifestations of the disease.

Biochemical research has indicated a series of metabolic disorders including:

(a) changes in cartilage metabolism, mainly affecting chondroitin sulfate and proteoglycan;

(b) changes in clinical chemistry, including the plasma enzymes (e.g., alkaline phosphatase, glutamate-oxalo- acetate transaminase, beta-hydroxybutyrate dehydrogenase), and the urinary excretion of creatinine and hydroxyproline;

(d) characteristics of low-selenium status, including changes in glutathione peroxidase activity, tocopherol content, and lipid peroxides in plasma.

3. Preventive measures

The following techniques for the prevention of Kashin-Beck disease have been studied by Chinese scientists and were discussed at the meeting. All these approaches have been used in studies on subpopulations of varying sizes, and positive results from individual studies were reported at the meeting.

Page 47 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

(a) Comprehensive prevention

Encourage the children to diversify their foods (variety and source), offer two servings/week of soup containing soybeans, sea weed, and multiple vitamins. Purify drinking-water (use deep well water with precipitation) and improve personal hygiene. It has been reported that, with this scheme, the prevalence of X-ray changes and of changes in the metaphysis decreased.

(b) Selenium intervention

Three different measures have been used for selenium supplementation including:

(i) oral administration of sodium selenite at 1 mg/week or 2 mg/week to children of 7 - 10 years and 11 - 13 years, respectively;

(ii) selenized table salt containing a sodium selenite concentration of 16.7 mg/kg; and

(iii) spraying sodium selenite solution on wheat crops at the rate of 15 g/ha (1 g/mu)a.

Using these measures, incidence rates dropped within one year, and recovery from changes in the metaphysis was noted.

Water can be purified by precipitation, filtration, and chlorination. This technique and/or the use of deep groundwater have both been associated with an improvement in clinical symptoms and a reduction in incidence rate.

(d) Change of grains

Changing the locally produced grain for grains produced in other areas led to the prevention of new cases, a decreased incidence of X-ray changes, and better recovery from meta-physeal changes.

In endemic areas of the USSR, significant reductions in the prevalence rates and the severity of Kashin-Beck disease were connected with special governmental and public health measures. The most effective measures were the organization of population migration from endemic to non-endemic regions, the importation of food from non-endemic areas, thermal treatment of local products and water, annual check-ups on children and adult populations in endemic regions by public health personnel, and the development of appropriate health education programmes. On the basis of the theory of biogeochemical provinces linking certain endemic diseases with the geochemical characteristics and quality of the soil and water in certain areas, studies in the USSR related very high phosphate and manganese contents in the soil, food, and drinking- water in the endemic area with Kashin-Beck disease. Reducing the contents of these chemicals in food and water is part of the present preventive measures in the USSR. As mentioned in the discussion, differences in selenium levels were not detected between the affected and non-affected areas studied in the USSR.

4. Conclusions and recommendations

(a) Kashin-Beck disease is a highly disabling disease of permanent character that mainly affects children. In China

Page 48 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

alone, about 2 million people are affected, at present, and more than 30 million people living in the endemic areas are at direct risk of acquiring the disease. All the evidence indicates that the disease is caused by a certain quality of the environment, specific for the endemic regions, which

------a 1 mu = 0.0667 ha. From: Beijing Language Institute (1979) Chinese English Dictionary, Beijing, Commercial Press.

encompass a large part of China and parts of neighbouring countries. Several studies indicate the involvement of certain chemicals in food and water (manganese, phosphorus, mycotoxins, microbial toxins, humic acid, etc.), as well as nutritional imbalance, in particular, selenium deficiency, as factors contributing to the etiology of this disease.

A coordinated effort is needed to elucidate the etiology of this disease, which is endemic in a very specific area of Asia. Cooperation among countries affected by the disease should be supported by international organizations with the aim of developing uniform diagnostic criteria and protocols for epidemiological studies to test the above hypotheses and elucidate more precisely the cause of the disease and contributing factors. Exchange of information on effective measures for the prevention of the disease should be another component of such intercountry cooperation, as well as the training of personnel needed for preventive, diagnostic, and therapeutic activities. As a first step in this direction, exchange of scientists between the countries with endemic regions should be supported as well as the development of methods and their use in the above-mentioned cooperative efforts.

Such activities would significantly contribute towards chemical safety and the solution of a problem affecting populations in several neighbouring member states.

(b) Kashin-Beck disease primarily affects the joints. However, the relation with other osteoarthroses is not clear, and the possible etiological factors involved are not known.

Thus, in cooperation with non-governmental and other organizations, a meeting should be convened to define specific characteristics, identify risk factors, and consider possibilities for the prevention and control of this disease and its possible relationship with other osteoarthroses.

(c) In spite of the public health significance of Kashin-Beck disease, the disease does not seem to be sufficiently recognized in the medical literature. In this respect, the data presented at this meeting was considered highly informative.

It was recommended that the co-sponsoring organizations should explore the possibility of publishing the full proceedings of this meeting as a monograph. The possibility of making the video tape that was prepared in the endemic regions for the meeting more widely available should also be explored.

ANNEX TO APPENDIX III. THE IPCS/WHO/PRC MEETING ON KASHIN-BECK DISEASE

Participants

Page 49 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

Professor L. Amin-Zaki, Consultant Paediatrician, Central Hospital, Abu Dhabi, United Arab Emirates

Professor E.A. Bababunmi, Director, Laboratory of Biomembrane Research, Department of Biochemistry, University of Ibadan, College of Medicine, Ibadan, Nigeria

Dr R.V. Bhat, National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India

Dr J. Borgoño, Professor of Preventive Medicine, University of Chile, Chief, Department of International Affairs, Ministry of Health, Santiago, Chile

Dr Y. Egashira, Honorary Staff, National Institute of Health, Hatano Research Institute, Food and Drug Research Centre, Hatanoshi, Kanagawa-ken, Japan

Dr R.A. Goyer, Deputy Director, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA

Dr P. Grandjean, Institute of Community Health, Department of Environmental Medicine, Odense University, Odense, Denmark

Dr V.N. Ivanov, Director, Chita Medical Institute, Chita, USSR

Dr V.V. Ivanov, Chief, Department of Pathophysiology, Krasnoyarsk Medical Institute, Krasnoyarsk, USSR

Dr R.D. Kimbrough, Research Medical Officer, Toxicology Branch, Clinical Chemistry Division, Bureau of Laboratories, Center for Environmental Health, Centers for Disease Control, Atlanta, Georgia, USA

Professor P. Krogh, Department of Microbiology, Royal Dental College, Copenhagen, Denmark

Dr O.A. Levander, Vitamin and Mineral Nutrition Laboratory, Nutrition Institute, US Department of Agriculture, Beltsville, Maryland, USA (Co-Rapporteur)

Dr K.R. Mahaffey, Division of Standards Development and Technology Transfer, National Institute for Occupational Safety and Health, Cincinnati, Ohio, USA

Dr G. Martin-Bouyer, Conseiller Technique, Ministère des Affaires Sociales et de la Solidarité Nationale, Direction Générale de la Santé, Paris, France

Professor V.A. Nasonova, Director, Institute of Rheumatology, Academy of Medical Sciences of the USSR, Moscow, USSR (Vice-Chairman)

Professor H.D. Tandon, formerly Director, All India Institute of Medical Sciences, New Delhi, India

Professor H. Yanagawa, Department of Public Health, Jichi Medical School, Minamikawachi, Tochigi-ken, Japan

Dr Bai Shicheng, The Basic Medical Institute of Liaoning Province, Shenyang, People's Republic of China

Professor Chen Chunming, Director, China National Center for

Page 50 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

Preventive Medicine (CNCPM), Beijing, People's Republic of China (Chairman)

Dr Chen Junshi, Deputy Director, Department of Nutrition and Food Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China (Rapporteur)

Dr Chen Xiaoshu, Deputy Director, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

Dr Deng Jiayun, The Health and Antiepidemic Station of Sichuan Province, Chengdu, People's Republic of China

Dr Geng Jinzhong, Deputy Director, Division of Environmental Hygiene, Ministry of Public Health, Beijing, People's Republic of China

Dr Guo Xiong, Research Laboratory of Endemic Bone Disease, Xian Medical University, Xian, People's Republic of China

Professor Li Fangsheng, The Basic Medical Institute of Liaoning Province, Shenyang, People's Republic of China

Dr Li Guangshen, Institute of Endemic Disease, Norman Bethune University of Medical Sciences, Changchun, Jilin Province, People's Republic of China

Professor Li Jiyun, Northwestern Institute of Soil and Water Conservation, Chinese Academy of Sciences, Shaanxi, People's Republic of China

Dr Li Sheng, Deputy Director, Division of Technical Assistance and Development, China National Center for Preventive Medicine, Beijing, People's Republic of China

Dr Li Chongzheng, Institute of Prevention and Cure of Endemic Diseases, Gansu Province, People's Republic of China

Dr Liang Shutang, Institute of Prevention and Cure of Endemic Diseases, Shaanxi Province, People's Republic of China

Dr Ma Tai, Tianjin Medical College, Tianjin, People's Republic of China

Dr Mo Dongxu, Research Laboratory of Endemic Bone Diseases, Xian Medical University, Xian, People's Republic of China

Dr Niu Guanghou, The Health and Antiepidemic Station of Heilongjiang Province, Haerbin, People's Republic of China

Professor Niu Shiru, Director, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China (Vice-Chairman)

Professor Peng An, Institute of Environmental Chemistry, Chinese Academy of Sciences, Beijing, People's Republic of China

Professor Ren Hongzao, The Basic Medical Institute of Liaoning Province, Shenyang, People's Republic of China

Dr S. Shibata, Director, Division of Clinical Research, National Medical Centre of Japan

Page 51 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

Dr Sun Xi, Head, Division of Science and Technology, Office for Endemic Diseases (CPCCC), Shenyang, People's Republic of China

Professor Tan Jianan, Institute of Geography, Chinese Academy of Sciences, Beijing, People's Republic of China

Dr Xi Guangzeng, The 323 Hospital of the People's Liberation Army, Xian, People's Republic of China

Dr Xu Guanglu, Research Laboratory of Keshan Disease, Xian Medical University, Xian, People's Republic of China

Professor Yang Fuyu, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China

Professor Yang Guangqi, Department of Nutrition and Food Hygiene, Institute of Health, China National Centre for Preventive Medicine, Beijing, People's Republic of China

Professor Yang Jianbo, Haerbin Medical University, Haerbin, People's Republic of China

Professor Yang Tongshu, Institute of Endemic Disease, Normal Bethune University of Medical Sciences, Changchun, People's Republic of China

Dr Yin Peipu, Institute of Endemic Bone Diseases, Xian Medical University, Xian, People's Republic of China

Dr Ying Mingxin, Haerbin Medical University, Haerbin, People's Republic of China

Professor Yu Weihan, Haerbin Medical University, Haerbin, People's Republic of China

Dr Zhai Shusheng, The Second Institute of Prevention and Cure of Endemic Diseases, Jilin Province, People's Republic of China

Dr Zhao Tieli, Deputy Director, Office for Prevention of Endemic Diseases (CPCCC), Shenyang, People's Republic of China

Dr Zhou Zhenglong, Institute of Prevention and Cure of Endemic Diseases, Shanxi Province, People's Republic of China

Representatives from Other Organizations

Dr R. Hoffmann, Senior Programme Officer, United Nations Children's Fund (UNICEF), Beijing, People's Republic of Chinaa

Dr Zhang Naizheng, International League Against Rheumatism (ILAR), Capital Hospital, Chinese Academy of Medical Sciences, Beijing, People's Republic of China

Secretariat

Dr Eric H.T. Goon, WHO Representative and Programme Coordinator, Beijing, People's Republic of China

Dr M. Mercier, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland

Dr M. Mitrofanov, Division of Noncommunicable Diseases, World Health Organization, Geneva, Switzerland

Page 52 of 53 Diseases of suspected chemical etiology and their prevention, principles of studies on (EHC 72, 19...

Dr J. Parizek, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary)

Dr Xu Genlin, Director, Office of Research Management, Institute of Health, Beijing, People's Republic of China

Dr Yao Peipei, Division of Pneumoconiosis, Institute of Health, Beijing, People's Republic of China

Dr Liu Yuying, Division of Industrial Toxicology, Institute of Health, Beijing, People's Republic of China

Dr Wang Zhiwu, Institute of Kashin-Beck Disease of Heilong-jiang Province, Haerbin, People's Republic of China

------a Attended the opening of the meeting.