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.