Biological Effects of Proteolytic Enzyme Detergents1

Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from Thorax (1976), 31, 621.

Biological effects of proteolytic enzyme detergents1

A potentially serious respiratory hazard during later. A majority of the work presented was the manufacture of enzyme detergents was first unpublished. reported by Flindt (1969) and Pepys et al. (1969). In the eight years since the hazard was identified much effort has been put into investigating the PRODUCTION AND DUST CONTROL extent and nature of the effects and removing Van Veizen (Gist-Brocades, Netherlands) des- their cause. A feature of the work has been the cribed the manufacture of the enzyme by a fer- national and international exchange of medical mentation process, using Bacillus subtilis. The and industrial hygiene information between pro- purification steps need extremely careful quality ducing companies and the help provided by control. This is achieved by a batch process of academic departments of immunology and respira- manufacture. After purification (germ removal), tory disease to assess the biological effects. These a concentrated enzyme powder is produced by have included serial observations to monitor the precipitation, filtration, and drying. To ensure a efficacy of the rapidly improving dust conditions clean environment, the relatively dusty powder is achieved by the engineers. handled in a closed system. As in the detergent In 1974 Professor Hans Weill, of Tulane Uni- industry, great attention is now paid to engineer- versity, New Orleans, and Dr. John C. Gilson, ing detail where spillages or emissions of dust may Director of the MRC Pneumoconiosis Unit, sug- occur; for example, an ingenious pneumatic copyright. gested that 1976 would be an opportune time to double seal is used in the bagging of the powder. review the scene to obtain a general picture of In the detergent factory the heat-sensitive en- progress and of the lessons learnt which might zyme has to be incorporated in a powder base have applications elsewhere. The opportunity which is manufactured by spray drying in a large given by such a meeting was welcomed by the tower at elevated temperature. The manufacture companies in the USA and in Europe making the of the powder base was described by Davies enzymes and the enzyme detergent products. (Lever Brothers, UK) to give an indication of the http://thorax.bmj.com/ Topics covered included: making the enzymes scale of the manufacturing process (tons per and the detergents; toxicological and immuno- hour). After addition of the heat-sensitive en- logical studies in animals; clinical features of the zymes and other chemicals, the product is mixed effects in small groups and in individuals; immuno- and then put into cartons in the packing room. logical surveillance of exposed working groups; This was previously a labour-intensive area where and cross-sectional and longitudinal studies of exposures readily occurred. Great attention has respiratory symptoms and lung function of pro- been and is now continuing to be paid to reducing duction workers in several countries. There was these exposures. The dustiness of the product has on October 2, 2021 by guest. Protected an excellent opportunity, therefore, for engineers, been reduced by 'encapsulating' the enzymes. It epidemiologists, immunologists, occupational was glued to a coarse (>150 ,u) granule of phos- physicians, respiratory physiologists, and statisti- phate base by a tacky non-ionic detergent (an cians to contribute to the general assessment of ethoxylated linear aliphatic alcohol). The enzyme current knowledge in this field. Papers prepared manufacturers had now made further improve- for the meeting will be appearing in appropriate ments by forming 'marumes' and 'prills'. These journals. A list of the papers precirculated is given are beads containing the enzyme embedded in (p. 631) to assist in tracing the published articles non-dusty matrix. The 'prill' is produced by spraying a molten mixture of the enzyme with an 'Report of a symposium held on 4-5 May 1976 at the MRC organic material (non-ionic) into a tower in which Pneumoconiosis Unnit, Cardiff, sponsored by the Medical Research Council, UK, and the Soap and Detergent Industries Association the droplets cool to form regularly shaped non- (SDIA), UK. For the list of participants with their affiliations and the titles of papers presented, see p. 630. The report was prepared by J. C. dusty solid beads. By this means the enzyme is Gilson, C. P. Juniper, R. B. Martin, and H. Weill with the assistance incorporated into a less friable matrix than in the of Rapporteurs-D. R. Davies, J. H. Edwards, C. P. Juniper, W. E. Parish, and C. E. Rossiter. earlier methods of encapsulation. 621 Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from 622 Biological effects of proteolytic enzyme detergents

Bruce (Procter & Gamble, UK) described the much less marked. The haemorrhagic response in- advances in dust monitoring. The SDIA had duced by high concentration of enzymes could developed a high-volume (700 1 /min) Galley also be demonstrated on the exposed vascular sampler with a performance similar to the MSA bed of the rat cremaster muscle preparation. Heat- Fixt Flo. They also used a Casella personal ing the enzyme preparation prevented pulmonary sampler (2 1/min). At low dust concentrations and cremaster muscle injury. the instruments agreed well; at high levels In the guinea-pig repeated weekly exposures to (>02 Gu/m3; Gu=Glycine units, see Glossary aerosols at a level which did not induce pul- of Terms, p. 628) the personal sampler gave values monary damage at a single exposure resulted in of up to 10-fold greater. The reasons for this were systemic sensitization after the third or fourth not yet clear. A continuous sampler was devel- exposure. Mediation of the immune system was oped; this is useful in detecting peak emissions of shown by an immediate skin reaction, positive enzyme. The use of an Anderson sampler had Schultz-Dale reaction, and passive cutaneous shown that about 50% of the dust collected was anaphylaxis reactions (PCA). The latter reaction respirable (<7,u), and 70% of the enzyme dust established that the predominant circulating anti- was in this respirable fraction. However, there body was IgGla. With continuing exposures at was some difficulty in interpreting the results of weekly intervals, the respiratory response de- an Anderson sampler with a dust consisting of creased and was virtually absent after 20 ex- fragile particles which might be fractured into posures. This was associated with an increase in smaller pieces during passage through the the total level of specific enzyme inhibitor in the instrument. serum. Davies used the results of dust assessments from The difference in responses of isolated normal one factory from 1969 (when the first measure- or 'sensitized' guinea-pig lungs, when challenged ments were made) to 1975 to show the secular with enzyme preparations, confirmed that the trend of improvement achieved by following the induced in normal lung was bronchospasm copyright. SDIA recommendations (SDIA, 1969). During the associated with the release of histamine, whereas six years there had been a six-fold reduction in the effect on sensitized lung was associated with the average atmospheric dust concentration in a greater release of histamine and SRS-A. In vivo the packing rooms of the four UK detergent fac- studies using specific antagonists confirmed these tories (1200-*200 tLg/m3), and in the same period findings. the amount of associated enzyme dust had been The effect of detergents on the immune re- reduced by a factor in excess of 30 (2-3 Gu/m3) sponse was seen only at high detergent levels. http://thorax.bmj.com/ in 1969 to 0 05-0l1 Gu/m3 in 1975. Further When guinea-pigs were exposed to very high levels improvements had been achieved by the use of of a detergent aerosol (anionic or non-ionic; 'marumes' and 'prills' (SDIA Medical Review 70 mg/m3) in the presence of an enzyme aerosol, 1969-75). Atmospheric enzyme levels were now which alone did not produce sensitization at the present limits of analytical detectability. (6000 Gu/m3), a 40% incidence of sensitization occurred. Exposure for a total of 3500 hours TOXICOLOGY (18 h/day, 4 days/week, for 1 year) to dust eluted from enzyme washing powder product, at levels

Cambridge (Unilever, UK), described the effects on October 2, 2021 by guest. Protected of heavy exposures in guinea-pigs and rats to of about 2 mg/m3, with enzyme activity 1 Gu/m3, aerosols of solutions of enzyme preparations. produced sensitization in only 1 of 16 guinea- The effects were dose-related. Guinea-pigs ex- pigs. posed to 80 000 Gu/m3 for one hour (enzyme Ritz (Procter & Gamble, USA) reported on activity 1100 Gu/mg) showed respiratory distress the relative anaphylactogenicity and immuno- and hypothermia with some fatalities. Histologi- genicity of a number of commercial enzyme pre- cally, intra-alveolar haemorrhage with eosinophil parations-Alcalase, Rapidase, and Monsanto and neutrophil infiltration was seen. At a quarter DA-lO-together with an experimental Alkaline of the dose for half the time only eosinophil- Protease. Guinea-pigs were given enzyme intra- neutrophil infiltration was seen. Similar effects tracheally weekly or biweekly in the range of were observed in animals exposed to dry enzyme 10 ng-10 ,ag (protein nitrogen) per dose. The powder. The rat was much more resistant to the pulmonary response was graded in severity on a effect of exposure to enzyme aerosols; even at the five-point scale. Serum samples were examined by highest concentration alveolar haemorrhage was PCA for the presence of IgGl and by immuno- Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from Biological effects of proteolytic enzyme detergents 623 diffusion for precipitating antibodies against the greater than that of human, rabbit, or horse lung, enzyme. Dose response relationships were ob- so that cellular destruction in the guinea-pig served for each enzyme preparation, symptoms of might produce less emphysema. Wagner (PU, anaphylaxis being produced in general at about UK) said that the marked focal peribronchial 1 ,ug dose. At 10 jug, all animals exhibited symp- reaction, seen in the very high levels of detergent, toms often after the second dose, and there was was similar to that seen in the earlier response to little difference between the preparations. How- some inorganic dusts such as asbestos. ever, in terms of immunogenicity Alcalase induced Repeated low doses of enzyme, except that of lower levels of IgGI and precipitating antibodies the very lowest dose, produced sensitization than did the other enzymes. Augmentation of the mediated by IgGla. Tolerance was acquired after anaphylactic and immune responses to Alcalase repeated exposures, probably due to the presence by anionic and non-ionic detergents was seen at of other specific antibodies to the enzyme block- high detergent levels. ing the IgGla mediated response. Cambridge Brown (Procter & Gamble, Belgium) had said that, in general, the enzymes were less studied in detail in Cynomolgus monkeys the allergenic than egg albumin but more so than effect of inhalation of enzyme and detergent trypsin. All studies had shown a potentiating effect alone, and in combination at varying doses. The of the enzymes by the detergents. This had been enzyme was a mixture of Novo-Alcalase and known for many years (Parish) but the mechanism Milezyme 8X. The experiment lasted for six of potentiation was not understood. Little was months and exposures were to a highly (95%) yet known about the effect of particle size on respirable dust. The effects were studied by be- the production of sensitization. havioural changes, clinical signs, pulmonary func- In extrapolating the results of animal studies to tion tests, and/or histopathological alterations. man, Parish emphasized the marked species dif- Extensive haematology, clinical chemistry, and ference in the 'shock organ'. The immediate allergic responses affected different organs, for urine analysis were also made. copyright. At levels of 1 mg/m3 detergent dust with example, in the guinea-pig the lung, and in some 200 yg/m3 enzyme, no histological, pulmonary monkeys the gut. It was also necessary to function, or biochemical effects were observed. recognize the big differences in the exposures as Higher dust levels, 10-100 mg/m3, produced compared to man. In a majority of the animal functional and histological effects but were pri- studies, these had been relatively short term and marily related to the detergent level. The histo- at high concentrations. pathologic effects produced by exposure to high http://thorax.bmj.com/ levels of detergent (10-100 mg/m3) were similar IMMUNOLOGY to those seen after exposure to known lung In the Third Session the extensive knowledge now irritants (03, SO2, and NO2) but were partly acquired about the immunological effects based reversible on cessation of exposure. Lung function on clinical studies of individual cases and of tests suggested bronchiolar constriction; the groups of enzyme detergent workers was dis- histology showed some alveolar fibrosis and cussed. The only proven allergic response in man bronchiolar epithelial hyperplasia. Pulmonary to the enzyme detergent dusts is an IgE-mediated function was reversible after a of no ex- period asthma, or rhinitis, and very occasionally con- on October 2, 2021 by guest. Protected posure. An immunological response (precipitating junctivitis. The enzymes, like common environ- antibodies) was produced at all levels of enzyme mental antigens, for example, grass pollens, dust exposures. stimulate formation predominantly of IgE These varied experiments have defined a num- antibodies. ber of the responses, toxicological and immuno- The enzyme-induced allergy or allergic dis- logical, produced by enzymes and detergents and orders are almost limited to factory workers their interactions. The destruction of lung tissue exposed to the dusts usually for fairly long periods. with the development of emphysema, such as is An exception was one sensitized housewife (case seen with the enzyme papain, was not a feature. presented by Dijkman, Netherlands) who was Dolovich (Ontario) suggested that this was due exposed to antigen contaminating the clothes and to the detergent enzyme having no elastase the dusty hair of her husband, an enzyme deter- activity which was necessary for the production gent factory worker. The incidence of sensitiza- of emphysema. Parish (Unilever, UK) commented tion depends upon the amounts of enzyme antigen also that the elasticity of the guinea-pig lung was in the work rooms, duration of exposure, and, to Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from 624 Biological effects of proteolytic enzyme detergents some extent, the susceptibility of the workers. likely to induce inflammatory responses resem- When dust levels are high all persons, atopic and bling Arthus reactions in the absence of appro- non-atopic, are at risk, though not all become priate antibody (Parish). Some persons who show sensitized. At low dust levels atopic persons are such reactions on one test do not do so 6 or 12 more susceptible than non-atopics. If only traces months later (Juniper, Unilever, UK; Parish). This of the dust occur, as in some factories now, very was not typical of Arthus sensitivity in persons few people, if any, become sensitized. continually exposed to an antigen. Detection of specific hypersensitivity is by the The results of RAST to detect IgE antibodies to prick test and RAST (see Glossary of Terms); a enzymes carried out independently in different positive skin test or RAST may occur before laboratories were very consistent (Pepys; Wilson, clinical symptoms are apparent. Clinical effects Procter & Gamble, USA; How). Stored frozen of the dust are more readily interpreted with the sera must be centrifuged to remove aggregates in help of the results of immunological tests. order to obtain reproducibility of duplicate tests For reliable prick test results, a standard gentle in the RAST for Alcalase (Pepys). There was a procedure and concentrations of antigen that do statistically significant correlation between the not induce antigen-nonspecific irritation are size of prick test wheals of 3 mm diameter or essential (Pepys, London). The crude enzyme greater and the IgE antibody measured by RAST: preparations contain components of culture [r=0 5 (Wilson) and r=0 6 (How)]. As would media, cell debris, and some bacterial spores, so be anticipated, this does not apply when RAST a standard skin prick test antigen containing all counts are high. The correlation is not sufficiently the enzyme allergens is essential. This was pre- high for prediction in the individual. Furthermore, pared by Unilever Research and made available to important evidence was presented that the demon- the SDIA and internationally (How, Unilever, stration of, or increase in, serum IgE antibody UK). When using this antigen there was 99% may occur one or two years after appearance of positive prick tests (Wilson), and nearly 25% of agreement between negative skin tests and a nega- copyright. tive RAST (How). Zachariae (Demnark) reported skin test positive employees never did develop the use of skin prick tests with crystalline Alcalase elevated IgE levels. This shows that monitoring in Rumania before enzyme detergents were in use. allergy in factory workers by RAST only Six per cent of atopic and other patients (280) (Zachariae; Witmeur. Novo, Denmark) may miss were positive, but only one had positive RAST. early cases of hypersensitivity, but 'false positives' He thought the skin test might be non-specific were avoided. in some instances, and that RAST was useful to There was evidence about reversion of prick http://thorax.bmj.com/ confirm an allergy. In a study by Pepys, several tests in those no longer exposed to the enzymes. thousand atopic persons were routinely tested In the United States none of those sensitized for with enzyme antigen when attending an allergy two or more years to Monsanto DA-lO enzyme clinic, whether or not they used enzyme detergents mixture but 7% of those sensitized for at least at home; they showed no evidence of allergy to two years to Alcalase reverted to prick test nega- these enzymes. A similar survey of over 300 tive (Wilson). In the United Kingdom, 36 out of housewives reported by Dijkman led to the same 564 (6-4%) workers sensitized to Alcalase/ conclusion. The essential need for careful Maxatase reverted to prick test negative (Juniper). standardization of the prick test procedure and A six-year serial study in one US factory on October 2, 2021 by guest. Protected the antigen was repeatedly stressed. (Wilson) had shown that, starting in 1969, the pro- Prick tests have now been repeated in many portion of workers becoming prick test positive hundreds of people. There was good evidence that within six months ranged from 0 to 39%, de- this did not lead to sensitization of those tested. pendent on the job, but by 1970 the reduction in Indurated (firm oedematous) skin prick test dust levels and the less frequent production of responses occurring 6 to 8 hours after test, and the product had greatly reduced the incidence. occasionally severe, were reported by Newhouse No one starting work after 1970 had been sensi- (London) and Martin (Procter & Gamble, UK). tized. In this factory there was no statistically There was considerable discussion about this type significant difference in sensitization rates of the of response. It had features comparable with an atopics and non-atopics as measured by either Arthus reaction, but there is no immunological skin or RAST tests. proof. This response may not even be immuno- In tests for antibodies in other immunoglobulin logical. Bacterial substances are particularly classes, no precipitating antibody was seen in Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from Biological eftects of proteolytic enzyme detergents 625

Ouchterlony double diffusion agar plates, though test reagent described by How was effective and non-immunological complexes of enzyme with safe in use. The early clinical picture became al anti-trypsin and a2 macro-globulin were formed modified over the next two years or so with the by most sera (How). The very discriminating absence of haemoptysis and chest pain. The techniques, capable of detecting extremely small severity of asthma and dyspnoea diminished from amounts of antibody, RID (see Glossary of Terms, an incapacity lasting days or weeks in 1968/69 p. 628), showed anti-enzyme antibodies which were to a milder process lasting hours and requiring identified as IgG in 43 of 65 sera, IgA in 21 of minimal treatment. These changes coincided with 65 sera, and a few with IgM by indirect radio- environmental improvement. Analysis of case immunoelectrophoresis, IRIEP (see Glossary of histories revealed three clinical types of asthma, Terms), (Pepys). The occurrence of IgG anti- immediate, non-immediate, and dual. Immediate enzyme by IRIEP was confirmed (How). An even asthma occurred 10 to 15 minutes after exposure more discriminating technique, a solid phase and lasted 1 to 3 hours. In other subjects, asthma assay, also detected IgG antibodies to enzyme developed slowly at about 6 hours to a maximum (Pepys), as did the 'sandwich' procedure used to at 8 hours and lasted up to 24 hours. Clinical test IgG fractions of sera (How). features included sweating, pallor, and anxiety IgG antibodies were detected only in the sera with marked tachycardia and dyspnoea. There of persons exposed to enzyme dusts in factories was cough with minimal sputum. Generalized and were found in almost all sera containing IgE rhonchi were usually heard but the degree of antibodies (Pepys). They were usually present in bronchospasm was less than would be expected very small amounts, possibly less than is required from the dyspnoea observed. Crepitations were to mediate conventional Arthus reactions. They not a striking feature, although occasionally are evidence of exposure to the antigens, not of present. The aetiology, particularly of the late allergy, and probably reflect the natural formation asthmatic episode, was easily missed, so steps of antibody as occurs in response to numerous were taken to alert local practitioners. Asthma at copyright. environmental antigens. work due to non-industrial antigens was also seen It was strongly recommended that in order to and could cause confusion. The atopics tended to achieve comparable results in all laboratories have immediate asthma, whereas the non-atopics standard reagents should be available and tended to respond 6 to 8 hours later. The time standardized procedures used. Important technical taken for recovery of respiratory function after points, for example, the effect on the in vitro an attack of bronchospasm, produced by pro- techniques of storage of sera, should be published teolytic enzyme, depended upon the duration of http://thorax.bmj.com/ and reference sera made available to standardize stimulation and the dose. Examples were given of the results. This would ensure more general con- full recovery and of the way an individual may formity in investigations in different factories react to other irritant dusts immediately after internationally. an attack of allergic asthma. Attacks of nocturnal asthma on succeeding nights following the initial CLINICAL episode were described and the similarity to the picture seen with other antigens was noted Juniper described the clinical pattern of disease (Pepys). Allergy due to this antigen is primarily arising from allergy to proteolytic enzymes pro- type I, and the clinical picture had become pro- on October 2, 2021 by guest. Protected duced by Bacillus subtilis, starting with the obser- gressively milder as the environment improved. vations of Flindt and Pepys in 1969. The features of dyspnoea, wheezing, and cough, with minimal Over 2800 employees in UK factories had been sputum, were accompanied by haemoptysis and reviewed under the SDIA recommendations for chest pain in some patients. Conjunctivitis when medical surveillance. No cases of fibrosing respira- it occurred was transitory and there were no tory disease had been detected. Many thousands sequelae. Dermatitis due to sensitization was not of serial chest radiographs had been reviewed seen in detergent factories although a specific without revealing any permanent occupationally erythematous and desquamatory condition affect- related disease associated with the enzymes or ing the palmar surfaces of the hands was observed detergent dust. One man developed an acute in workers producing the concentrated enzyme alveolitis with subsequent recovery. During the (Zachariae). Ihe 1969 SDIA Code of Practice had seven-year period there were no indications that recommended screening procedures and regular serial skin prick testing caused sensitization. medical examinations. The specific skin prick Thirty-six subjects who were prick test positive on

B Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from 626 Biological efjects of proteolytic enzyme detergents two or more occasions reverted to and remained immediate response subsided substantially within prick test negative. Thirteen subjects who had 4 hours, to be followed at approximately 5 to 8 immediate skin prick test responses also de- hours after inhalation by a 'late' reaction. This veloped a reaction which began about 6 hours 'late' reaction was accompanied by fever and after the test. This non-immediate reaction was leucocytosis lasting many hours or days, but there characterized by a firm oedema, was larger (10 was no evidence of alveolar involvement. Im- to 20 cm) than the immediate reaction, and lasted munodiffusion studies failed to demonstrate the up to 24 hours. These reactions could cause tem- specific precipitins to extracts of proteolytic porary inconvenience (Newhouse). The discussion enzymes from Bacillus subtilis. revealed that their significance was not yet In the discussion it was clear that the understood. mechanism of production of the non-immediate Ogilvie (Liverpool, UK) gave the results of a reaction to the enzyme (and other antigens) was seven-year follow-up of the early heavily exposed not fully understood, but that it did not necessarily group of 12 workers described by Flindt (Man- imply a cellular response at the alveolar level. chester, UK). Lung function studies were carried out in 1968, 1971, and 1975. At the initial EPIDEMIOLOGY examination abnormalities included increased At the Session on epidemiology, six papers were residual volume, increased transfer factor (TLco), concerned with and increased diffusion co-efficient (Kco). Twelve presented, mainly continuing medical surveillance of workers engaged in pro- were seen again in 1971 and nine in 1975. No and in significant deterioration occurred in any aspect of ducing proteolytic enzymes manufacturing lung function. The enzyme detergents. Witmeur reported on experi- residual volume and transfer ence in the of the factors tended to revert to normal; those initially production enzymes. Definite high respiratory symptoms were present in 2% of the had come down, those low had risen. 554 workers examined in May 1975. Positive This copyright. important study revealed no evidence RAST tests to Alcalase occurred in that relatively heavy exposure to proteolytic 3% of enzymes in workers in 1970 and in 2% in 1974, but the rates detergent powders caused, in those were much in the removed from further exposure, any higher atopic workers (up to progressive 27%). There was no relation between forced bronchopulmonary dysfunction due to airways volume levels and duration of obstruction, emphysema, or fibrosis over the expiratory (FEV1) employment or level of exposure to enzymes. In seven-year period. In 1975, 10 of the original 12 172 workers examined at intervals up to six years, http://thorax.bmj.com/ who could be traced were at work. Only one of the the nine tested had any although atopics had lower levels of FEV1 respiratory symptoms. He there was no excess decline over the in had a history of bronchitis before exposure to the period any group of workers and no relation to exposure. enzymes. The chest radiographs were normal. The Lainhart interpretation of the transfer factor (Procter & Gamble, USA) reported a changes was study of prick test results and lung function of discussed. It seemed possible that some subjects 4578 in four had had a predominantly asthmatic and others an people European plants making alveolar reaction. It enzyme detergent products over a six-year period. was concluded that if pro- There had been a teolytic enzymes caused an asthmatic response decrease in sensitization rates with an for new employees from 5% in 12 months in 1970 on October 2, 2021 by guest. Protected increased diffusing capacity, an associated to in 1974. alveolar reaction could not be excluded solely by 3% Although significant differences did occur between plants, there was no evidence the finding of a normal diffusing capacity. that function Dijkman lung (FEV1, FVC) was reduced or described the effects in six male declined faster among atopics, those with a workers exposed and sensitized in 1969 to deter- positive history of response to exposure, or those gent containing proteolytic enzymes. Nasal irrita- test tion prick positive. The survey was based on past was a presenting symptom, followed by records. It revealed differences which may well wheezing which in three men was initially have arisen from the way the information was nocturnal only. Skin prick tests were carried out first recorded and collected. It showed the with common allergens which revealed slight value of reactions in potential systematic surveillance records three subjects, and to the enzyme in a large multinational company. In future the reagent in five. In these five, bronchial challenge methods of recording will be better standardized. with the enzyme produced a dual asthmatic Pham reaction, (Nancy, France) described a detailed and in one only a late reaction. The cross-sectional comparison of two detergent Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from Biological eflects of proteolytic enzyme detergents 627 enzyme factories by studies of representative similar rates, but the mean FEV1 of the prick samples of 130 and 150 production workers. Al- test positive group was initially and throughout though the proportion with bronchitis (9%) or significantly lower than the predicted level. The who were prick test positive (11 %) were very FVC results were above predicted for both groups, similar in the two plants, asthma (7% and 1%) whereas the FEV1 /FVC ratio was lower in the and rhinitis (18,' and 3%) were much more positive group. These subjects were a subgroup of prevalent in one plant. Although there were some the total population reported by Juniper and by significant changes in lung function after acetyl- Lainhart. in choline, particularly intermediate flow rates Juniper presented the SDIA Medical Review and FEV, there was no relation of lung function 75 to skin test 1969/July (SDIA 1976). He emphasized that changes positivity or duration of this was a study of the whole industry in the UK employment. Pham recommended that acetyl- (2865 people). Spirometry and skin prick testing choline provocation tests should be added to the were carried out on the tests routinely carried out in whole population at six- the industry to monthly intervals. With falling dust levels con- improve sensitivity of functional measurement. version to skin prick test positive had also fallen Weill (New Orleans, USA) reported on longi- from the original high values of 85% in atopics tudinal studies of a representative sample of 230 and up to 35% in non-atopics. Atopics were later workers exposed to detergent enzyme dust in four excluded from working with these products and US plants; these studies started in January 1970 the rate for non-atopics had fallen to around shortly after the enzyme was first added to the 20 %. detergent. There had been a marked reduction in dust exposure since 1970, partly because of im- The improved environment had led to a reduc- proved environmental tion in the number of workers transferred for control, as in Europe, but medical reasons also because enzyme-containing products were no from enzyme dust exposure-an longer made on all working shifts. Positive prick average of 25 per year for 1968 to 1971 to less copyright. tests were very common, ranging from 40% to than 10 per year for 1972 to 1975. 75% in the four plants. (This high rate was in part Serial studies of lung function in 2000 workers due to the method of selecting subjects to include showed that there was no change in FEV, ex- those with higher exposure.) Definite correlation pressed as percent of the predicted, in relation to exists between atopic status and prick test re- either reaction to the enzyme prick test or to the sults; between exposure to enzyme dust and degree of contact with enzymes. The same findings sensitization rate, between respiratory symptoms applied to the FEV/FVC ratio. The rate of de- http://thorax.bmj.com/ and atopicity but not additionally with smoking; cline of FEV was about 45 ml per annum in all and between job-related symptoms and atopicity, groups; it was similar for other groups within the prick test results, and dust exposure. industry not involved in enzyme detergent pro- Lung function changes have been assessed from duction, for new entrants to the industry, and for the start of the study, ie, after some exposure. workers in unrelated industries in the same After initial decline before 1972, TLC, FEV1, geographical areas. FVC have all increased during the five years of The discussion on the studies of lung function study. The transfer factor had fallen slightly but was opened by Cotes (PU, UK). He said that the on October 2, 2021 by guest. Protected still exceeded 100% predicted in three out of the findings presented showed that the present risk to four plants in 1975. The net effect is that the health of current exposure levels of proteolytic final values for lung function all fall well within enzymes was likely to be small. One of the prob- the normal range. lems was, were the best tests of lung function Newhouse had studied since 1969 at six-monthly being used? He suggested that FEV, was the intervals a group of men who had already been most relevant and reproducible test, but some heavily exposed to the enzymes. Fifty-five were additional measures of flow rate needed to be prick test positive and 47 prick test negative. studied in more detail, eg, FEV25 75. There was Both groups had worked in the same departments support for this, especially as the flow rates could and had similar lengths of exposure. High IgE now be readily measured at the same time as the levels were found in men with very active skin FEV. He also showed that, even for the most reactions and clinical symptoms. Over the period repeatable tests of lung function, measurement the mean FEV1 of the prick test positive and error is about 5% and prediction error about negative groups were similar and declined at 15%, so that reference values have little useful- Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from 628 Biological effects of proteolytic enzyme detergents

ness except in the detection of marked disease. cigarette smoke. It was, however, argued that To assess accurately a rate of decline, it would be results on such people had been presented in the necessary to measure 100 people for five years, and papers. It was unfortunate that lack of time pre- even then this may detect only a 50% change in vented further discussion of this, which might rate of fall of lung function. have led to further mutual understanding. He referred to the reports from Australia that the elastic recoil pressure in the lung might be CONCLUSIONS reduced in those with heavy exposure in the past. A number of general points came out of the Musk (Australia) confirmed that a difference, meeting: which was apparently related to the past intensity (1) The early detection and publishing of the of exposure, had been observed in a follow-up of potentially serious hazard from a new industrial these subjects. No new information on this aspect process led to rapid and effective collaborative was reported from the studies in Europe and USA. effort to identify the nature of the problem and General discussion centred round three main bring it under control. topics. First, lung function-it was eventually (2) The engineering control of the dust and the agreed that generalized statements about lung medical surveillance of the employees have pre- function levels in terms of prediction relations vented much serious disability. It has also enabled could be misleading because of instrumental, those with a positive skin prick test to continue at operator, and naturally occurring intergroup dif- work without developing respiratory symptoms. ferences. The observations of Ogilvie and Weill (3) When lung function and immunological on the fall of transfer factor from above normal tests are used for serial observations in large to normal levels were thought consistent with groups of individuals in several factories and in clinical observations on ordinary cases of asthma different countries, the validity of the findings is responding to treatment, but good documentation critically dependent upon precise standardization of this change was not produced, although high of the tests. In the future much more attentioncopyright. values for transfer factor during asthmatic has to be given to this aspect. episodes have been previously reported. The dis- (4) The skin prick test, when performed cor- cussion also brought out the difficulty in ensuring, rectly, is a safe, sensitive, specific, and useful when using tests of lung function in the long measure for biological monitoring of the response term, that technical changes did not introduce a level to the enzyme. This observation could be of bias. use in other situations. There was less eventual agreement about ter- (5) The enzymes used in detergents can produce http://thorax.bmj.com/ minology for prick test reactions. 'Arthus-like' was an IgE mediated asthma which is dose related. objected to strongly, particularly in close tem- Exposure levels during the manufacture of the poral association with a discussion on alveolar enzyme and the enzyme detergents can now be changes, although the same discussant confessed kept so low that very few people will become to the use of 'non-immunological Arthus-like' in sensitized. Occasional cases of sensitization are, print. Suggestions were made for the use of however, likely to occur due to a combination of descriptive terms such as 'immediate', 'non- circumstances, such as accidental spillage and immediate', eg, '6-8 hour' reactions. This was highly susceptible subjects. on October 2, 2021 by guest. Protected clearly a topic needing further research leading (6) Knowledge acquired over the last eight years to better definitions. indicates that respiratory disease other than asthma is unlikely to arise from the use of these A strong demand was made for the separation enzymes, but continued surveillance of those ex- and reporting of results on the few 'responders' to posed is still required. detergent enzymes. The general picture of fewer (7) The risk of respiratory and skin test sensi- workers becoming sensitized, and the absence of tization to the enzyme detergents during their any average deterioration of lung function re- domestic use is extremely small. lated to exposure in large groups, was reassuring but there were still a few workers who had to be removed from further exposure. Little had been GLOSSARY OF TERMS said about the extent of their disability in a wider GLYCINE UNIT sense, for example, possible increased sensitivity This is a measure of the enzyme activity. It is to common antigens and other pollutants such as measured by the release of amino acid from a Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from Biological effects of proteolytic enzyme detergents 629 standard protein substrate (a substituted casein). a dual response following a wheal and flare. They The amino acid is estimated colorimetrically. A may be indurated, erythematous, with a surround- number of units are in use and are related as ing zone of oedema, or more extensive areas of follows: soft ill-defined oedema, with or without central 1 Anson Unit=623 000 Glycine Units= 243 000 induration. The reactions vary greatly according Delft Units. to the degree of hypersensitivity and concentra- 1 ,ug of 15 Anson Units of protease activity as tion of antigen. Intradermal tests may induce received from the enzyme manufacturers is severe congested or haemorrhagic reactions. approximately equivalent to 1 Glycine of Unit/mg enzyme concentrate. RAST This radio-allergosorbent test was devised to de- IgE tect the very small amounts of in Ig IgE antibody designates immunoglobulin, or globulin with sera. Antigen is coupled to inert particles or antibody activity. IgE is the reagin, the antibody discs, and antibody in the serum being tested conferring immediate-type hypersensitivity and binds to the coupled antigen. The antigen is then mediating asthma, hay fever, and the wheal and treated with anti-IgE globulin labelled with a flare of the prick test. It is present in extremely radioactive isotope. The amount of radioactivity small amounts in the serum and cannot easily be bound, usually expressed as counts per minute, detected by the conventional precipitin and agglut- bears a direct relation to the amount of IgE anti- inin techniques. IgE is usually detected in vitro in the test serum that is bound by RAST. body to the antigen. In the case of Alcalase, aggregates occur in the serum stored for more than one to two GUINEA-PIG ANAPHYLACTIC ANTIBODIES years. These may combine with the enzyme Two classes of antibody cause immediate-type coupled to the test substrate. The aggregates allergy in the guinea-pig. One is a subclass of contain IgE and cause poor reproducibility of copyright. IgG, designated IgGI which occurs in two forms, RAST. Removal of aggregates by centrifuging IgGla and IgGIb. These are the antibodies overcomes this. usually formed after sensitization in the laboratory. IgGla is mentioned in this report. The other class of OUCHTERLONY DOUBLE DIFFUSION PRECIPITATION antibody is IgE, which has properties This test for similar to those of human IgE. precipitating antibody is carried out

in thin agar gels, in which antibody in one hole http://thorax.bmj.com/ or well diffuses into the gel to meet antigen IgG AND IgM diffusing from another well. If the antibody reacts Immunoglobulins G and M are present in much with the antigen, lines or bands of precipitation larger amounts in serum than IgE. They are occur where the antibody meets the antigen in precipitating antibodies and can activate com- sufficient concentration. This is a standard pro- plement and may mediate Arthus reactions. They cedure to detect precipitating antibodies as are (particularly IgG) are associated with extrinsic found, for example, in patients with extrinsic fibrosing alveolitis, eg, farmer's lung. Their allergic alveolitis. presence is evidence of exposure, not necessarily on October 2, 2021 by guest. Protected of disease, as persons may have precipitins without disease. RID (radioimmunodiffusion) The procedure is the same as that above, except that the antigen is labelled with a radioactive IgA isotope, and the precipitation lines are detected Immunoglobulin A is present in serum and in a by exposure to a photographic plate. This tech- modified form on mucosal surfaces. It is believed nique detects amounts of precipitating antibody to protect by masking or covering the deter- too small to be seen macroscopically. minants of antigens, preventing allergic responses. It is not known to mediate allergy. IRIEP (indirect radioimmunoelectrophoresis) The test serum is separated in an agar gel, and the ARTHUS REACTIONS positions of the various immunoglobulins are These are skin test responses occurring 4 to 8 detected by antisera to each of them (eg, anti- hours after antigen challenge, either alone or as IgG). Antigen labelled with a radioactive isotope Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from 630 Biological effects of proteolytic enzyme detergents

is then added to the plates to see if any of the Dr. A. G. van Velzen precipitated globulin (eg, IgG) is antibody for the Gist-Brocades nv research & development, PO Box particular antigen, and binds it. This technique 1, Wateringseweg 1, Delft detects very small amounts of antibody. UNITED KINGDOM SOLID PHASE ASSAY TEST FOR ANTIGEN UPTAKE Dr. J. C. Gilson, Dr. J. E. Cotes, Dr. J. H. Edwards, BY IgG ANTIBODIES Dr. P. D. Oldham, Mr. C. E. Rossiter This is a procedure for the detection and measure- MRC Pneumoconiosis Unit, Llandough Hospital, ment of extremely small amounts of IgG antibody, Penarth, S. Glamorgan CF6 lXW and of antibodies in the IgG subclasses or other Mr. R. E. Blofeld, Mr. C. F. Bruce, Dr. R. B. Martin immunoglobulin classes (Jacoby and Procter & Gamble Limited, PO Box lEE, Gos- Pepys). forth, Newcastle upon Tyne NE99 lEE Dr. G. Cambridge, Dr. M. J. How, Dr. W. E. Parish SANDWICH' RADIOIMMUNE ANALYSIS Unilever Research, Colworth/Welwyn Laboratory, This technique detects any antibody in a serum Unilever Limited, Colworth House, Sharnbrook, that binds to antigen. If the serum is separated Bedford MK44 ILQ into its various immunoglobulin fractions, it is Dr. D. R. Davies, Mr. A. K. Kinshott possible to detect antibody in each class. It is a Lever Brothers, Port Sunlight, Wirral, Merseyside complicated procedure and open to technical L62 4XN errors not occurring in the solid phase assay test. Dr. C. P. Juniper, Mr. J. Philp Unilever Limited. PO Box 68, Unilever House, London EC4P 4BQ LIST OF PARTICIPANTS Dr. M. L. H. Flindt, Professor W. R. Lee BELGIUM University of Manchester, Department of Occu- Miss N. M. Brown pational Health, Stopford Building, Oxford Road, Procter & Gamble, Professional and Regulatory Manchester M13 9PT Services Department, European Technical Centre, Professor T. S. Scott Temselaan 100, B 1820-Strombeek-Bever Chairman, Standing Committee, SDIA, 15 Dene-copyright. wood Court, Victoria Road, Wilmslow, Cheshire SK9 5HP CANADA Professor J. Pepys Dr. J. Dolovich Department of Clinical Immunology, The Brompton McMaster University, Department of Pediatrics. Hospital, London SW3 6HP 1200 Main Street West, Hamilton, Ontario, Dr. C. Ogilvie L8S 4J9 Regional Cardio-thoracic Centre, Broadgreen http://thorax.bmj.com/ Hospital, Thomas Drive, Liverpool L14 3LB DENMARK Dr. M. L. Newhouse Dr. 0. Witmeur, Dr. H. Zachariae TUC Centenary Institute of Occupational Health, University of Aarhus, Marselisborg Hospital, London School of Hygiene and Tropical Medicine. Skanderborgvej 9, 8000 Aarhus C Keppel Street (Gower Street), London WC1E 7HT Dr. P. Baxter, Dr. M. Greenberg Health & Safety Executive, Employment Medical FRANCE Advisory Service, Baynards House, 1 Chepstow Dr. Q. T. Pham Place, Westbourne Grove, London W2 4TF. Institut National de la Sante et de la Recherche Mr. P. E. Lovering on October 2, 2021 by guest. Protected Medicale, Unite de Recherche de Physio-pathologie Health and Safety Executive, HM Factory In- Respiratoire, U. 14 Inserm-Plateau de Brabois, spectorate, Occupational Hygiene Section Fl 9, 54500 Vandoeuvre-les-Nancy 403-405 Edgware Road, Cricklewood, London NW2 6LN Dr. R. W. Chalmers, Dr. J. P. W. Hughes, Mr. A. C. GERMANY Shepherd, Dr. A. P. Timney Dr. C. Gloxhuber Albright & Wilson Ltd, 1 Knightsbridge Green, Henkel & Cie GmbH, 4 Dusseldorf-Holthausen, London SWIX 7QD Henkelstrasse 67

UNITED STATES OF AMERICA THE NETHERLANDS Dr. W. S. Lainhart, Dr. K. D. McMurrain, Dr. H. Professor J. H. Dijkman Ritz, Dr. G. H. Scott, Dr. E. R. Wilson Department of Internal Medicine, University The Procter & Gamble Company, Ivorydale Hospital, Nijmegen Technical Center, Cincinnati, Ohio 45217 Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from Biological efjects of proteolytic enzyme detergents 631

Professor H. Weill Q. T. PHAM Tulane University School of Medicine, Department Respiratory manifestations and skin reagency of Medicine, Pulmonary Diseases Section, 1700 among workers exposed to enzymes in the house- Perdido Street, New Orleans, La 70112 hold detergents industry Dr. S. De Salva HANS WEILL, JOHN DIEM, HENRY GLINDMEYER, and Colgate-Palmolive Company, Research and CARMEL WAGGENSPACK Development Department, 909 River Road, Longitudinal studies of workers exposed to deter- Piscataway, NJ 08854 gent enzyme dust in four United States plants Dr. W. Musk Harvard University School of Public Health, MURIEL L. NEWHOUSE Department of Physiology, 665 Huntington Avenue. Longitudinal studies of prick test and lung function Boston, Massachusetts 02115 C. P. JUNIPER The medical review of workers in the enzymatic washing product industry in the United Kingdom: 1969 to July 1975 LIST OF PAPERS PRESENTED SDIA, April 1976 A. G. VAN VELZEN Production of detergent enzymes J. H. DIJKMAN Clinical investigations in B. subtilis allergy D. R. DAVIES The manufacture of biological detergents C. F. BRUCE BIBLIOGRAPHY Dust monitoring and analysis Axelsoon, J. (1972). The Union demands-prohibit D. R. DAVIES and C. F. BRUCE the Enzymes. Fabriks-arbetarem. Environmental improvements Belin, L. (1973). Characterization of the allergenic G. W. CAMBRIDGE components of Bacillus subtilis enzymes used The pathology and pharmacology in laboratory in detergents (abstract). Journal of Allergy and Clinical Immunology, 51, 119. animals of the allergic and proteolytic responses copyright. following inhalation of Bacillus subtilis proteases Belin, L. (1974). Enzyme sensitivity testing. Presented H. L. RITZ and E. R. FLETCHER at the 47th Annual SDA Industry Convention, Immunological responses in the guinea-pig Boca Raton, Florida, January 1974. NANCY M. BROWN, WILLIAM B. COATE, WILLIAM M. Belin, L., Hoborn, J., Falsen. E., and Andre, J. BUSEY, WARREN H. SCHOENFISCH, and ELVIN A. NEWMANN (1970). Enzyme sensitisation in consumers of Inhalation toxicity using Cynomolgus monkeys enzyme-containing washing powder. Lancet, 2,

1153. http://thorax.bmj.com/ C. P. JUNIPER Allergy due to Bacillus subtilis Bernstein. I. L. (1971). Preliminary investigations of sensitization phenomena in populations not ex- COLIN OGILVIE posed to high concentrations of enzyme dust Seven-year follow-up of an early exposed group (abstract). Journal of Allergy, 47, 97. J. PEPYS Bernstein. I. L. (1972). Enzyme allergy in populations Clinical immunology and methods of assessment exposed to long-term, low-level concentrations of (paper not precirculated) household laundry products. Journal of Allergy E. ROYCE WILSON and PAMELA JONES DANNEMAN and Clinical Immunology, 49, 219.

Longitudinal study of IgE and skin test response Berson, S. A., Yalow. R. S., Saito, T. O., and on October 2, 2021 by guest. Protected related to exposures Selikoff, I. J. (1971). Antibodies to 'alcalase' M. J. HOW after industrial exposure. New England Journal Skin tests and serological tests in the diagnosis of of Medicine, 284, 688. allergic reactivity to Bacillus subtilis enzyme Bolam, R. M., Hepworth, R., and Bowerman, L. T. preparations (1971). In-use evaluation of safety to skin of H. ZACHAR IAE enzyme-containing washing products. British Prick- and radioallergosorbent tests (RAST) to Medical Journal, 2, 499. Alcalase in a population not exposed to enzyme di Bosco, M. M. (1974). Allergic respiratory diseases detergents from enzymes present in biodetergents. Rivista 0. WITMEUR degli Infortuni e delle Malatti Professionali, 61, Medical experience in enzyme production 411. WILLIAM S. LAINHART and E. ROYCE WILSON Burger. M. M. (1970). Proteolytic enzymes initiating Six-year retrospective study of four enzyme deter- cell division and escape from contact inhibition gent production plant employees of growth. Nature, 227, 170. Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from 632 Biological efJects of proteolytic enzyme detergents

Carter, R. 0. and McMurrain, K. D., Jr. (1970). Con- Gandevia, B.. Musk, A. Q., and Kalyannasundaram, sumer safety of enzyme detergents (letter). V. (1973). Survey of Colgate-Palmolive employees Journal of the A merican Medical A ssociation, and ex-employees formerly exposed to proteolytic 211, 2017. enzymes in the detergent industry. Supplementary Davies, R. J. (1974). Occupation and asthma. Health. Report. 11, 11. Goodwin. B. F. J. and How, M. J. (1976). RAST Debanne, M. T. and Dolovich, J. (1974). Antigenic using cruide and purified anti-IgE. Clinical reactivity of inactivated Bacillus subtilis enzyme Allergy, 6, 441. preparation. Journal of Allergy and Clinical Gothe, C. J., Nilzen. A.. Holmgren, A., Szamosi. Immunology, 53, 205. A., Werner, J., and Wide. L. (1972). Medical Dijkman, J. H. (1975). Observations on biphasic problems in the detergent industry caused by bronchial reactions due to inhalation of enzymes proteolytic enzymes from Bacillus subtilis. Acta of Bacillus subtilis. Clinical Allergy, 5, 25. Allergologica, 27, 63. Dijkman, J. H., Borghans, J. G. A., Savelberg, P. J., Greenberg, M., Milne, J. F., and Watt, A. (1970). and Arkenbout, P. M. (1973). Allergic bronchial Survey of workers, exposed to dusts containing reactions to inhalation of enzymes of Bacillus derivatives of Bacillus subtilis. British Medical subtilis. American Review of Respiratory Disease, Journal, 2, 629. 107, 387. Hendricks, M. H. and Carter R. 0. (1974). Consumer Dixon, B. (1971). Risks and benefits in perspective. safety of enzyme laundry. Soap and Chemical New Scientist, 51, 238. Specialities, June. Dixon, B. (1974). When is the risk worthwhile? How. M. J. and Cambridge, G. W. (1971). Prick-tests World Medicine, 10 April, p. 30. and serological tests in the diagnosis of allergic Dolovich, J., Hargreave, F. E.. Chalmers, R., Shier, reactivity to enzymes used in washing products. K. J., Gauldie, J., and Bienenstock, J. (1973). British Journal of Industrial Medicine, 28, 303. Late cutaneous allergic responses in isolated Jacoby. B. and Pepys, J. (1976). The antigen-binding IgE-dependent reactions. Journal of Allergy and capacity of serum IgG. An immunosorbent Clinical Immunology, 52, 38. method applied to subtilisin as antigen. Journalcopyright. Dolovich, J. and Little, D. C. (1972). Correlates of of Immutnological Methods, 11, 37. skin test reactions to Bacillus subtilis enzyme Juniper. C. P. (1975). Respiratory allergies in in- preparations. Journal of Allergy and Clinical dustry: a review of recent advances and current Immunology, 49, 43. clinical practice. Journal of the Society of Dubos, R. (1971). Toxic factors in enzymes used in Occupational Medicine, 25, 50. laundry products. Science, 173, 259. Leapman, M. (1971). Symptoms after exposure to Falleroni, A. E. and Schwartz, D. P. (1971). Im- enzymes. 7'he Tinmes. 24 February. http://thorax.bmj.com/ mediate hypersensitivity to enzyme detergents (letter). Lancet, 1, 548. Lichtenstein, L. M. (1971). Human experimentation Federal Trade Commission (1972). FTC closes Enzyme by industry: the case of 'enzyme' detergents. Safety Enquiry but urges further study. 14 April. Annals of Internal Medicine, 75, 964. Flindt, M. L. H. (1969). Pulmonary disease due to Little, D. C. and Dolovich, J. (1973). Respiratory inhalation of derivatives of Bacillus subtilis disease in industry due to B. subtilis enzyme containing proteolytic enzyme. Lancet, 1, 1177. preparations. Canadian Medical A ssociation Journal, 108, 1120. Franz, T.. McMurrain, K. D., Brooks, S. and Bern- stein, 1. L. (1971). Clinical, immunologic, and McMurrain, K. D., Jr. (1970). Dermatologic and on October 2, 2021 by guest. Protected physiologic observations in factory workers ex- pulmonary responses in the manufacturing of posed to B. subtilis enzyme dust. Journal of detergent enzyme products. Journal of Occu- Allergy, 47, 170. pational Medicine, 12, 416. Fulwiler. R. D., Abbott, J. C., and Darcy, F. J. Malley, A. and Baecher, L. (1972). Hypersensitivity to (1972). Evaluation of detergent enzymes in air. bacterial enzymes. I. Atopic hypersensitivity A merican Industrial Hygiene A ssociation induced by rhesus monkeys. Journal of Allergy Journal, 33, 231. and Clinical Immuinology, 49, 36. Gaffuri, E. (1970). Respiratory effects of biological Mitchell, C. A. and Gandevia, B. (1971). Acute detergents (letter). British Medical Journal, 4, bronchiolitis following provocative inhalation of 52. 'alcalase' a proteolytic enzyme used in the Gandevia, B., Musk, A. Q., and Kalyannasundaram, detergent industry. Medical Journal of Australia, V. (1973). Survey of Colgate-Palmolive employees 1, 1363. and ex-employees formerly exposed to proteolytic Mitchell, C. A. and Gandevia, B. (1971). Respiratory enzymes in the detergent industry. symptoms and skin reactivity in workers exposed Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from Biological ef0ects of proteolytic enzyme detergents 633

to proteolytic enzymes in the detergent industry. detergent workers (abstract). Journal of Allergy, American Review of Respiratory Disease, 104, 1. 47, 97. Mitchell, C. A. and Gandevia, B. (1973). Loss of pul- Soap and Detergent Industry Association (1971). monary elastic recoil due to heavy occupational Recommended operating precedures for UK exposure to proteolytic enzymes used in the factories handling enzyme materials. A nnals of detergent industry. In Proceedings of the IVth Occupationzal Hygiene, 14, 71. International Pneumoconiosis Conference (ILO). Soap and Detergent Industry Association. Reports of Bucharest, p. 515. Apimondia, Bucharest (Sum- the Soap and Detergent Industry Association, mary, correspondence: Medical Journal of First report, October 1969; Second report, May Australia (1971) 1, 1032). 1970; Third report, Novcmber 1970; Fourth Musk, A. W. and Gandevia, B. (1976). Loss of pul- report, April 1973; Fifth report. April 1976. monary elastic recoil in workers formerly ex- Obtainable from the Secretary of the Soap and posed to proteolytic enzyme (alcalase) in the Detergent Industry Association. detergent industry. British Journal of Industrial Teculescu, D. (1974). Detergent enzymes-a new Medicine. 33, 158. challenge for the respiratory tract. Revue Rou- Nagy. S. M., Lichtenstein, L. M., Ball, W. C., et al. maine de Medecine Interne, 11, 185. (1971). Acute and chronic respiratory disease in Tolos, W. P.. Richards, D. E.. and Scheel, L. D. factory workers due to the inhalation of B. (1975). Histamine induction and release follow- subtilis extracts used in the manufacturing of ing proteolytic enzyme exposure. A merican In- "enzyme' detergents. Presented at the 27th dustrial Hygiene Association Journal, 36, 272. Annual Meeting of the American Academy of Allergy. Chicago, February 1971. Tuma, J.. Parker, L., and Braun, D. C. (1973). The Newhouse, M. L., Tagg, B., Pocock. S. J., and proteolytic enzymes and the prevalence of signs McEwan. A. C. (1970). An epidemiological study and symptoms in US cotton textile mills. Journal of workers producing enzyme washing powders. of Occupational Medicine, 15, 409. Lancet. 1, 689. US Department of Health, Education and Welfare New Yorker (1971). The enigmatic enzyme. 16 (1971). Enzyme containing laundering compcunds copyright. Januarv. p. 42. and consumer health. Press release. 17 November. Pepys, J., Longbottom, J. L.. Hargreave, F. E.. and Valer. M. (1975). Skin irritancy and sensitivity to Faux, J. (1969). Allergic reactions of the lungs laundry detergents containing proteolytic to enzymes of Bacillus subtilis. Lancet, 1, 1181. enzymes. Part II. Berufsdermatosenz, 23, 96. Pepys, J., Wells, I. D.. D'Souza. M. F., and Green- Watt, A., Morley. R., Greenberg. M., and Fox, A. J. M. (1973). and (1973). Follow-up of a group of workers exposed berg, Clinical immunological http://thorax.bmj.com/ responses to enzymes of Bacillus subtilis in factory to dusts containing derivatives of Bacillus sub- workers and consumers. Clinical Allergy, 3, 143. tilis. Clinical Allergy, 3, 133. Richards, D. E., Scheel, L. D.. and Groth, D. H. Webster, J. R., Jr.. Cugell, D. W., Bazley, E. S., (1975). An evaluation of the inhalation toxicity Cunningham, L., and Booth, B. (1972). Deter- of one commercial proteolytic enzyme prepara- gent worker lung-a hypersensitive pulmonary tion. A merican Industrial Hygiene A ssociation disease? Clinical conference from Northwestern Journal, 36, 266. University-McGaw Medical Center. Chest, 61, Robertson, D. S. F. (1972). Enzyme detergents. 174.

Occupational Health. I February. Weill, H.. Waddell, L. C., and Ziskind, M. (1971). A on October 2, 2021 by guest. Protected Royal Industrial Safety Commission (1972). Directives study of workers exposed to detergent enzymes. regarding work with enzymes. Journal of the A merican Medical A ssociation, 217, 425. Shapiro, R. S. and Eisenberg, B. C. (1971). Sensi- tivity to proteolytic enzymes in laundry Weill, H., Waggenspack, C., De Rouen, T., and detergents. Journal of Allergy, 47, 76. Ziskind, M. (1973). Respiratory reactions to B. Shore, N. S.. Greene, R., and Kazemi, H. (1971). subtilis enzymes in detergents. Journal of Occu- Lung dysfunction in workers exposed to Bacillus pational Medicine. 15, 267. subtilis enzyme. Environmental Research, 4, 512. Weill, H., Waggenspack, C., De Rouen, T., and Simesen, B.. Wilde, L., and Zachariae, H. (1976). Prick- Ziskind. M. (1974). Follow-up observations of and radioallergosorbent tests (RAST) to Alcalase workers exposed to enzyme detergents. A nnals in a population not exposed to enzyme deter- of the New York Academy of Sciences, 221, 76. gents. A cta A llergologica, 31, 71. Weitzman, R. H. and Wilson, A. F. (1974). Diffusing Slavin, R. G. and Lewis. C. R. (1971). Enzyme capacity and over-all ventilation: perfusion in asthma: an occupational disease of laundry asthma. American Journal of Medicine, 57, 767. Thorax: first published as 10.1136/thx.31.6.621 on 1 December 1976. Downloaded from 634 Biological effects of proteolytic enzyme detergents

Wicher, V. and Dolovich, J. (1972). Proteinase- Translated from Schweizerische medizinische dependent false positive precipitin tests. Journal Wochenschrift, 100, 1908. of Allergy and Clinical Immunology, 49, 59. Zachariae, H., Thomsen, K., and Rasmussen, 0. G. Witmeur, O., Wolf-Jurgensen, P., Hoegh-Thomsen, (1973). Occupational enzyme dermatitis. Results 0. and Zachariae. J., Rasmussen, G., Wide L., of patch testing with alcalase. A cta Dermato- H. (1973). Medical experience in enzyme pro- Venerologica, 53, 145. duction. Acta Allergologica, 28, 250. Wuthrich, B. and Schwarz-Speck, M. (1970). Zetterstrom, 0. and Wide, L. (1974). IgE-antibodies Bronchial asthma after occupational exposure and skin test reactions to a detergent-enzyme to proteolytic enzymes (Bacillus subtilis proteases). in Swedish consumers. Clinical Allergy, 4, 273. copyright. http://thorax.bmj.com/ on October 2, 2021 by guest. Protected