EXPOSURE, SENSITIZATION AND ALLERGY TO INDUSTRIAL ENZYMES Markku Vanhanen People and Work Research Reports 46 Finnish Institute of Occupational Health Department of Pulmology, Helsinki University Central Hospital Helsinki 2001 1 Cover Design Susanna Virtanen Layout Vammalan Kirjapaino Oy ISBN 951-802-453-7 ISSN 1237-6183 Vammalan Kirjapaino Oy Vammala 2001 2 To Sanna-Leena, Ilkka, Sini and Jukka 3 CONTENTS SUMMARY .................................................................................... 6 ACKNOWLEDGEMENTS ............................................................... 8 ABBREVIATIONS........................................................................... 9 LIST OF ORIGINAL PUBLICATIONS.............................................. 10 1. INTRODUCTION ....................................................................... 11 2. REVIEW OF THE LITERATURE ................................................. 12 2.1. What enzymes are............................................................. 12 2.2. History of enzyme use and technology ............................. 12 2.3. Modern production of enzymes by microbes.................... 13 2.4. Classification of enzymes .................................................. 13 2.5. Applications of industrial enzymes.................................... 14 2.6. Health effects of industrial enzymes ................................. 17 2.6.1. Respiratory allergies caused by enzymes................ 17 2.6.1.1. Detergent industry.................................. 17 2.6.1.2. Pharmaceutical industry, health care and related occupations................................ 18 2.6.1.3. Baking industry ....................................... 19 2.6.1.4. Enzyme-producing industry..................... 20 2.6.1.5. Other industries....................................... 21 2.6.2. Dermatitis due to enzymes .................................... 21 2.6.3. Allergy to enzymes among consumers................... 21 2.6.4. Determinants of sensitization ................................ 28 2.7. Characterization of enzyme allergens ............................... 29 2.8. Diagnosing enzyme-induced asthma with a challenge test 29 2.9. Monitoring of enzymes in the workplace air..................... 30 2.9.1. Catalytic methods.................................................. 30 2.9.2. Immunologic methods........................................... 34 2.10.Exposure guidelines for enzymes...................................... 35 3. AIMS OF THE STUDY............................................................... 40 4. MATERIAL AND METHODS .................................................... 41 4.1. Workplaces and subjects................................................... 41 4.2. Total dust and enzyme measurements.............................. 43 4 4.2.1. Sampling................................................................ 43 4.2.2. Analysis.................................................................. 44 4.3. Assessment of work-related symptoms............................. 44 4.4. Assessment of sensitization............................................... 45 4.4.1. Skin prick test ........................................................ 45 4.4.2. Immunoglobulin E measurements.......................... 46 4.5. Characterization of enzyme allergens ............................... 46 4.6. Lung function tests and testing bronchial hyperreactivity (study V)............................................................................ 46 4.7. Specific challenge tests (study V)....................................... 46 4.8. Statistical methods............................................................ 47 5. RESULTS ................................................................................... 48 5.1. Enzyme and total dust measurements (studies I–IV).......... 48 5.2. Sensitization to enzymes (studies I–IV).............................. 50 5.3. Sensitization to flours and storage mites .......................... 51 5.4. Sensitization to environmental allergens........................... 51 5.5. Relation of atopy and smoking to sensitization to enzymes, flours and storage mites ................................................... 51 5.6. Work-related symptoms.................................................... 56 5.7. Specific challenge tests (study V)....................................... 57 5.8. Characterization of enzyme allergens ............................... 58 6. DISCUSSION............................................................................. 61 6.1. Air concentration of dust and enzymes............................. 61 6.1.1. Total dust............................................................... 61 6.1.2. Enzymes................................................................. 62 6.2. Sensitization and allergy to enzymes................................. 65 6.3. Role of atopy in the sensitization to enzymes................... 67 6.4. Diagnosing enzyme-induced asthma using specific bron- chial provocation test........................................................ 68 6.5. Characterization of enzyme allergens ............................... 69 6.6. Validity issues.................................................................... 69 6.6.1. Study design and selection of study populations..... 69 6.6.2. Validity of the methods......................................... 70 6.6.2.1. Assessment of sensitization..................... 70 6.6.2.2. Assessment of symptoms........................ 70 6.6.2.3. Assessment of exposure.......................... 71 6.7. Prevention of allergies to enzymes.................................... 71 7. CONCLUSIONS......................................................................... 74 8. REFERENCES............................................................................. 76 ORIGINAL PUBLICATIONS (I–V)................................................... 87 5 SUMMARY The production and use of industrial enzymes have increased markedly during the last few decades. Today, enzymes are used, for example, in the detergent, food, feed, textile and pulp and paper industries. Respiratory allergies to powdered microbial enzymes surfaced in the late 1960s in the detergent industry. With improvements in industrial hygiene, the problem abated. Since the 1980s, allergies have emerged in other industries however, notably in bakeries. A series of studies on enzyme allergy was performed in 1992–1997. The aim was to assess exposure and allergy to enzymes in Finnish enzyme manufacturing and industries using enzymes. Investigations were performed in four bakeries, one flour mill, one rye crisp factory, one detergent factory, four animal feed factories, one biotechnical research laboratory and one biotechnical plant having both research and production units. For determining α-amylase, a catalytic method was used which detects also the inherent amylase of flour. For protease detection both a catalytic method and a more specific immunologic procedure were used. Cellulase and xylanase were measured with an immunologic method. Powdered enzyme-containing additives were used in the bakeries, where high α-amylase levels, up to 6.6 µg/m3, were found during dough making. In other locations, the levels were generally lower, below 0.2 µg/m3. In addition, xylanase concentrations of 2– 200 ng/m3 (mean 65 ng/m3) were found, possibly also due to inherent xylanase. Enzyme-containing additives were mixed in the four mill, and α-amylase concentrations up to 1.1 µg/m3 and cellulase concentrations up to 180 ng/m3 were determined at the mixing sites. In the rye crisp factory the α-amylase levels were lower than in the bakeries (mean value 0.1 µg/m3 for personal samples and 0.03 µg/m3 for stationary samples). The cellulase concentrations ranged from 25 to 160 ng/m3 in different phases of the mixing, dough making and bread forming. At the same sites, lower levels (7– 40 ng/m3) of xylanase were measured. In the animal feed factories, the nonspecific assay showed high levels of protease (up to 0.4–2.9 µg/m3) and α-amylase (up to 0.2 µg/m3), which coincided with the high total dust levels but not with the amount of added enzyme. In the detergent factory, the protease levels, measured with a catalytic method, were generally below 50 ng/m3, but at the enzyme mixing site very high concentrations, above 1000 ng/m3, were found. The analysis with an immunologic method gave results of the same 6 order, indicating that the main origin of the protease was the added enzyme. Few measurements prior this study from the enzyme manufacturing industry had indicated cellulase concentrations on the order of 50 ng/m3 in laboratory work. Judging from job descriptions, much higher enzyme concentrations probably occurred occasionally during the mixing, drying and packing of enzymes. The prevalence of sensitization to enzymes, assessed by skin prick testing, was 7.8% in the bakeries, 4.8% in the flour mill and 2.7% in the rye crisp factory. When the office personnel was excluded, the figures were 8.4%, 5.3% and 3%, respectively. In the animal feed industry the corresponding prevalences were 4.6% and 7.1%, and in the detergent industry 11.8% and 22.5%. In the biotechnical research laboratory 11.7% of the workers and in the biotechnical plant 12.6% of the workers were sensitized. In the category of research, laboratory and enzyme manufacturing work, the rates were 12.6% and 15.4%, respectively. A statistically significant exposure-response linear trend was demonstrated among the biotechnical workers. Atopy, as demonstrated using skin prick testing, increased the risk of sensitization three to five times among the workers studied, except in the detergent factory. Sensitization to enzymes was associated with work-related respiratory symptoms in all the industries studied. Several cases of specific occupational asthma or rhinitis due to enzymes were diagnosed later, thus verifying the causal connection of sensitization to clinical allergy. The bronchial challenge method used proved to be practical for challenges with powdered enzymes. Sensitization was found for previously well-known enzymes, such as protease in the detergent industry and α-amylase in the bakeries. Lipase and cellulase were also shown to be allergens in the detergent industry. In addition, it was found that phytase causes sensitization in enzyme production and the animal feed industry. Sensitization to cellulase and xylanase was common due to the increasing manufacture of these enzymes in Finland. Immunoblotting showed that the antigens of α-amylases of bacterial and fungal origin differed from each other, as the sera from persons sensitized to fungal amylase did not bind to bacterial amylase, and vice versa. Development and international standardization are urgently needed to establish methods for measuring air concentrations of enzymes. For the prevention of sensitization to enzymes and allergic diseases caused by them, the risk of allergy has to be recognized at workplaces, and exposure to enzymes must be kept to a minimum. 7 ACKNOWLEDGEMENTS This work was carried out at the Finnish Institute of Occupational Health, Helsinki. I wish to thank Professor Jorma Rantanen, Director General of the Institute, and also the directors of the Department of Occupational Medicine during 1991–2001, Professor Vesa Vaaranen, Professor Kaj Husman, and Professor Helena Taskinen, for providing excellent working facilities for this project. In addition, I wish to express my gratitude to the following persons: docent Henrik Nordman, MD, my supervisor and co-author, for giving me the idea for the study and for his expertise and advice, docent Timo Tuomi, PhD, my second supervisor and co-author, for his co- work, help and untiring support, my co-authors Outi Tupasela, MSc, and Ulla Tiikkainen, PhLic, especially for their expertise in the immunologic studies, my other co-authors: Peter C. Holmberg, MD, Heikki Hokkanen, MSc, Maija Hytönen, MD, Professor Lasse Kanerva, Helena Keskinen, MD, Professor Matti Leisola, Ritva Luukkonen, PhD, Marja Miettinen, MD, Pertti Mutanen, MSc, Kyllikki Tarvainen, MD, Anneli Tuomainen, PhD, Matti Tuppurainen, MD and Risto Voutilainen, MD, docents Antti Ahonen, MD, and Erkki Yrjänheikki, PhD, for their critical review of the manuscript, my untiring co-workers Ms Riitta Valio and Ms Terttu Mäkelä in the allergy investigations at numerous workplaces, Mr Reima Kämppi for the measurements of dusts and enzymes, Arne Ståhl, MSc, for the immunologic determination of protease, my present and former supervisors and colleagues over the years, especially Mari Antti-Poika, MD, Brita Grenquist-Nordén, MD, Heikki Koskinen, MD, Tuula Estlander, MD, Riitta Jolanki, DTech, Ilmari Böss, MD, and Riitta Sisko Koskela, PhD, the staff of the Department of Occupational Medicine, the directors and employees at the workplaces studied, the staffs of the occupational health units of the workplaces, especially Ms May Roth- Edelmann, and Georgianna Oja, ELS, for revising the language. I owe my warmest thanks to my wife Sanna-Leena and our children Ilkka, Sini and Jukka for their patience and love during this work. The work was supported financially by the Finnish Work Environment Fund, the Finnish Society of Allergology and Immunology and the Allergy Research Foundation, which I acknowledge gratefully. 8 ABBREVIATIONS ELISA enzyme-linked immunosorbent assay FIOH Finnish Institute of Occupational Health FEV forced expiratory volume in 1 second 1.0 IgE immunoglobulin E kDa kilo Dalton MW molecular weight OA occupational asthma OEL occupational exposure limit PEFR peak expiratory flow rate py person-year RAST radioallergosorbent test TLV threshold limit value SPT skin prick test wt/vol weight/volume 9 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original articles, which are referred to in the text by the Roman numerals indicated below: I Vanhanen M, Tuomi T, Hokkanen H, Tupasela O, Tuomainen A, Holmberg PC, Leisola M, Nordman H. Enzyme exposure and enzyme sensitization in the baking industry. Occup Environ Med 1996;53:670–676. II Vanhanen M, Tuomi T, Nordman H, Tupasela O, Holmberg PC, Miettinen M, Mutanen P, Leisola M. Sensitization to industrial enzymes in enzyme research and production. Scand J Work Environ Health 1997;23:385–391. III Vanhanen M, Tuomi T, Tiikkainen U, Tupasela O, Voutilainen R, Nordman H. Risk of enzyme allergy in the detergent industry. Occup Environ Med 2000;57:121–125. IV Vanhanen M, Tuomi T, Tiikkainen U, Tupasela O, Tuomainen A, Luukkonen R, Nordman H. Sensitisation to enzymes in the animal feed industry. Occup Environ Med 2001;58:119–123. V Vanhanen M, Tuomi T, Tupasela O, Keskinen H, Tuppurainen M, Hytönen M, Tarvainen K, Kanerva L, Nordman H. Cellulase allergy and challenge tests with cellulase using immunologic assessment. Scand J Work Environ Health 2000; 26:250–256. 10