<b>Bijsluiter</b>. De hyperlink naar het originele document werkt niet meer. Daarom laat Woogle de tekst zien die in dat document stond. Deze tekst kan vreemde foutieve woorden of zinnen bevatten en de opmaak kan verdwenen of veranderd zijn. Dit komt door het zwartlakken van vertrouwelijke informatie of doordat de tekst niet digitaal beschikbaar was en dus ingescand en vervolgens via OCR weer ingelezen is. Voor het originele document, neem contact op met de Woo-contactpersoon van het bestuursorgaan.<br><br>====================================================================== Pagina 1 ======================================================================

<pre>             Health Council of the Netherlands
          Fungal alpha-amylase
          (derived from the fungus
          Aspergillus oryzae)
             Health-based recommended occupational exposure limit
2014/25
</pre>

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<pre>Fungal alpha-amylase
(derived from the fungus
Aspergillus oryzae)
     Health-based recommended occupational exposure limit
</pre>

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<pre></pre>

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<pre>Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp              : aanbieding advies schimmel alfa-amylase
Uw kenmerk             : DGV/BMO/U-932542
Ons kenmerk            : U-8232/JR/cn/459-S70
Bijlagen               :1
Datum                  : 6 november 2014
Geachte minister,
Graag bied ik u hierbij aan het advies over de gevolgen van beroepsmatige blootstelling aan
schimmel alfa-amylase.
In het advies Preventie van werkgerelateerde luchtwegallergieën (rapportnr. 2008/03) heeft
de Gezondheidsraad een werkwijze voorgesteld voor het afleiden van gezondheidskundige
advieswaarden of voor het vaststellen van op risico gebaseerde referentiewaarden voor
allergene stoffen. Het voorliggende advies over schimmel alfa-amylase maakt gebruik van
deze werkwijze. De commissie heeft de concentratie schimmel alfa-amylase in de lucht
berekend waarbij een werknemer een extra kans van één procent gedurende zijn arbeids-
zame leven heeft om door beroepsmatige blootstelling gesensibiliseerd te raken ten
opzichte van de kans hierop in de niet beroepsmatige blootgestelde algemene bevolking.
      De conclusie van het advies is opgesteld door de Commissie Gezondheid en beroepsma-
tige blootstelling aan stoffen (GBBS) van de Gezondheidsraad. De commissie heeft daarbij
gebruik gemaakt van commentaren die zijn ontvangen op een openbaar concept van dit
advies en van de oordelen die intern zijn ingewonnen bij de Beraadsgroep Gezondheid en
omgeving.
Ik heb dit advies vandaag ter kennisname toegezonden aan de staatssecretaris van Infra-
structuur en Milieu en aan de minister van Volksgezondheid, Welzijn en Sport.
Met vriendelijke groet,
prof. dr. J.L. Severens,
vicevoorzitter
Bezoekadres                                                      Postadres
Rijnstraat 50                                                    Postbus 16052
2515 XP Den Haag                                                 2500 BB Den Haag
E - m a il : jm .r i j n ke ls @ g r.n l                         w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 6 6 3 1
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<pre></pre>

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<pre>Fungal alpha-amylase
(derived from the fungus
Aspergillus oryzae)
Health-based recommended occupational exposure limit
Dutch Expert Committee on Occupational Safety,
a Committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2014/25, The Hague, November 6, 2014
</pre>

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<pre>The Health Council of the Netherlands, established in 1902, is an independent
scientific advisory body. Its remit is “to advise the government and Parliament on
the current level of knowledge with respect to public health issues and health
(services) research...” (Section 22, Health Act).
     The Health Council receives most requests for advice from the Ministers of
Health, Welfare & Sport, Infrastructure & the Environment, Social Affairs &
Employment, Economic Affairs, and Education, Culture & Science. The Council
can publish advisory reports on its own initiative. It usually does this in order to
ask attention for developments or trends that are thought to be relevant to
government policy.
     Most Health Council reports are prepared by multidisciplinary committees of
Dutch or, sometimes, foreign experts, appointed in a personal capacity. The
reports are available to the public.
                 The Health Council of the Netherlands is a member of the European
                 Science Advisory Network for Health (EuSANH), a network of science
                 advisory bodies in Europe.
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. Fungal alpha-amylase (derived from the fungus
Aspergillus oryzae) - Health-based recommended occupational exposure limit. The
Hague: Health Council of the Netherlands, 2014; publication no. 2014/25.
all rights reserved
ISBN: 978-94-6281-017-4
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<pre>   Contents
   Samenvatting 9
   Executive summary 13
   Scope 17
.1 Background 17
.2 Committee and procedure 17
.3 Data 18
   Identity, properties and monitoring 19
.1 Identification 19
.2 Identity 20
.3 Physical and biochemical properties 20
.4 EU classification and labelling 21
.5 Validated analytical methods 21
.6 Summary 25
   Sources 27
.1 Natural sources 27
.2 Man-made sources 27
   Contents                               7
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<pre>    Exposure 29
 .1 General population 29
 .2 Working population 29
    Kinetics 33
    Mechanism of action 35
 .1 Immunological responses 35
 .2 Non-immunological responses 36
 .3 Conclusion 37
    Effects 39
 .1 Observations in humans 39
 .2 Animal studies 46
 .3 Summary 48
    Existing guidelines, standards and evaluation 49
 .1 General population 49
 .2 Occupational population 49
    Hazard assessment 51
 .1 Hazard identification 51
 .2 Selection of the study suitable for risk estimation 53
 .3 Quantitative hazard assessment 54
 .4 Recommendation for a health-based reference value 55
 .5 Groups at extra risk 56
    References 57
    Annexes 65
A   Request for advice 67
B   The Committee 69
C   The submission letter (in English) 71
D   Comments on the public review draft 73
E   Prevalence of sensitisation to alpha-amylase and of respiratory symptoms 75
F   Exposure-response relationships 87
G   Meta-regression analysis 95
    Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>Samenvatting
Op verzoek van de minister van Sociale Zaken en Werkgelegenheid leidt de
Commissie Gezondheid en beroepsmatige blootstelling aan stoffen, een commis-
sie van de Gezondheidsraad, gezondheidskundige advieswaarden af voor stoffen
in de lucht waaraan mensen blootgesteld kunnen worden tijdens hun beroepsuit-
oefening. Deze advieswaarden vormen vervolgens de basis voor grenswaarden
waarmee de gezondheid van werknemers beschermd kan worden. Dit advies gaat
over de gevolgen van blootstelling aan schimmel alfa-amylase, een enzym dat
afkomstig is van de schimmel Aspergillus oryzae. Vooral mensen die in bakke-
rijen of meelfabrieken werken kunnen hiermee te maken hebben. De conclusie
van de commissie is gebaseerd op wetenschappelijke publicaties die vóór augus-
tus 2014 zijn verschenen.
Fysische en chemische eigenschappen
Het enzym alfa-amylase van de schimmel Aspergillus oryzae, is een glycoprote-
ene met 478 aminozuren. In zuivere vorm is het een witgeel hygroscopisch poe-
der. Het enzym zet langketenige koolhydraten om tot maltose en glucose.
Extracten van de schimmel worden vooral toegepast als deegverbeteraar bij de
bereiding van bakkerijproducten.
Samenvatting                                                                   9
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<pre>  Blootstelling
  Blootstelling aan schimmel alfa-amylase in de lucht kan worden gemeten als de
  hoeveelheid allergeen in inhaleerbaar stof en is voornamelijk gemeten in bakke-
  rijen. Gemiddelde concentraties in de inademingslucht over een volledige werk-
  dag, lopen uiteen van de detectielimiet tot enkele honderden ng/m3, met maxima
  tot 30 µg/m3. De hoogste blootstellingen zijn gemeten tijdens het wegen en men-
  gen van meel en bij werkzaamheden met deeg. Omdat deegverbeteraars in ver-
  schillende hoeveelheden worden gebruikt voor brood en gebak, is de
  blootstelling ook afhankelijk van de soort bakkerij. Gedurende een interventie-
  programma tussen 2000 en 2007 is de blootstelling aan alfa-amylase in Neder-
  landse industriële bakkerijen en meelfabrieken afgenomen, maar toegenomen bij
  producenten van bakingrediënten.
  Kinetiek
  Blootstelling aan schimmel alfa-amylase vindt plaats via stofdeeltjes of aeroso-
  len. Er zijn geen specifieke gegevens over de kinetiek voor enzymen, maar men
  verwacht dat deze enzymen zich net zo gedragen als andere deeltjes. De plaats
  van depositie in het luchtwegsysteem is afhankelijk van de deeltjesgrootte. In
  bakkerijen is alfa-amylase voornamelijk aangetoond in stofdeeltjes met een aero-
  dynamische diameter groter dan 5 µm. De aerodynamische diameter van meer
  dan de helft van de stofdeeltjes was zelfs groter dan 9 µm. De meeste van deze
  stofdeeltjes zullen waarschijnlijk in de neus, mond en bovenste luchtwegen
  terecht komen.
  Effecten
  Inademing van schimmel alfa-amylase veroorzaakt zowel immunologische als
  niet-immunologische reacties. Immunologische reacties leiden tot sensibilisatie
  die bij voortdurende blootstelling allergische luchtwegreacties kan veroorzaken.
  Mensen krijgen dan bijvoorbeeld astma of ontstekingen in neus, luchtwegen en
  ogen. Deze symptomen kunnen echter ook worden veroorzaakt door irritatie, een
  niet-immunologisch mechanisme. De enige manier om deze twee reacties te
  onderscheiden is de mensen te testen op sensibilisatie voor schimmel alfa-amy-
  lase. Uit de beschikbare onderzoeken kan verder worden opgemaakt dat speci-
  fieke sensibilisatie binnen enkele maanden na het begin van de blootstelling kan
0 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>optreden, maar dat het ook jaren kan duren voordat allergische symptomen zich
ontwikkelen.
    De meeste gegevens over effecten van beroepsmatige blootstelling aan
schimmel alfa-amylase zijn afkomstig van onderzoek onder medewerkers van
bakkerijen en meelfabrieken. Het aantal gevallen van sensibilisatie onder deze
medewerkers loopt uiteen van 1 tot 30 procent, afhankelijk van het arbeidsverle-
den, de werkomstandigheden en persoonlijke kenmerken. Ter vergelijking: het
aantal gevallen van sensibilisatie door blootstelling aan schimmel alfa-amylase
in de algemene, niet-blootgestelde bevolking ligt tussen de 1 en 2 procent.
    Er zijn geen onderzoeken uitgevoerd naar mogelijke andere schadelijke
gezondheidseffecten onder werknemers. Ook zijn er nauwelijks diergegevens uit
proefdieronderzoek gepubliceerd.
Evaluatie en advies
Voor het afleiden van een advieswaarde hecht de commissie de meeste waarde
aan gegevens over sensibilisatie. Iemand die gesensibiliseerd raakt, loopt bij
voortdurende blootstelling een grote kans om allergisch klachten te krijgen. Aan-
gezien sensibilisatie niet omkeerbaar is zal deze persoon voor de rest van zijn of
haar leven gesensibiliseerd zijn en bij blootstelling allergische klachten kunnen
krijgen. In bakkerijen worden behalve alfa-amylase ook tarwemeel en andere
additieven gebruikt. Ook deze stoffen kunnen luchtwegklachten veroorzaken die
niet zijn te onderscheiden van de klachten die schimmel alfa-amylase veroor-
zaakt. Met speciale immunologische tests kan dat onderscheid bij sensibilisatie
wel gemaakt worden. De commissie heeft geen bewijs gevonden dat luchtweg-
klachten bij een lagere blootstelling optreden dan sensibilisatie. Dit betekent dat
een advieswaarde die gebaseerd is op gegevens van sensibilisatie tevens lucht-
wegklachten helpt te voorkomen.
    Om een gezondheidskundige advieswaarde te kunnen afleiden, zijn gegevens
nodig over de relatie tussen blootstelling en het optreden van sensibilisatie. In
veel onderzoeken is zo’n relatie niet goed onderzocht vanwege een gebrek aan
blootstellingsgegevens. In twee afzonderlijk van elkaar uitgevoerde onderzoeken
zijn echter wel zulke relaties bestudeerd. In beide gevallen gaat het om medewer-
kers in bakkerijen, is de blootstelling uitgedrukt in allergeengehalte in de stof in
de lucht (bepaald met dezelfde techniek) en zijn gegevens over het optreden van
sensibilisatie gerapporteerd. De uitkomsten zijn iets verschillend, maar de com-
missie ziet dit als normale statistische variatie bij een verschillende samenstel-
ling van deelnemers.
Samenvatting                                                                         11
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<pre>       De commissie is van mening dat voor het effect ‘sensibilisatie’ geen drem-
  pelwaarde is aan te wijzen. In de algemene (niet beroepsmatig blootgesteld)
  bevolking komen al gevallen van sensibilisatie voor schimmel alfa-amylase
  voor; er is dus geen nulsituatie. Dit laatste impliceert dat blootstelling, ongeacht
  de hoogte, een risico geeft op het ontwikkelen van sensibilisatie en dat daarom
  het beste een referentiewaarde kan worden afgeleid. Een referentiewaarde is een
  concentratie van alfa-amylase in de lucht waarbij beroepsmatige blootstelling
  leidt tot een vooraf bepaalde extra kans op het optreden van luchtwegsensibilisa-
  tie ten opzichte van het aantal gevallen in de (niet blootgestelde) algemene
  bevolking.
       Vanwege de grote overeenkomsten tussen de twee onderzoeken heeft de
  commissie besloten de gegevens van beide samen te voegen en daaruit een refe-
  rentiewaarde te berekenen met behulp van een lineair regressiemodel. De refe-
  rentiewaarde is gebaseerd op 1 procent extra kans (additioneel risico van 1
  procent). Voor schimmel alfa-amylase heeft de commissie een waarde afgeleid
  van 0,9 ng/m3.
  Gezondheidskundige referentiewaarden voor sensibilisatie
  De commissie beveelt een referentiewaarde van 0,9 ng enzym/m3 aan voor
  beroepsmatige blootstelling aan schimmel alfa-amylase, gemiddeld over een
  achturige werkdag. Bij deze concentratie hebben werkers ten opzichte van de
  algemene bevolking een extra kans van 1% op sensibilisatie voor schimmel alfa-
  amylase allergenen.
2 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
</pre>

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<pre>Executive summary
Scope
At request of the Minister of Social Affairs and Employment, the Dutch expert
Committee on Occupational Exposure Safety (DECOS), one of the permanent
committees of experts of the Health Council, proposes health-based
recommended occupational exposure limits for chemical substances in the air in
the workplace. These recommendations serve as basis in setting legally binding
occupational exposure limits by the minister.
    In this advisory report, the committee evaluates the consequences of
exposure to fungal alpha-amylase, an enzyme from the fungus Aspergillus
oryzae. Workers in bakeries and flour mills may be exposed to this enzyme. The
committee’s conclusions are based on scientific papers published before August
2014.
Physical and chemical properties
The enzyme alpha-amylase from the fungus Aspergillus oryzae, is a 478 amino
acid glycoprotein. In pure form it is whitish-yellow and a hygroscopic powder.
The enzyme catalyses the hydrolysis of long-chain carbohydrates to maltose and
glucose. Fungal alpha-amylase extracts are primarily used as a dough improver
in the preparation of bakery products.
Executive summary                                                              13
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<pre>  Exposure
  Exposure to airborne fungal alpha-amylase should be monitored as the content of
  the allergen in inhalable dust, and has mainly been determined in bakeries.
  Average concentrations in full-shift inhalable air ranged between the detection
  limit and a few hundred ng/m3, with maxima up to 30 µg/m3. The highest
  exposures were observed for workers involved in weighing and mixing, and
  dough handling activities. Because different quantities of dough improver are
  used for bread and pastry, exposure is also dependent on the type of bakery.
  During an intervention programme between 2000 and 2007 in the Netherlands,
  exposure to alpha-amylase decreased in industrial bakeries and flour mills, but
  increased at ingredient producers.
  Kinetics
  Exposure to alpha-amylase occurs from dust particles or liquid aerosols. There
  are no kinetics data specifically relating to enzymes, but they are considered to
  behave as other particles. The place of deposition in the airway system depends
  on the size of the particle. In bakeries, fungal alpha-amylase was predominantly
  present in dust particles with an aerodynamic diameter larger than 5 µm, with
  over fifty percent associated with particles larger than 9 µm. The majority of
  these particles are therefore likely to be deposited in the nose, mouth and upper
  airways.
  Effects
  Inhalation of fungal alpha-amylase elicits immunological as well as non-
  immunological responses. Immunological responses lead to sensitisation, which
  may induce – at continuing exposure – allergic respiratory symptoms, such as
  asthma, rhinitis, and rhinoconjunctivitis. The respiratory symptoms can also be
  caused by irritation, a non-immunological response. The only way to distinguish
  between the two types of responses is to test people on being sensitised to fungal
  alpha-amylase. The available studies suggest that sensitisation may occur within
  months after starting of exposure, but it can take several years to develop
  symptoms.
      Most data on the effects of occupational exposure to fungal alpha-amylase
  are retrieved from studies on bakery workers and flour millers. The number of
  cases of specific sensitisation among these workers varies between 1 to 30
4 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
</pre>

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<pre>percent. For comparison, the number of cases in the general, non-exposed
population varies between 1 and 2 percent.
     No studies have been performed on other possible adverse health effects in
humans, nor were there relevant animal studies reported.
Evaluation and recommendation
In deriving a health-based OEL, the committee considered data on sensitisation
as the most relevant. Somebody who is sensitised has a high risk in developing
allergic reactions at continuing exposure. Because sensitisation is an irreversible
effect, the person in question will be sensitised for the rest of his or her life, and
at exposure, may show allergic symptoms. In bakeries not only fungal alpha-
amylase is used, but also wheat flour and other additives. Also these substances
can induce respiratory symptoms, which are not distinguishable from the
symptoms described for alpha-amylase exposure. Such a distinguish can be made
for sensitisation by using special immunological tests. Further, the committee did
not find evidence that respiratory symptoms occur at lower exposure levels than
sensitisation. This means that an occupational exposure limit based on data on
sensitisation also prevents the development of respiratory symptoms.
     In deriving a health-based occupational exposure limit, data are needed on
exposure-response relationships. However, in most studies such a relationship
was not investigated due to lack of exposure data. In two independent studies an
exposure-response relationship was assessed. Both studies were carried out
among bakery workers, used airborne enzyme levels as exposure parameter
(measured by the same technique), and tested on sensitisation to fungal alpha-
amylase. The outcomes differ somewhat, but according to the committee this is
explained by normal statistical variation due to differences in the composition of
the population under study.
     The Committee is of the opinion that for the effect ‘sensitisation’ no
threshold level can be assessed. The reason being that in the general (not
occupationally exposed) population already cases of sensitisation to fungal
alpha-amylase have been reported. That implies that exposure, irrespective the
level, gives a risk in developing sensitisation, and that the setting of reference
values is warranted. A reference value is a concentration of alpha-amylase in the
air, at which occupational exposure leads to a predefined accepted level of risk of
allergic airway sensitisation, compared to the background risk in the general,
non-exposed population.
     Since the two studies show large similarities, the committee decided to
combine the data and to use a linear regression model to derive a reference value.
Executive summary                                                                      15
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<pre>  The reference value is based on an additional sensitisation risk of 1 percent. For
  fungal alpha-amylase, the committee derived a value of 0.9 ng/m3.
  Health-based recommended reference values for sensitisation
  The committee recommends a reference value of 0.9 ng enzyme/m3 for
  occupational exposure to fungal alpha-amylase, as an eight-hour time-weighted
  average concentration. At this concentration workers have an additional
  sensitisation risk for fungal alpha-amylase of 1 percent compared to the
  background risk in the general population.
6 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
</pre>

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<pre> hapter 1
        Scope
1.1     Background
        At request of the minister of Social Affairs and Employment, the Dutch expert
        Committee on Occupational Safety (DECOS), a committee of the Health Council
        of the Netherlands, performs scientific evaluations on the toxicity of substances
        that are used in the workplace (Annex A). The purpose of these evaluations is to
        recommend health-based occupational exposure limits, which specify levels of
        exposure to airborne substances, at or below which it may be reasonably
        expected that there is no risk of adverse health effects.
        In this advisory report, such an evaluation and recommendation is made for
        alpha-amylase from the fungus Aspergillus oryzae.
1.2     Committee and procedure
        The present document contains the assessment of DECOS, hereafter called the
        committee. The members of the Committee are listed in Annex B. The
        submission letter to the Minister can be found in Annex C.
             In 2014, the President of the Health Council released a draft of the report for
        public review. The individuals and organisations that commented on the draft are
        listed in Annex D. The Committee has taken these comments into account in
        Scope                                                                                17
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<pre>    deciding on the final version of the report. The received comments, and the
    replies by the Committee, can be found on the website of the Health Council.
1.3 Data
    The Committee’s recommendations on the health-based occupational exposure
    limit or reference values of alpha-amylase from A. oryzae are based on scientific
    data, which are publicly available. Published literature was retrieved from the
    on-line databases Medline and Toxline, supplemented with subject searches in
    journals and internet sources. The final search was carried out in August 2014.
 8  Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
</pre>

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<pre> hapter 2
        Identity, properties and monitoring
2.1     Identification
2.1.1   Fungal alpha-amylase
        Alpha-amylase is a naturally occurring enzyme that catalyses the hydrolysis of
        starch into sugars. It is present in the saliva of humans and some other mammals,
        in seeds containing starch as a food reserve (e.g. wheat), and is secreted by
        bacteria and many fungi. It is also produced industrially, both from bacteria (e.g.
        Bacillus spp. for use in the detergent industry) and fungi (e.g. Aspergillus spp. for
        use in the food industry).1
            Regarding occupational exposure, the most used alpha-amylase is the
        enzyme produced by the fungus Aspergillus oryzae. Since techniques are
        available to monitor specifically the alpha-amylase from this fungus, and data do
        not indicate allergenic cross-reactions among the different types of alpha-
        amylases, the committee restricted this advisory report to the fungal alpha-
        amylase from Aspergillus oryzae.
2.1.2   Combined exposure
        Fungal alpha-amylase is mainly used in the bakery industry. The dust in the
        bakery industry may contain several other ingredients used in the bakery process,
        such as flour dust from wheat and rye, other enzymes (malt enzymes), and
        Identity, properties and monitoring                                                   19
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<pre>    ingredients (e.g. baker’s yeast, egg powder, sugar). The committee is aware that
    these ingredients might contribute to the biological effects of fungal alpha-
    amylase. For instance, flour dusts from wheat and rye are known to have
    sensitizing properties.2,3 However, the present risk evaluation is restricted to
    fungal alpha-amylase.
2.2 Identity
    Until the 1960’s, the food industry mainly employed acid hydrolysis for
    processing starch. Since then, this traditional method was replaced by enzymes,
    mainly fungal alpha-amylase from Aspergillus oryzae. Alpha-amylases (EC
    3.2.1.1) catalyze the hydrolysis of internal α-1,4 glycosidic bonds in long-chain
    carbohydrates yielding maltose and glucose. Commercial flour additives contain
    up to 0.3% w/w alpha-amylase, usually from Aspergillus oryzae.4 In bread
    production, amylases are added to baking flour at 0.001 to 0.02% w/w, to
    compensate for the low natural amylase content.5-7 The liberation of yeast-
    fermentable carbohydrates (dextrins and maltose) by alpha-amylase stimulates
    the growth of yeast, improving the rising of the dough and the quality of the
    bread. Alpha-amylase is also used to delay the progressive hardening and drying
    in time, thereby prolonging the shelf life of bread.8
    Name                       :  alpha-amylase from Aspergillus oryzae
    EC number                  :  232-565-6
    CAS number                 :  9000-90-2; 9001-19-8
    Enzyme Commission no.      :  3.2.1.1
    Synonyms                   :  1,4-alpha-D-glucan glucanohydrolase; Asp o 2; Fungamyl;
                                  Taka-amylase
    Appearance                 :  whitish-yellow, very hygroscopic powder
    Molecular weight           :  53 kD
    pH-optimum                 :  7.0
2.3 Physical and biochemical properties
    Alpha-amylase from A. oryzae is a 478 amino acid glycoprotein.9 The molecule
    consists of three structural domains; the structure is stabilized by four disulfide
    bonds. Binding to calcium is necessary for enzyme activity; two calcium binding
    sites are located near the enzymatically active site cleft of the enzyme.10 By
    purifying the commercial alpha-amylase extract from Aspergillus oryzae, Baur et
    al. (1994) demonstrated that the enzyme is the main allergenic component of the
    commercial baking enzyme products.5 The enzyme activity rapidly declines
 0  Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>      upon heating, but the antigenic properties are more resilient. Heating the enzyme
      for 20 minutes at 99.8°C (the interior temperature of dough and bread during
      baking) caused a partial decrease of the IgE-binding capacity.11
2.4   EU classification and labelling
      In the European Union, fungal alpha-amylase is classified as a category 1
      respiratory sensitiser and labelled with H334 (May cause allergy or asthma
      symptoms or breathing difficulties if inhaled).12
2.5   Validated analytical methods
2.5.1 Environmental monitoring
      Several methods are available for determining airborne fungal alpha-amylase,
      namely as non-specific, inhalable dust which is sampled using gravimetric
      techniques (calculated using the mass of the sampled particles and the air volume
      monitored), and by measuring the amount of alpha-amylase in inhalable dust.
      Inhalable dust sampling
      In The Netherlands, inhalable dust is usually sampled with the Dutch 'PAS-6'
      sampling head, but other methods are also available. For determining the
      aerodynamic particle size of dust, personal air samplers can be used. The
      Institute of Occupational Medicine (IOM) in Edinburgh, Scotland, developed the
      IOM inhalable dust sampling head and cassette to meet the sampling criteria for
      inhalable particulate mass.2 Within Europe, size fractions for measurement of
      airborne particles in workplace atmospheres have been standardized since 1993
      (European Standard EN 481:1993). In this standard three size fractions have
      been defined (inhalable, thoracic and respirable).13
      Alpha-amylase content in inhalable dust
      Basically, there are two ways to monitor the presence of alpha-amylase in
      inhalable dust. One is measuring its allergen content by using an immunoassay,
      the other is measuring the enzyme activity.
          In the case of alpha-amylase, the most relevant adverse health effect is
      specific sensitisation, an immunological reaction which may lead to allergic
      symptoms (see Chapter 7 and 9). Such an immunologic reaction starts with the
      Identity, properties and monitoring                                               21
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<pre>  recognition of epitopes by the immune system. Epitopes are components
  (antigenic determinants) which are present in the protein of the enzyme. By
  means of immunoassays using antibodies, low levels of epitopes can be
  recognized. In addition, these antibodies are unique in that they only recognize
  certain epitopes in the fungal alpha-amylase, and not the epitopes in alpha-
  amylase from other sources. Thus by using such an immunoassay, the
  concentration of fungal alpha-amylase can be measured, even in the presence of
  alpha-amylase from other sources (e.g. the alpha amylase as natural content of
  wheat flour), and therefore the technique may be considered both sensitive and
  specific.
      The other technique, measuring enzyme activity, is not preferred, because
  inactive or denaturated enzyme molecules may still be allergenic and therefore
  have adverse health properties, whereas these are not measured enzymatically.
  Furthermore, by measuring enzyme activity no distinction can be made between
  the different sources of alpha-amylase.14
      Overall the committee concludes that it is best to use an immunoassay in
  determining exposure levels of airborne fungal alpha-amylase. Details on the
  methods available are summarized below.
  Immunoassays. Houba et al. (1997) developed a method using an enzyme
  immunoassay with affinity-purified polyclonal rabbit IgG antibodies to fungal
  alpha-amylase (sandwich-immunoassay).15 In addition, Sander et al. (1997) and
  Elms et al. (2001) developed an enzyme (inhibition) immunoassay using
  monoclonal antibodies to fungal alpha-amylase (ELISA-immunoassay).16,17
  Both types of immunoassays are specific for fungal alpha-amylase and do not
  detect cereal alpha-amylases occurring naturally in flour.
      In an interlaboratory comparison study, Lillienberg et al. (2000) compared
  the outcomes of both types of immunoassays using 80 representative whole-shift
  inhalable dust samples (PAS-6 sampler), taken in four bakeries in Sweden,
  Germany and The Netherlands.18 Extracts of the samples were distributed to
  laboratories in the three countries. Two sandwich-immunoassays were used,
  using polyclonal antibodies from two different origins. Also two other ELISA-
  immunoassays were used, one based on a polyclonal antibody, the other on a
  monoclonal antibody. The geometric means for samples analysed by the
  sandwich-immunoassays varied about a factor 2 among laboratories; the
  geometric means of alpha-amylase determined by the other ELISA-
  immunoassay was 3-6 times higher. The detection limit was 65-150 pg/mL for
  the sandwich-immunoassay, and 600 pg/mL for the other ELISA-immunoassay.
2 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>     With the aim to develop an international standard for measuring exposure to
fungal alpha-amylase, Sander et al. (2007) conducted a study to compare the
sandwich- and the ELISA-immunoassays, and to establish optimal elution and
assay conditions.19 Inhalable dust was collected in bakeries and mills in
Germany, England, The Netherlands and Spain. The assays for measuring fungal
alpha-amylase in dust extracts correlated well for log-transformed values of
positive samples (Pearson’s correlation coefficient ≥ 0.88), but the absolute
concentrations differed up to 5.8-fold. Of the extraction methods employed,
phosphate-buffered saline with 0.05% Tween-20 proved to be the best. Storage
of dust/filter extracts at -20°C resulted in an approximate loss of 40% in alpha-
amylase antigenic activity after 3-4 months.
     In epidemiological studies, usually those in which the sandwich-
immunoassay was used, airborne dust was sampled on Teflon (PTFE)
filters.14,15,20-22 In other studies, glass fibre filters were used.16,23-25 The effect of
filter type on the yield of alpha-amylase has not yet been examined.
     More recently, a semiquantitative lateral flow immunoassay was developed
for fungal alpha-amylase, allowing rapid on-site detection of the allergen in
airborne dust. The detection limit for this method is 320 pg/mL.26,27
Correlation between inhalable dust concentration and fungal
alpha-amylase content
The correlation between inhalable flour dust and fungal alpha-amylase is highly
variable. A significant correlation was found in some studies (Page et al. 201028,
Bulat et al. 200420), whereas the correlation in other studies was poor (Houba et
al. 199715, Elms et al. 200329, Baatjies et al. 201030). A possible explanation for
the variability between inhalable dust and alpha-amylase concentration are the
variable enzyme contents of the premix products used in the bakeries.30 For
example, cake and pastry baking differ from bread baking in that flour improvers
containing alpha-amylase do not play a vital part in the process.31 For these
reasons, the committee is of the opinion that dust exposure measurements cannot
be used as a surrogate for exposure to fungal alpha-amylase. Cereal flour
allergen concentrations in airborne dust are also an inadequate surrogate for
exposure to fungal alpha-amylase, because there is hardly any cross-reactivity
between cereal flour allergens and fungal alpha-amylase.(Houba et al. 1996,
Sandiford et al. 1994, Nieuwenhuijsen et al. 1999).32-34
Identity, properties and monitoring                                                        23
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<pre>2.5.2 Biological effect monitoring
      Tests are available to screen for sensitised persons against specific allergens. A
      useful clinical method for determining a rough approximation of the person’s
      sensitivity to an allergen is the skin prick test. In this test, allergens are
      introduced into the skin, after which the extent of local inflammation (wheal and
      flare diameter (mm)) is measured, as a result of the pharmacological effects of
      mediators, such as histamine, on the blood vessels in the skin. Skin prick tests
      resulting in a wheal diameter of at least 3 mm larger than the negative diluent
      (saline) control after fifteen minutes are usually considered positive for
      sensitisation.
          Alternatively, analysis of the presence of relevant specific IgE-antibodies, for
      instance in blood and nasal secretions, may be carried out. Serum concentrations
      of IgE antibodies to fungal alpha-amylase can be determined by
      radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). In
      both assays, fungal alpha-amylase is conjugated to commercially available
      cyanogen bromide activated cellulose or paper discs. In the Radio-Allergo-
      Sorbent-Test (RAST) developed by Brisman and Belin (1991), an 125I-labelled
      anti-human IgE preparation is used as a tracer.35 Baur et al. (1994) used an
      enzyme-allergo-sorbent-test (EAST) for determining alpha-amylase specific IgE
      in sera.5 In this assay, an anti-IgE-beta-galactosidase conjugate is used for
      detection. The EAST and RAST techniques were found to correlate to a high
      degree. The standard cut-off level for considering a test positive is ≥ 0.35 kU/L.
      More recently a commercial test has become available for quantifying specific
      IgE antibodies to fungal alpha-amylase in blood, employing a highly sensitive
      enzyme-enhanced chemiluminescent enzyme immunoassay (CAP-FEIA,
      Page et al. 2010, Baatjies et al. 2009).28,36 In contrast to the standard cut-off level
      generally used of ≥ 0.35 kU/L, in this commercial the test cut-off level is
      0.10 kU/L.
          Specific bronchial challenge testing is employed for the assessment of
      bronchial hyperresponsiveness, and if occupational asthma is suspected. The test
      provoces a physical response (rhinitis, asthma), and involves inhalation of a low
      dose of an allergen. Since there is serious risk of the patient suffering a serious
      asthmatic attack, it is important to perform the test in a good clinical setting.
 4    Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>2.6 Summary
    In the present report alpha-amylase from the fungus Aspergillus oryzea is
    evaluated. It is an enzyme that catalyses long-chain carbohydrates yielding
    maltose and glucose. Alpha-amylase is a glycoprotein that is composed of
    antigenic determinants, so-called epitopes, which can be recognized by the
    immune system. In this way specific sensitisation followed by allergic reactions
    can be provoked. Fungal alpha-amylase content in inhalable dust can be
    monitored by using an immunoassay. Monitoring inhalable dust as parameter for
    alpha-amylase exposure is not preferred, because of the poor correlation between
    inhalable dust and alpha-amylase content. Persons can be tested on sensitising
    responses by a skin or blood test.
    Identity, properties and monitoring                                              25
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<pre>6 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)</pre>

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<pre> hapter 3
        Sources
3.1     Natural sources
        Alpha-amylase form Aspergillus oryzea is a naturally occurring enzyme that
        catalyses the hydrolysis of starch into sugars. It is present in the saliva of humans
        and some other mammals, in seeds containing starch as a food reserve (e.g.
        wheat), and is secreted by bacteria and many fungi.
3.2     Man-made sources
3.2.1   Production
        Fungal alpha-amylase is also produced industrially, both from bacteria (e.g.
        Bacillus spp. for use in the detergent industry) and fungi (e.g. Aspergillus spp. for
        use in the food industry).1 The enzyme is manufactured for the food and feed
        industry.37,38 Production volumes are not available.
3.2.2   Use
        Bakers use fungal alpha-amylase as a supplement to cereal flour to improve
        carbohydrate fermentation by yeasts. This supplement can be added to the cereal
        flour by the bakers themselves, or is added already during flour milling to the
        cereal flour, which then is sold to the bakeries as a ready-for-use flour product.
        Sources                                                                               27
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<pre>  Inhalable dust in the atmosphere of the enzyme producing industry, bakeries,
  flour mills and manufacturers of dough improvers is the main sources of
  occupational exposure to the enzyme. Because it is added to flour from finely
  milled or otherwise processed cereal grains, fungal alpha-amylase is usually
  associated with flour dust in bakeries and related facilities. The most common
  tasks associated with alpha-amylase exposure involve dust-generating activities
  such as dispensing, sieving, weighing and mixing.39,40
      Fungal contamination of cereals as a possible source of alpha-amylase was
  proposed by Moneo et al. (1994).41 In a study of 259 millers in Spain, skin tests
  and specific serum IgE to fungal alpha-amylase were obtained in 12 of the 73
  workers. Since the workers did not use fungal alpha-amylase, the authors suggest
  that the enzyme is secreted by moulds growing on wheat or wheat flour.
  However, Houba et al. (1996) reported having detected only low concentrations
  of airborne fungi in (Dutch) bakeries, of which only a small fraction appeared to
  originate from A. oryzae.14
8 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre> hapter 4
        Exposure
4.1     General population
        Almost no studies have been published concerning exposure levels to fungal
        alpha-amylase in non-occupational settings. Vissers et al. (2001) measured wheat
        and fungal alpha-amylase allergens in floor dust samples, collected in the private
        homes of 34 bakers in The Netherlands.42 The concentration of fungal alpha-
        amylase allergens (geometric mean) ranged from 10.5 ng eq/m2 (living room) to
        76.7 ng eq/m2 (baker’s bedroom). The highest levels were found in houses that
        could be reached directly through the bakery, in houses with textile floor covers,
        and if bakers brought their work clothes into the house.
            Fungal alpha-amylase was shown to retain to some extent its antibody
        binding capacity and allergenicity after baking. In comparison with the content
        of fungal alpha-amylase in dough, between 0.1 and 20% of the antibody-binding
        capacity remained in bread and rolls.43 Fungal alpha-amylase may be added to
        certain washing powders, but exposure from dust from these agents in
        households is probably negligible.44
4.2     Working population
        Characteristic exposure data for fungal alpha-amylase are available for bakeries,
        flour mills and bakery ingredient producers in the Netherlands.14,15,18,45 The
        results of full-shift personal measurements of fungal alpha-amylase in inhalable
        Exposure                                                                           29
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<pre>              dust show exposures similar to those found in studies from other countries
              (Table 1).
able 1 Full-shift personal exposure to airborne inhalable dust-associated fungal alpha-amylase in various industries.
ndustry and country                AM                 GM                 GSD              Range              Reference
                                   (ng/m3)            (ng/m3)            (ng/m3)          (ng/m3)
 andwich-immunoassay (polyclonal antibodies)
 4 Bakeries, The Netherlands; 454 1.9 - 39.4a         0.7 - 18.1a        -a               ND - 221.8         Houba et al.
 ersonal samples                                                                                             199614
 1 Bakeries, The Netherlands;                                                                                Houba et al.
 large bakery: 406 personal                           ND - 18.1a         -a                                  199715
   samples
 small bakery: 101 personal                           ND - 0.3a          -a
   samples
 Bakeries (5 small, 2 large),      22.0               2.8                10.4             ND - 307.1         Burstyn et al.
 anada; 96 personal samples                                                                                  199821
 lour mills, packing stations and  0.7 - 46.7a                                                               Nieuwenhuijsen
arge bakeries (n=7), the UK; 256                                                                             et al. 199922
 ersonal samples
 Bakeries, UK; 26 personal                                                                ND - 29.8          Elms et al.
amples                                                                                                       200116
 akeries, mills and flour improver                                                                           Elms et al.
 roducer/packer, UK;                                                                                         200329
 8 small; 27 personal samples                         1.7e                                <0.4 - 173
 5 medium; 28 personal samples                        1.4e                                <0.4 - 12.1
 6 large; 41 personal samples                         4.2e                                <0.4 - 1,370
 2 mills; 17 personal samples                         1.5e                                <0.4 - 11.1
 1 improver producer/packer;                          455.9e                              71.2 - 1,178
   4 personal samples
 akeries, Belgium;                                                                                           Bulat et al.
 4 industrial; 54 personal                            0.2 - 0.5          -a               0.1 - 136.2a       200420
   samplesb
 66 traditional; 218 personal                         0.5 - 0.6          -a               0.1 - 51.1a
   samplesb
 akeries, The Netherlands;                                                                                   Meijster et al.
 65 traditional; 169 personal                         0.8                6.5              0.1 - 115          200745
   samples
 20 industrial; 344 personal                          0.4                6.0              0.1 - 910
   samples
 7 flour mills; 142 personal                          8.4                9.7              0.2 - 30,009
   samples
 7 ingredient producers; 113                          33.5               22.4             0.2 - 889,054
   personal samples
 akery, USA; 83 personal samples 0.1 - 2.1f                                               0.02 - 11,000      Page et al.
                                                                                                              200946, 201028
 7 Small bakeries, Scotland; 114   -c                 -c                 -c               3 - 710            HSE 199931
 ersonal samplesc
 0            Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre> LISA-immunoassay (monoclonal antibodies)
  Conventional bakery, Germany; 335                  13                  -c               ND - 4,836         Sander et al.
 7 personal samples                                                                                          199825
  “Vollwertbäckerei”, Germany; 14 -                  -                                    ND                 Sander et al.
 ersonal samples                                                                                             199825
  akeries in:                                                                                                Lillienberg et al.
 Sweden; 29 personal samples 7.2 - 22.0d              0.8 - 4.7d         5.0 - 9.0d       0.1 - 237d         200018
 Germany; 29 personal samples 9.5 - 30.1d             0.1 - 614.6d       0.6 - 3.8d       5.0 - 9.0d
 The Netherlands;                   7.9 - 25.6d       0.1 - 165.5d       1.3 - 6.2d       5.0 - 10.0d
   22 personal samples
 0 Supermarket bakeries, UK;        -                -                   -                ND - 12.4          Brant et al. 200523
 9 personal samples
  akeries (n=55), UK;                                 5.9                -c               <0.78 - 33.7       Elms et al. 200647
 95 personal samples
 8 Supermarket bakeries,            0.29             0.13                2.08             0.1 - 19.6         Baatjies et al.
 outh-Africa; 211 personal samples                                                                           200936, 201030
AM: arithmetic mean; GM: geometric mean; GSD: geometric standard deviation; ND: below the detection limit.
 No overall AM and/or GM was presented in the original publication, but only for each occupational title; the means presented
 re the lowest and highest AM and GM for the occupational titles;
 Calculated from the reported fraction of alpha-amylase in inhalable dust;
 This information could not be obtained from the original publication;
 Interlaboratory comparison study. The range of values determined by the 4 participating laboratories is shown;
 Only the median was reported;
 GM for entire population not reported; separate values for current low exposure (range 0.019 - 1.2 ng/m3) and for current and
 ast higher exposure (range 0.095 - 11,000 ng/m3).
              In bakeries, the highest exposures to fungal alpha-amylase were observed for
              workers involved in weighing, mixing, pouring and dough handling activities, as
              well as during the production of croissants, puff-pastry and bread-bun.4,21,29,48
                  In a study in supermarket bakeries in the UK, fungal alpha-amylase was
              detected in whole-shift personal respirable dust samples of only three of nine
              sensitised individuals, showing the difficulty of capturing relevant sensitising
              exposures even in whole-shift, personal measurements.23 Elms et al. (2001)
              analysed four consecutive 15-minute inhalable dust samples from eight bakery
              workers, and found a large inter-sample variation of the alpha-amylase
              concentration, revealing alternating periods of high and low exposure.16 Short-
              term peak exposures should therefore be taken into consideration for sensitised
              individuals who have low exposure levels if measured over the full shift period.
              Short-term exposure measurements during performance of dust-generating tasks
              were reported by Brisman and Belin (1991).35 Using an enzymatic method for
              analysing fungal alpha-amylase in inhalable airborne dust in a Swedish factory
              producing bread mixes and dough improvers, they determined a concentration of
              30 µg/m3 in a 30-60 min personal sample for a worker at a packing station.
                  In The Netherlands, an intervention programme was enrolled as part of a
              covenant in the flour processing industry (industrial bakeries, flour mills and
              Exposure                                                                                                       31
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<pre>             ingredient producers). Worker’s health was monitored through the instalment of
             a sector-wide health surveillance system and all employers and employees were
             informed about the risks of occupational exposure and were provided
             information on good work practices and control measures. The campaign
             included site visits by a trained consultant and distribution of a specially
             developed dust control manual. The effect of the programme on exposure to
             airborne inhalable fungal alpha-amylase was investigated in a large study using
             exposure data from studies conducted in bakeries, flour mills and ingredient
             producing companies between 1993 and 2007 (see Table 2).14,15,24,32,45
                 Despite several changes in work practices (e.g., the increased use of liquid
             instead of powdery fungal alpha-amylase containing bread improver), only
             limited effect on exposure levels was observed from the sector-wide intervention
             programme. The overall trends in exposure, not corrected for co-variables,
             indicated a slight downward trend for fungal alpha-amylase in bakeries. Per
             sector, a significant yearly downward trend was observed of 8% for fungal alpha-
             amylase among the population of bakery workers. However, trends varied
             substantially between job categories: a downward trend was observed for pastry
             bakers and cleaners, a strong yearly increasing exposure trend was observed
             (>20%) for general bakers, whereas no trend was observed for bread bakers,
             dough makers, maintenance workers and low-exposed jobs. In flour mills, no
             trend was observed in fungal alpha-amylase exposure over time. On job level, a
             significant decreasing trend was shown only for maintenance workers. For
             workers in the ingredients production industry, there was a strong increase in
             occupational exposure to fungal alpha-amylase for all job titles. Overall, a non-
             significant increasing trend of almost 30% annually was observed over the
             covenant period for this sector (2001-2007). There was no apparent explanation
             for the exposure increase to fungal alpha-amylase, as information from the sector
             did not indicate an increased use or changed work practices. The authors stated
             that the results should be interpreted with caution because data per job were
             limited for this industry.24
 able 2 Average exposure level to fungal alpha-amylase (ng/m3) per sector for the period 2000-2001 and 2007.49
 ector                    2000 - 2001                                      2007
                          N                GM             GSD              N                GM             GSD
ndustrial bakeries        175               1.0            3.3             131                0.4           7.1
 lour mills               143               8.0            9.8               88               6.3          15
ngredient producers       114              32             22                 89             177            16
N, number of companies; GM, geometric mean; GSD, geometric standard deviation.
 2           Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>hapter 5
       Kinetics
       Occupational exposure to fungal alpha-amylase occurs from inhalation of dust or
       liquid aerosols. There are no data on absorption, distribution, metabolism, and
       excretion specifically relating to enzymes, but they are considered to behave as
       other particulate matter. Therefore, below the kinetics of particles (with no or
       very low toxic potential) is summarized.
       Deposition
       Upon inhalation, particles are deposited on the mucous membranes of the
       airways. The place of deposition in the airways is dependent on the size of the
       particle.38 Based on the aerodynamic diameter, particles are divided in inhalable,
       thoracic and respiratory fractions (see European Standard EN 481:1993).
       Inhalable particle (or dust) fractions are defined as fractions in which 50% of the
       particles have an aerodynamic diameter of 100 µm. These particles are mainly
       deposited in the nose and nasopharyngeal region of the respiratory tract. In
       thoracic fractions about 50% of the particles have an aerodynamic diameter of 10
       µm, and these can be found the trachea and bronchial region of the respiratory
       tract. Finally, particles in the respiratory fraction are the smallest, and may reach
       the lungs (particles with an aerodynamic diameter of 3.5-4 µm or smaller). In
       crispbake and small bakeries in The Netherlands, fungal alpha-amylase was
       predominantly present in dust particles with an aerodynamic diameter larger than
       Kinetics                                                                              33
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<pre>  5 µm, with over 50% associated with particles larger than 9 µm, and less than
  10% associated with particles with an aerodynamic diameter lower than 5 µm.15
      Dust particles are cleared from the lungs by macrophages and the
  mucociliary system. However, heavy exposure may lower the ability of
  macrophages to eliminate particles, which may result in penetration of dust
  particles into the interstitium. The individual (anatomic) characteristics of an
  exposed person are also of importance in the development of disease.2,3
4 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre> hapter 6
        Mechanism of action
        Respiratory exposure to fungal alpha-amylase elicits immunological (allergic)
        and non-immunological (irritation) responses, such as rhinitis, rhinoconjunc-
        tivitis, asthma. The symptoms of irritation, are similar to allergic symptoms. For
        interpreting the symptoms and its consequences on health, it is important to
        make a distinction between the non-immunological and immunological
        responses. In practice, symptoms are ascribed to irritation if immunological
        responses are ruled out
6.1     Immunological responses
        Sensitisation is an immunological mechanism (type I hypersensitivity reaction),
        which may occur at a first exposure, and is characterised by little or no response
        against the sensitising agent, in this case alpha-amylase. However, when a person
        is sensitised, subsequent exposure may cause intense responses, such as asthma,
        rhinitis and conjunctivitis. This may occur at low exposure concentrations. The
        responses may be life threatening and may have an immediate or delayed onset.
        The key mechanism of sensitisation is the formation of specific IgE-antibodies
        against alpha-amylase. These IgE-antibodies are incorporated at the surface of
        mast cells. Following a second encounter with the same allergens, mast cells may
        overreact when these allergens bind to the antibodies presented at the surface of
        the mast cells. Mast cells form the starting point of a cascade of chemical
        reactions resulting in clinical symptoms. Specific IgE-antibodies against alpha-
        Mechanism of action                                                                35
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<pre>    amylase have been demonstrated in workers who were sensitised after inhalation
    of dust, by in vivo and in vitro tests. Workers who have become sensitised to a
    particular agent may also exhibit cross-reactivity to other agents with similar
    chemical structures. However, cross-reactivity was not found between fungal
    alpha-amylase and pancreas alpha-amylase from pigs (Losada et al. 1992)50 or
    cereal alpha-amylase (Nieuwenhuijsen et al. 1999; Sandiford et al. 1994).22,34,51
         A special group among the population are the atopics, that are persons with a
    family history of similar clinical symptoms or persons who are allergic to
    common allergens. In general, atopic individuals may be slightly more prone to
    sensitisation to alpha-amylase than non-atopic individuals. In the human
    population it is estimated that 5.1 to 20.8% are atopic. Most investigators found
    that atopic workers were more vulnerable to develop work-related respiratory
    symptoms than non-atopic workers.
         In addition to being atopic, other factors may increase the risk to become
    sensitised to work-related allergens, such as the level of airborne allergens. In
    none of the available studies involving fungal alpha-amylase, a positive
    association was found between sensitisation to the enzyme and smoking habits
    (Brisman and Belin 1991; Cullinan et al. 1994, 2001; Houba 1996; Vanhanen et
    al. 1997),35,52-55 except in atopic workers (Droste et al. 2003; Harris-Roberts et
    al. 2009; Page et al. 2010),28,40,56 although others did not always confirm this.
         Age has not been reported to be a determinant of sensitisation to fungal
    alpha-amylase. Gender was a potential confounder in one study but the authors
    did not stratify their results (Brisman et al. 2004).57
6.2 Non-immunological responses
    A strong association between exposure to fungal alpha-amylase and respiratory
    symptoms has been shown in a number of studies (see Chapter 7), of which up to
    70% of work-related asthmatic symptoms in bakery employees has been
    attributed to non-specific irritation to dust.7 This led Smith et al. (1999) to
    suggest that sensitisation to alpha-amylase is not a relevant endpoint for the
    prevention of bakery-related respiratory disease.58
         As is written in Section 5, fungal alpha-amylase is considered to behave as
    dust particles. In general, exposure to large dust particles, irrespective of its
    chemical activity properties, may lead to local irritation to the eyes, nose and
    ears. In addition, inhalable dust particles may lead to irritation and inflammation
    of the bronchioles, alveolar ducts and alveoli. When dust particles are deposited
    in the respiratory system, the body tries to clear the material, in which the
    mucociliary defence system, and/or inflammatory cells, such as macrophages,
 6  Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>    are involved. Macrophages produce inflammatory mediators, which induce
    inflammatory responses with symptoms of irritation.
6.3 Conclusion
    Respiratory exposure to fungal alpha-amylase elicits immunological and non-
    immunological responses. Immunological responses, primarily IgE-mediated,
    lead to sensitisation and allergic respiratory symptoms, such as rhinitis,
    rhinoconjunctivitis, asthma, or in some cases to contact dermatitis (on dermal
    exposure). The non-immunological responses are most likely due to irritation of
    the upper and lower airways. Irritation may result in similar symptoms as
    described for allergic responses.
    Mechanism of action                                                             37
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<pre>8 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)</pre>

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<pre> hapter 7
        Effects
7.1     Observations in humans
        Observations in humans are limited to effects in the skin, eyes, airways and
        lungs. No data are available on effects on other organ systems, nor on
        carcinogenic effects and reproduction toxicity.
7.1.1   Irritation
        In a prospective cohort study, involving 300 UK bakery and flour mill workers
        not previously exposed to flour, only 15% of the workers with respiratory
        symptoms or eye, nose or skin symptoms, were sensitised to wheat flour or
        alpha-amylase (as revealed by positive skin prick tests), suggesting that a high
        proportion of the work-related symptoms did not have an immunologic basis
        (Cullinan 1994, 2001).52,53
             Houba et al. (1996) performed a survey among 393 Dutch bakery workers.54
        Twenty-three percent of these workers reported work-related rhinitis and/or chest
        tightness, but only 30% (7% of all bakery workers) showed an immunological
        response to wheat flour or fungal alpha-amylase. Although the authors noted that
        the sensitivity of the IgE test was not perfect, and that there are also other
        potential bakery allergens that have not been tested, they considered a non-
        specific reaction to the dusty environment in the bakeries as one of the likely
        explanations for this finding. Most workers with work-related symptoms, but
        Effects                                                                           39
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<pre>      without an immunologic response (sensitisation) to wheat flour or fungal alpha-
      amylase, had IgE antibodies to non-occupational allergens (58%), had a history
      of allergic symptoms to common allergens (27%), or reported chronic respiratory
      symptoms outside the job environment (55%).
          Smith and Lumley (1996) screened 3,450 workers in a large food company in
      the UK using a questionnaire.7 The overall prevalence of work-related asthmatic
      symptoms was 4.4%. After an examination by a physician, only 9% of this group
      had occupational asthma. The majority of the complaints was thought to have
      arisen from irritation or aggravation of pre-existing asthma.
          In a group of workers (n=679) from 18 different UK flour mills, Smith et al.
      (2000) examined the prevalence of respiratory symptoms and their relationship
      to sensitisation to wheat flour allergens and fungal amylases.59 Of the examined
      workers, 20.5% described upper respiratory tract symptoms of an occasional
      nature, which the investigators related to short-term exposures to high levels of
      dust. Of the workers, 0.9% was sensitised to fungal alpha-amylase (positive skin
      prick test); three individuals had symptoms due to wheat flour allergy. Total
      inhalable dust exposure was measured for personnel exposed to flour dust at 10
      different sites (116 samples). Median levels of 6.2 mg/m3 were measured (range
      1-10 mg/m3; TWA 8 h) for production personnel, and a median of 18.7 mg/m3
      for hygiene operatives. The authors concluded that the principal causation of
      symptoms experienced by workers were non-specific irritant effects related to
      short-term exposures to high levels of total inhalable dust.
          The committee noted that the investigators of the studies above did not
      describe (extensively) relationships between exposure levels and occurrence of
      symptoms.
7.1.2 Sensitisation
      Prevalence
      General population
      In two studies among apprentice bakers in Canada and Poland, the prevalence
      early in the training was 0% (0/230, Gautrin et al. 199760; 0/287, Walusiak et al.
      200461). In a study among trainee bakers in Italy (n=144, 81 attending the first
      year and 63 attending the second year), the prevalence for sensitisation to fungal
      alpha-amylase was 0.7% (1 case, also sensitised to wheat and with a personal
      history of allergy). In the same study, no persons sensitised to fungal alpha-
 0    Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>amylase were found in the reference group (81 graphic artist students; De Zotti
et al. 1995).62
    The prevalence of fungal alpha-amylase specific IgE antibodies in US blood
donors was 1.0% (5/529).63 The prevalence of sensitisation to fungal alpha-
amylase among Dutch laboratory animal workers (positive skin prick test),
reported by Houba et al. (1996), was 1.7% (7/416).14 The prevalence of
sensitisation among workers in a Belgian petrochemical plant was 0.8%
(2/251).56
    These studies suggest that there is at least some background level of
sensitisation to fungal alpha-amylase (0 - 1.7%) in the general population. That
in the general population people may be sensitised to fungal alpha-amylase could
be explained by consumption of alpha-amylase containing food products, and the
use of ready for use flour products (containing fungal alpha-amylase as
supplement) for preparing bread and other bakery products themselves. Other
reasons may be: cross-reactivity with as yet unknown other allergens; atopy; a
lack of standardised methods for testing specific sensitisation resulting in false-
positive outcomes; and, the use of anonymous blood donors, which may have
been employed in companies using fungal alpha-amylase.
Working population
In Table 3 data from varies studies (mainly cross-sectional studies) are shown on
the percentages of workers who are sensitised to fungal alpha-amylase,
irrespective if they showed also symptoms of respiratory allergy or not. Detailed
descriptions on study design, and results are given in Annex E. The most
common tests used to determine the presence of specific sensitisation to fungal
alpha-amylase were the skin prick test, and the radioallergosorbent test (RAST)
to measure serum specific IgE levels.
    Overall, prevalence rates vary between 1 and 19% among workers in
bakeries and flour mills. Higher prevalence rates (up to 31%) have been reported
for workers in enzyme producing and handling companies. The variability in
prevalence rates could be explained by differences in exposure levels, duration
and frequency of exposure, and the use of different protocols in testing
sensitisation (e.g., variations in origin of fungal alpha-amylase, test
concentrations, and cut-off points). In spite of these differences, the available
studies clearly show that immunological sensitisation to fungal alpha-amylase is
common.
Effects                                                                             41
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<pre>  Table 3 Sensitisation rates in workers exposed to fungal alpha-amylase. See Annex E for study
  details.
  Reference                               Type of        N             Skin prick test Specific serum
                                          industry                     (% positive) IgE
                                                                                       (% positive)
  Bakeries
     Houba et al 199614                   bakery         169             9             8
     Smith et al 199744                   bakery         385           16
     Baur et al 199864                    bakery         88/89         19              19
     Smith and Wastell Smith 199865       bread bakery 392             16
     Smith and Wastell Smith 199865       cake bakery 77                 1
     Jeffrey et al 199966                 bakery         205                           15
     Smith et al 200059                   flour mill     678             1
     Droste et al 200356                  bakery         222             8
     Walusiak et al 200461                baker’s        287                           11 (third year)
                                          training
     Storaas et al 200567                 bakery         183             7             2
     Brant et al 200523                   supermarket 233/210                          4
                                          bakery
     Page et al 200946                    bakery         96                            4 (7 at low
                                                                                       threshold)
     Harris-Roberts et al 200940          bakery         160                           6
     Baatjies et al 200936                supermarket 507/513            4             4
                                          bakery
  Flour milling
     Cullinan et al 199452                bakery + flour 256             5
                                          mill
  Enzyme producing industry and other sources
     Brisman and Belin 199135             dough          20            30              5
                                          improver
     Johnsen et al 199768                 enzyme         140             4
                                          producer
     Vanhanen et al 199755                enzyme         173             3             3
                                          producer
     Biagini et al 199669                 biotechnology 36               6
                                          plant
     Losada et al 199250                  pharmaceutical 83            31
  Dose-response relationships
  A few studies have been performed in which exposure-response relationships to
  sensitisation were investigated. These include one longitudinal cohort study
  among bakery and flour mill workers in the United Kingdom, and three cross-
  sectional studies performed in bakeries in the Netherlands, Belgium, and the
  USA. Details on study design and results are given in Annex F. All used the
2 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>               sandwich-immunoassay to determine airborne exposure levels of fungal alpha-
               amylase. Furthermore, the majority of the studies used a skin prick test to assess
               specific sensitisation to the enzyme; only two studies reported (also) on serum
               specific IgE levels. Since studies were performed in bakeries, co-exposure to
               other potential allergens was inevitable.
               Two studies showed a statistically significant increase in number of sensitised
               workers with increasing exposure: Nieuwenhuijsen et al. (1999), and Houba et
               al. (1996).22,70 Data from these studies are shown in Table 4.
 able 4 Summary of dose-response relationships between fungal alpha-amylase exposure and specific sensitisation with
 tatistical significance.
  tudy characteristics        Exposure levels                  Sensitisation                            Reference
                              (fungal alpha-amylase, ng/m3)
 nitial cross-sectional       Method: Sandwich-immunoassay. Method: Skin prick test.                    Nieuwenhuijsen et
 hase of cohort; 264 new      Exposure at time of study        Prevalence, exposure at time of study:   al. 199922, 199471
workers                       (AM±SD):                         • low: 3.1% (7/225)
                              • low: 0.7±0.8 ng/m3             • medium: 16.7% (3/18)                   Cohort details:
Cohort, 3 bakeries,           • medium: 10.7±2.2 ng/m3         • high: 15.4% (2/13)                     Cullinan et al.
  flour packing factory,      • high: 46.7±16.6 ng/m3          Prevalence, highest exposure ever:       199452
 nd 3 mills, UK               Highest exposure ever worked in: • low: 2.5% (5/203)
                              • low: 0.8±0.8 ng/m3             • medium: 9.5% (2/21)
                              • medium: 10.5±2.3 ng/m3         • high: 29.4% (5/17)
                              • high: 48.0±16.6 ng/m3          Prevalence ratio, highest exposure ever:
                                                               • medium: 3.9 (95% CI, 0.8-20.2)
                                                               • high: 9.9 (95% CI, 2.8-34.6)
  ross-sectional,             Method: Sandwich-immunoassay     Prevalence, skin prick test:             Houba et al. 199670;
 4 bakeries in                Highest exposure ever worked in  • low: 1.4% (1/71)                       Doekes et al. 19986
he Netherlands;               (GM±GSD):                        • medium:12.8% (5/39)
 78 workers                   • low: 0.7±4.0 ng/m3             • high: 30.4% (7/23)
                              • medium: 1.3±3.8 ng/m3          • indistinct: 8.3% (3/36)
                              • high: 18.1±4.6 ng/m3           • reference population: 1.7% (7/416)
                              • indistinct/variable:           Prevalence ratio, skin prick test:
                                 6.1±8.2 ng/m3                 • medium:8.6 (95% CI, 1.01-74)
                                                               • high: 15.9 (95% CI, 1.95-129)
                                                               • indistinct: 4.6 (95% CI, 0.48-45)
                                                               Prevalence, specific IgE:
                                                               • low: 2.5% (2/71)
                                                               • medium: 13% (5/39)
                                                               • high: 15% (4/23)
                                                               Prevalence ratio, specific IgE:
                                                               • medium: 4.6 (95% CI, 0.85-22)
                                                               • high: 3.9 (95% CI, 0.65-24)
AM, average mean; 95% CI, 95% confidence interval; GM, geometric mean; SD, standard deviation.
               Effects                                                                                                    43
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<pre>  The study by Nieuwenhuijsen et al. (1999) was a nested case-control study
  within a longitudinal cohort study among bakeries, flour mills and a flour
  packaging factory in the United Kingdom.22 Workers (n=264) were screened for
  the presence of sensitisation to wheat flour allergens, and fungal alpha-amylase.
  The investigators divided the workers in three exposure groups (AM±SD, values
  represent highest exposure ever worked in): 0.7±0.8 ng/m3 (low), 10.5±2.3 ng/
  m3 (medium), and 48.0±16.6 ng/m3 (high). The prevalence to sensitisation to
  fungal alpha-amylase increased by increasing exposure from 2.5% (low), 9.5%
  (medium) to 29.4% (high), resulting in prevalence ratios of: 3.9 (95% CI, 0.8-
  20.2; medium versus low exposure), and 9.9 (95% CI, 2.8-34.6; high versus low
  exposure). Analyses showed a significant exposure-response relationship to
  fungal alpha-amylase. Atopics showed a statistically non-significant increased
  risk of sensitisation compared to non-atopics. The authors noted that the number
  of workers in the medium- and high exposure group was small, the reason being
  that most workers were exposed to low concentrations of fungal alpha-amylase.
      Houba et al. (1996) carried out a cross-sectional study in bakeries in the
  Netherlands. Workers (n=178) were screened for the presence of sensitisation
  to common allergens, cereal flour allergens, bakers’ yeast, storage mites and
  fungal alpha-amylase.70 In this study the workers were divided into three
  exposure groups (GM±SD, values represent highest exposure ever worked in):
  0.7±4.0 ng/m3 (low), 1.3±3.8 ng/m3 (medium), and 18.1±4.6 ng/m3 (high).
  Increased percentages of sensitised workers (to fungal alpha-amylase) were
  found with increasing exposure: 1.4% (low), 12.8% (medium) to 30.4% (high).
  The corresponding prevalence ratios were: 8.6 (95% CI, 1.01-74, medium versus
  low), and 15.9 (95% CI, 1.95-129, high versus low). Sensitisation to fungal
  alpha-amylase was more common in atopic workers, which was shown by a
  strong exposure-response relationship, whereas in the group of non-atopic
  workers no such strong relationship was observed. For IgE sensitisation, the
  authors found a positive trend for alpha-amylase exposure, but it did not reach
  statistical significance. In the other studies no clear statistically significant
  positive trends were found, although the number of sensitised workers tended to
  increase with increasing exposure.
      From the same cohort-study as Nieuwenhuijsen et al., Brisman et al. (2004)
  reported on a nested case-control study using more recent data and making a
  distinction between different exposure groups.57 However, the committee noted
  the poor reporting, in that: it is not clear whether the persons in the groups are the
  same persons as in the Nieuwenhuijsen-study; no data are given on the number
  of persons in the groups; and, it is unclear whether data include new cases only
4 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>      or all (new and old) cases. For this reason the committee considers this study not
      useful for quantitative risk assessment.
           The committee noted that exposure categories based on ‘the highest exposure
      a worker ever worked in’ are preferred over exposure categories based on current
      exposure, because it is plausible that a worker may become sensitised when
      working at a high exposure level and develops respiratory symptoms later, even
      if relocated to a lower exposure area.
7.1.3 Allergic respiratory symptoms
      The committee is aware that respiratory symptoms may be induced by other
      factors than exposure to fungal alpha-amylase (see Section 7.1.1). Since in the
      majority of the studies co-exposure with other potential allergens, for instance in
      wheat and rye flour, was inevitable, these may have also induced respiratory
      symptoms. For this reason, associations between exposure to fungal alpha-
      amylase and respiratory symptoms should be interpreted with caution.
           Studies have been published on exposure to fungal alpha-amylase, on which
      respiratory symptoms (cough, sneezing, watery eyes, runny nose, rhinitis,
      shortness of breath, asthma) could not be attributed to other causes than exposure
      to the enzyme. For instance, a few cases of bakers’ asthma have been described
      in bakers who were sensitised to fungal alpha-amylase, and not to other potential
      allergens in bakeries (see Annex F and G). Also some studies described
      hyperresponsiveness after sensitised subjects underwent a specific bronchial
      provocation test (see Annex F).
7.1.4 Allergic contact dermatitis
      Allergic contact dermatitis in bakery workers, sensitised to fungal alpha-
      amylase, has been reported in a number of studies. For instance, Schirmer et al.
      (1987) reported a case of a German baker, sensitised to alpha-amylase in a dough
      additive.72 Specific IgE, IgG and IgM antibodies were detected in his serum. In a
      retrospective observational study, Hernández-Bel et al. (2011) confirmed sensi-
      tisation to fungal alpha-amylase by skin prick test in two Spanish bakers among
      27 cases of protein contact dermatitis on hands and forearms.73 Morren et al.
      (1993) examined 32 Belgian bakers with hand dermatitis, seven of whom had an
      immediate wheal-and-flare reaction to fungal alpha-amylase upon scratch
      chamber testing, and two also had delayed contact eczema.33 All were found
      positive to skin prick testing. In a cross-sectional study, Brisman and Belin
      Effects                                                                             45
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<pre>      (1991) found one person with contact dermatitis among six Swedish bakers,
      sensitised to alpha-amylase (skin prick test positive).35
7.1.5 Food allergy
      Heating of fungal alpha-amylase, simulating bread baking, only partially
      removes the allergenic properties of the enzyme.11 Allergenicity from enzyme
      residues in bread can therefore not be excluded. A case of a Spanish farmer with
      sneezing, cough and oral angioedema after eating bread was reported.74 Skin
      prick testing revealed sensitisation to olive tree pollen and to fungal alpha-
      amylase. Since the farmer had never worked in pharmaceutical industry or in a
      bakery-related job, the authors concluded that the only source of sensitisation to
      alpha-amylase was bread. No other studies were found on fungal alpha-amylase
      induced food allergies in regular bread consumers.
7.2   Animal studies
      No animal data on long-term exposure were available. Also no data were
      presented on carcinogenic and reproduction toxic properties.
7.2.1 Single exposure
      In Japanese studies, acute oral LD50’s in rat and mouse between 7.5 and 15 g/kg
      body weight, respectively, have been reported.75 The WHO/FAO working group
      reviewed several toxicity studies, and reported on acute oral LD50 in mouse of
      less than 20 g/kg bodyweight (data obtained from industry).76 In a Russian
      journal, the lowest concentration published for inhalation toxicity in mammals
      (species unspecified) was 15 mg/m3.75
7.2.2 Repeated exposure
      The WHO/FAO working group also reported on two dietary animal studies with
      repeated exposure to fungal alpha-amylase (data obtained from industry).76 The
      first is a 3-week toxicity study with groups of 5 male and 5 female Wistar rats,
      which received 0, 0.5, or 5% fungal alpha-amylase in the diet. Only minor
      differences were observed in body weight change and food intake. At study
      termination, no compound-related haematological effects, changes in organ weights
      or gross pathology were noted. The second is a 3-month study in Sprague-Dawley
      rats (10-20 male and female animals/group), which received diets containing 0%,
 6    Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>      5% or 10% of the enzyme (equivalent to 3.5 or 7 g enzyme/kg bw/day). No signs of
      toxicity were observed and no effect on body weight gain or food consumption.
      Differential blood counts were normal after 4 and 8 weeks. Furthermore, at study
      termination, haematology, organ weights, gross examination and histopathology
      revealed no compound-related effects.
7.2.3 Specific sensitisation to fungal alpha-amylase
      Robinson et al. (1998) studied the sensitisation potencies of enzymes, including
      commercial fungal alpha-amylase (Fungamyl), using the Guinea-pig
      intratracheal test (GPIT) and the Mouse intranasal test (MINT) models.77 In each
      dosing group, 10 female Hartley guinea-pigs, and five female BDF1 mice were
      used.
          Regarding the GPIT, alpha-amylase was administered intratracheally, once a
      week for 10 weeks. Sensitisation was determined by observing the animals for
      signs of respiratory distress. Intratracheal instillation of 0.3, 1 or 3 µg (per
      animal) of the enzyme caused sensitisation in guinea-pigs at the highest dose
      only.
          In the MINT, the enzyme was administered intranasally in a saline-detergent
      matrix on days 1, 3 and 10. Sensitisation was determined by measuring serum
      specific IgG1 on day 15. Intranasal administration of the enzyme resulted in an
      ED50 (the dose resulting in half of the maximum serum IgG titre) of 2.35 µg (per
      animal per installation). The lowest dose resulting in an increase of enzyme-
      specific serum IgG1 was 0.2 µg (per animal).
          Experiments in animals confirm the findings in humans that exposure to
      fungal alpha-amylase may lead to specific sensitisation and allergy. For instance,
      allergenicity of enzymes was studied by Sarlo et al. (1997) in an animal model,
      the Guinea-pig intratracheal test.78 The animals received alpha-amylase by
      intratracheal instillation, once per week for ten weeks. Passive cutaneous
      anaphylaxis antibody (IgG1) titres against the enzyme in sera of the treated
      animals were measured by intradermally injecting the sera in shaven naïve
      Guinea-pigs. The findings were compared with alcalase, an enzyme for which
      much information is available. The test was shown to have predictive value for
      IgE-mediated responses to inhalation of alpha-amylase by humans. Sarlo et al.
      compared antibody titres to alpha-amylase from Bacillus licheniformis with
      those from alcalase treatment, and found that the alfa-amylase showed a 10-fold
      higher allergenicity then alcalase.
      Effects                                                                            47
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<pre>7.3 Summary
    Most data on the effects of occupational exposure to fungal alpha-amylase are
    retrieved from human studies on bakery workers and flour millers. Effects are
    mainly described on the upper airways and lungs, such as asthma (baker’s
    asthma), rhinitis, and rhinoconjunctivitis. Part of the symptoms are explained by
    irritation responses, whereas another part of the symptoms by (specific IgE
    mediated) immune responses (sensitisation, allergy). Sensitisation to fungal
    alpha-amylase may occur soon after start of exposure (months), but it can take
    several years to develop symptoms.
         The percentage of workers who show irritation to the respiratory tract due to
    exposure to fungal alpha-amylase, is difficult to estimate. The reason for this is
    that allergic symptoms are comparable with the symptoms of irritation. Another
    factor is that it is inevitable that workers in bakeries and flour mills are exposed
    to other compounds as well, such as wheat and rye flour dust, and other
    additives, which are known to cause respiratory irritation (and sensitisation) as
    well. Based on a few studies in which also investigations were performed on
    specific sensitisation, it is estimated that a considerable proportion of the workers
    with respiratory symptoms are due to irritation only.
         Regarding sensitisation to fungal alpha-amylase, prevalence values of up to
    30% have been reported among workers in bakeries and flour mills. Also in the
    enzyme producing industry, workers were found to be sensitised to fungal alpha-
    amylase (3-31%). In the general population, prevalence values up to 1.7% were
    found.
         In a few epidemiological studies, investigators put effort in assessing
    exposure-response relationships between occupational exposure to fungal alpha-
    amylase and specific sensitisation and respiratory symptoms. The lowest
    exposure levels reported on were less than 1 ng alpha-amylase/m3. In two studies
    (Nieuwenhuijsen et al. 1999, and Houba et al. 1996) statistically significant
    positive trends were observed with increased exposure. None of the two studies
    showed an exposure level below which no signs of sensitisation or respiratory
    symptoms were observed.
         The number of animal studies on the toxicity of fungal alpha-amylase is very
    limited. In one study using guinea pigs and mouse, the allergenic potential of
    fungal alpha-amylase was confirmed.
 8  Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre> hapter 8
        Existing guidelines, standards and
        evaluation
8.1     General population
        Not available.
8.2     Occupational population
        No occupational exposure limits have been established for fungal alpha-amylase
        in The Netherlands or in other countries.
        Existing guidelines, standards and evaluation                                  49
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<pre>0 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)</pre>

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<pre> hapter 9
        Hazard assessment
        The committee limited this evaluation to alpha-amylase derived from Aspergillus
        oryzae.
9.1     Hazard identification
        Available epidemiological studies have shown that the main health effects of
        exposure to airborne fungal alpha-amylase are symptoms observed in the
        respiratory tract and the eyes, such as rhinitis, asthma (baker’s asthma), and
        conjunctivitis. Occasionally, upon skin contact, also dermatitis is recorded. Part
        of the symptoms have been shown to be of allergic origin, mediated by
        immunoglobulin E (IgE) antibodies to fungal alpha-amylase. However, the
        respiratory symptoms may also be explained by non-allergic irritation responses,
        as is shown in some studies among bakery workers and apprentices. The allergic
        potency of fungal alpha-amylase is confirmed in animals.
            No relevant human and animal data were available on other possible adverse
        health effects, nor were there data presented on the carcinogenic potential and
        reproduction toxicity.
        In bakeries and flour mills it is inevitable that workers are simultaneously
        exposed to dust of other compounds, such as cereal flour dust, and other
        additives. In particular cereal flour dust may induce respiratory symptoms which
        are indistinguishable from the symptoms caused by alpha-amylase exposure.2
        Hazard assessment                                                                  51
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<pre>  Also cereal flour dust contains allergens that may induce IgE mediated immune
  responses and thus sensitisation. However, tests are available (skin prick tests
  and IgE-based immunoassays) that can distinguish between sensitisation to
  alpha-amylase and other allergens. Furthermore, data do not indicate that persons
  who are sensitised to cereal flour allergens will also be sensitised to alpha-
  amylase without previous exposure to the enzyme (cross-reactivity). Overall, in
  assessing a health-based occupational exposure limit for fungal alpha-amylase,
  the committee prefers to use the data on sensitisation to the enzyme as critical
  endpoint, instead of allergic symptoms. In addition, another reason in using data
  on sensitisation rather than data on allergic symptoms is that sensitisation often
  precedes the onset of allergic symptoms, although sensitisation per se is not an
  illness.3 Since most persons who are sensitised will develop allergic symptoms at
  continuing exposure, the committee considers sensitisation a crucial step in
  developing an allergy: by preventing sensitisation also allergic symptoms will be
  prevented, whereas the other way round might not be the case.
  Overall, the prevalence of sensitisation to fungal alpha-amylase among workers
  reached up to 19% in bakeries and flour mills, and up to 31% in the enzyme
  producing factories. The variations within an occupational group is explained by
  differences in duration, frequency, the level of exposure, and the use of different
  protocols in testing sensitisation (variations in origin of alpha-amylase,
  concentrations, and cut-off points). Despite these variations, the studies show a
  clear association between exposure to fungal alpha-amylase and induction of
  sensitisation. In the background population, which is not occupationally exposed
  to the enzyme, the highest prevalence of sensitisation to alpha-amylase was
  found to be 1.7%.
  A few investigators assessed relationships between exposure levels and
  sensitisation to fungal alpha-amylase. Of most interest are the studies in which
  exposure was determined by measuring airborne fungal alpha-amylase using an
  immunoassay, instead of airborne dust levels. The reason being that the latter
  exposure parameter is less reliable, because measurements have shown a high
  variability of alpha-amylase content in dust.
      Focussing on airborne alpha-amylase levels and specific sensitisation, in two
  studies a statistically significant increase in number of sensitised workers was
  observed with increasing exposure (Nieuwenhuijsen et al. 1999; Houba et al.
  1996; see Table 4 in Chapter 7).14,22 Also other studies showed positive trends,
  but the relationships were weak and did not reach statistical significance
  (Brisman et al. 2004; Cullinan et al. 2001; Droste et al. 2003; Page et al.
2 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>    2010).28,53,56,57 The committee noted that in the studies by Brisman et al. (2004)
    and Cullinan et al. (2001) the same group of workers participated as in
    Nieuwenhuijsen et al. (1999) (data from a same cohort).
        The committee is aware that the results could have been influenced by bias
    and confounding which may have resulted in an underestimation or
    overestimation of the findings. For instance, in some studies potential sources of
    bias could not be ruled out, such as the healthy worker effect (the working
    population appears be more healthy, because workers who are ill have left the
    workplace and are not taken into account). This may explain why in higher
    exposure groups respiratory symptoms levelled off.14,22,44 Other bias are the
    presence of atopic workers (considered more vulnerable in developing a specific
    allergy), and missing data of an individual person on exposure in the past.
    Furthermore, exposure-response relationships are expressed as mean exposure
    levels during a full work-shift (8 hour time weighed average concentrations).
    However, it is reasonable to believe that peak exposure (high exposures in a short
    time (minutes)) may have occurred.16,23,35 Peak exposure is strongly related to
    short work activities, and plays a role in inducing sensitisation and work-related
    symptoms. As such it may have influenced the exposure-response relationships.
    On the other hand, no data are available to the committee on the influence of the
    height and frequency of these peak exposures on the exposure-response
    relationships. Consequently, the committee is not able to take this confounding
    quantitatively into account in assessing a health-based occupational exposure
    limit.
        Overall, despite the presence of bias, confounding, variations in study design,
    exposure monitoring and sensitisation tests, the outcomes of the studies support
    the suggestion that with increasing exposure the number of sensitised persons
    increases as well.
9.2 Selection of the study suitable for risk estimation
    Of interest in selecting a suitable study are the two cross-sectional studies, one
    performed in the United Kingdom and the other in the Netherlands, which
    showed statistically significant increases in prevalence to sensitisation with
    increasing exposure to fungal alpha-amylase: Nieuwenhuijsen et al. (1999), and
    Houba et al. (1996); see Table 4 and Annex G for study details).14,22
        There are many similarities between the studies with respect to: design
    (cross-sectional); exposure monitoring (sandwich-immunoassay; performed in
    the same laboratory); sensitisation testing (skin prick test for fungal alpha-
    amylase); division into groups (three exposure groups based on highest exposure
    Hazard assessment                                                                   53
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<pre>    ever worked in); and occupational group (bakery workers). Difference include:
    design (the Nieuwenhuijsen-study was part of a cohort study); the number of
    participating workers (264 and 178 workers in the Nieuwenhuijsen-study and
    Houba-study, respectively); occupational groups (the Nieuwenhuijsen-study also
    included workers from flour mills and a flour packing factory); mean duration of
    employment (Nieuwenhuijsen-study, 28 months (range: 1 month to maximal 4
    years); Houba-study, 10.2 ± 8.7 years (range 0.2 - 43 years)); and sensitisation
    testing (Houba et al. also used a serum specific IgE immune-assay). The
    committee noted the disproportional group sizes in the Nieuwenhuijsen-study, in
    that the majority of the workers were assigned in the low-exposure group. This
    phenomenon was however also noted in the Houba-study, although to a lesser
    extent. Overall, the committee concludes that there are many similarities
    between the two studies and that any difference in outcome are most likely
    explained by statistical variation due to differences in random test effects.
        Concerning the Nieuwenhuijsen-study, as is mentioned earlier, data are
    obtained from a cohort study on which also other investigators reported on
    exposure-response relationships (Brisman et al. 2004; Cullinan et al. 2001).53,57
    Using data from the same participants as Nieuwenhuijsen et al. (1999), Brisman
    et al. (2004) found trends of increasing sensitisation to fungal alpha-amylase
    with increasing exposure, but this did not reach statistical significance. However,
    the prevalence of chest symptoms statistically significantly increased with
    increasing exposure. Also Cullinan et al. (2001) observed positive trends for
    sensitisation and chest symptoms (using total dust inhalable dust as exposure
    measure). Overall, the findings by Brisman et al. (2004) and Cullinan et al.
    (2001) are in line with those by Nieuwenhuijsen et al. (1999).
        Overall, the committee has no preference for the Houba- or the
    Nieuwenhuijsen-study, and thus decided to use them both as point of departure in
    deriving a value.
9.3 Quantitative hazard assessment
    In none of the two studies a clear threshold level was observed, below which no
    cases of sensitisation to fungal alpha-amylase were found. In addition, in the
    general (not occupationally exposed) population already cases of sensitisation to
    fungal alpha-amylase have been reported. This means that an exposure level, at
    which sensitisation to airborne fungal alpha-amylase will not occur, cannot be
    identified; thus no threshold-based occupational exposure level can be attained.
    Earlier, the Health Council reported on this issue.3 The council concluded that in
    theory a threshold level exists for allergic sensitisation by inhaled allergens. This
 4  Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>    implies that a health-based recommended occupational exposure limit can be
    calculated, using the same procedures and methods as those for other non-
    carcinogenic substances. However, the council emphasized that for most
    allergens, in practice it will not be possible to calculate a reliable health-based
    recommended occupational exposure limit. The reason being that the threshold
    level will be too low to discern using the techniques presently available. For
    those allergens, the Health Council proposed an alternative approach, involving
    determination of reference values, i.e. concentration levels that correspond to
    predefined accepted levels of extra risk of allergic sensitisation. In addition, the
    Ministry of Social Affairs and Employment has requested the council to base a
    reference value on an additional sensitisation risk to fungal alpha-amylase of 1
    percent due to occupational exposure, compared to the background risk in the
    general population (zero exposure). In the case of fungal alpha-amylase such a
    reference value is derived.
    Since the Houba and Nieuwenhuijsen-studies have many similarities, the
    committee decided to combine their data and to perform a meta-regression
    analysis. By doing so, the power of the statistical analyses increases, improving
    the precision of the estimated effect. Details on the meta-regression analysis are
    shown in Annex G. Using a linear regression model, the committee derived an
    exposure level of fungal alpha-amylase of 0.9 ng enzyme/m3, which corresponds
    to an additional sensitisation risk of 1% compared to the background
    sensitisation rate.
        The committee discussed whether the reference value should be adjusted for
    inter-individual differences in vulnerability among humans. In case of
    developing allergies, a group of vulnerable people are the atopics. Since atopics
    were included in the study populations, no adjustments are needed.
    The available literature does not suggest that non-allergic symptoms occur at
    lower exposure levels than allergic symptoms. Therefore, the committee is of the
    opinion that a risk assessment based on sensitisation not only protects against
    allergic symptoms, but most likely against non-allergic symptoms as well.
9.4 Recommendation for a health-based reference value
    The committee recommends a reference value for occupational exposure to
    fungal alpha-amylase of 0.9 ng enzyme/m3, as an eight-hour time-weighted
    average concentration. At this concentration workers have an additional
    Hazard assessment                                                                    55
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<pre>    sensitisation risk for fungal alpha-amylase of 1 percent compared to the
    background risk in the general (not exposed) population.
9.5 Groups at extra risk
    Three groups of workers have or may have an increased risk when exposed to
    fungal alpha-amylase. First, workers sensitised to fungal alpha-amylase will
    likely develop symptoms after repeated exposure to low levels of the enzyme.
    Second, workers with an atopic status or an allergic constitution may have an
    increased risk to develop work-related allergic sensitisation and subsequently
    respiratory symptoms and lung function changes. Third, workers with pre-
    existing asthma or those with more general respiratory symptoms may also have
    an increased risk to develop symptoms most likely because of non-specific
    irritation.
 6  Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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8 Sarlo K, Fletcher ER, Gaines WG, Ritz HL. Respiratory allergenicity of detergent enzymes in the
  Guinea Pig Intratracheal Test: Association with sensitization of occupationally exposed individuals.
  Fundamental and Applied Toxicology 1997; 39(1)
9 Gautrin D, Ghezzo H, Infante-Rivard C, Malo J-L. Incidence and determinants of IgE-mediated
  sensitization in apprentices. American Journal of Respiratory and Critical Care Medicine 2000;
  162(4): 1222-1228.
0 Gautrin D, Ghezzo H, Infante-Rivard C, Malo JL. Incidence and host determinants of work-related
  rhinoconjunctivitis in apprentice pastry-makers. Allergy 2002; 57(10): 913-918.
1 Zotti R de, Bovenzi M. Prospective study of work related respiratory symptoms in trainee bakers.
  Occup Environ Med 2000; 57(1): 58-61.
2 Storaas T, Irgens A, Florvaag E, Steinsvag SK, Ardal L, Van Do T et al. Bronchial responsiveness in
  bakery workers: relation to airway symptoms, IgE sensitization, nasal indices of inflammation, flour
  dust exposure and smoking. Clin Physiol Funct Imaging 2007; 27(5): 327-334.
3 Storaas T, Ardal L, Van Do T, Florvaag E, Steinsvag SK, Irgens A et al. Nasal indices of eosinophilic
  and exudative inflammation in bakery-workers. Clin Physiol Funct Imaging 2007; 27(1): 23-29.
4 Baur X, Fruhmann G, Haug B, Rasche B, Reiher W, Weiss W. Role of Aspergillus amylase in baker's
  asthma. The Lancet 1986; 327(8471): 43.
5 Flindt MLH. Allergy to alpha-amylase and papain. Lancet 1979; 314(8131): 1407-1408.
2 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
</pre>

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<pre>6 Quirce S, Cuevas M, Díez-Gómez M, Fernández-Rivas M, Hinojosa M, González et al. Respiratory
  allergy to Aspergillus-derived enzymes in bakers' asthma. J Allergy Clin Immunol 1992; 90(6): 970-
  978.
7 Blanco Carmona JG, Juste PS, Garces SM. Occupational asthma in bakeries caused by sensitivity to
  alpha-amylase. Allergy 1991; 46(4): 274-276.
8 Birnbaum J, Latil F, Vervloet D, Senft M, Charpin J. Rôle de l'alpha-amylase dans l'asthme du
  boulanger. Rev Mal Respir 1988; 5(5): 519-521.
9 Houba R, Heederik D, Kromhout H. Grouping strategies for exposure to inhalable dust, wheat
  allergens and alpha-amylase allergens in bakeries. Annals of Occupational Hygiene 1997; 41(3):
  287-296.
0 Droste J, Vermeire P, Van SM, Bulat P, Braeckman L, Myny K et al. Occupational exposure among
  bakery workers: impact on the occurrence of work-related symptoms as compared with allergic
  characteristics. J Occup Environ Med 2005; 47(5): 458-465.
  References                                                                                         63
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<pre>4 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)</pre>

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<pre>A Request for advice
B The Committee
C The submission letter (in English)
D Comments on the public review draft
E Prevalence of sensitisation to alpha-amylase and of respiratory
  symptoms
F Exposure-response relationships
G Meta-regression analysis
  Annexes
                                                                  65
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<pre>6 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)</pre>

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<pre>nnex A
     Request for advice
     In a letter dated October 11, 1993, ref DGA/G/TOS/93/07732A, to, the State
     Secretary of Welfare, Health and Cultural Affairs, the Minister of Social Affairs
     and Employment wrote:
     Some time ago a policy proposal has been formulated, as part of the simplification of the
     governmental advisory structure, to improve the integration of the development of recommendations
     for health based occupation standards and the development of comparable standards for the general
     population. A consequence of this policy proposal is the initiative to transfer the activities of the
     Dutch Expert Committee on Occupational Standards (DECOS) to the Health Council. DECOS has
     been established by ministerial decree of 2 June 1976. Its primary task is to recommend health based
     occupational exposure limits as the first step in the process of establishing Maximal Accepted
     Concentrations (MAC-values) for substances at the work place.
     In an addendum, the Minister detailed his request to the Health Council as
     follows:
     The Health Council should advice the Minister of Social Affairs and Employment on the hygienic
     aspects of his policy to protect workers against exposure to chemicals. Primarily, the Council should
     report on health based recommended exposure limits as a basis for (regulatory) exposure limits for air
     quality at the work place. This implies:
     •    A scientific evaluation of all relevant data on the health effects of exposure to substances using a
          criteria-document that will be made available to the Health Council as part of a specific request
     Request for advice                                                                                        67
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<pre>      for advice. If possible this evaluation should lead to a health based recommended exposure limit,
      or, in the case of genotoxic carcinogens, a ‘exposure versus tumour incidence range’ and a
      calculated concentration in air corresponding with reference tumour incidences of 10-4 and 10-6
      per year.
  •   The evaluation of documents review the basis of occupational exposure limits that have been
      recently established in other countries.
  •   Recommending classifications for substances as part of the occupational hygiene policy of the
      government. In any case this regards the list of carcinogenic substances, for which the
      classification criteria of the Directive of the European Communities of 27 June 1967 (67/548/
      EEG) are used.
  •   Reporting on other subjects that will be specified at a later date.
  In his letter of 14 December 1993, ref U 6102/WP/MK/459, to the Minister of
  Social Affairs and Employment the President of the Health Council agreed to
  establish DECOS as a Committee of the Health Council. The membership of the
  Committee is given in Annex B.
8 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>nnex B
     The Committee
     •  R.A. Woutersen, chairman
        Toxicologic Pathologist, TNO Innovation for Life, Zeist; and professor of
        translational toxicology, Wageningen University and Research Centre,
        Wageningen
     •  P.J. Boogaard
        Toxicologist, Shell International BV, The Hague
     •  D.J.J. Heederik
        Professor in Risk Assessment in Occupational Epidemiology, Institute for
        Risk Assessment Sciences, Utrecht University, Utrecht
     •  R. Houba
        Occupational Hygienist, Netherlands Expertise Centre for Occupational
        Respiratory Disorders, Utrecht
     •  H. van Loveren
        Professor in Immunotoxicology, Maastricht University, Maastricht; and
        National Institute for Public Health and the Environment, Bilthoven
     •  T.M. Pal
        Occupational Physician; Netherlands Centre for Occupational Diseases,
        University of Amsterdam, Amsterdam
     •  A.H. Piersma
        Professor in Reproductive Toxicology, Utrecht University, Utrecht; and
        National Institute for Public Health and the Environment, Bilthoven
     The Committee                                                                69
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<pre>  •   H.P.J. te Riele
      Professor in Molecular Biology, VU University Amsterdam; and the
      Netherlands Cancer Institute, Amsterdam
  •   I.M.C.M. Rietjens
      Professor in Toxicology, Wageningen University and Research Centre,
      Wageningen
  •   F.G.M. Russel
      Professor in Molecular Pharmacology and Toxicology, Radboud University,
      Nijmegen Medical Centre, Nijmegen
  •   G.M.H. Swaen
      Eepidemiologist, Maastricht University, Maastricht
  •   R.C.H. Vermeulen
      Epidemiologist, Institute for Risk Assessment Sciences, Utrecht University,
      Utrecht
  •   P.B. Wulp
      Occupational Physician, Labour Inspectorate, Groningen
  •   J.J.A.M. Hendrix, advisor
      Social and Economic Council, The Hague
  •   J.M. Rijnkels, scientific secretary
      Health Council of the Netherlands, The Hague
  The Health Council and interests
  Members of Health Council Committees are appointed in a personal capacity
  because of their special expertise in the matters to be addressed. Nonetheless, it
  is precisely because of this expertise that they may also have interests. This in
  itself does not necessarily present an obstacle for membership of a Health
  Council Committee. Transparency regarding possible conflicts of interest is
  nonetheless important, both for the chairperson and members of a Committee
  and for the President of the Health Council. On being invited to join a
  Committee, members are asked to submit a form detailing the functions they
  hold and any other material and immaterial interests which could be relevant for
  the Committee’s work. It is the responsibility of the President of the Health
  Council to assess whether the interests indicated constitute grounds for non-
  appointment. An advisorship will then sometimes make it possible to exploit the
  expertise of the specialist involved. During the inaugural meeting the
  declarations issued are discussed, so that all members of the Committee are
  aware of each other’s possible interests.
0 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>nnex C
     The submission letter (in English)
     Subject          : Submission of the advisory report Fungal alpha-amylase
     Your Reference: DGV/MBO/U-932342
     Our reference : U-8232/JR/cn/459-S70
     Enclosed         :1
     Date             : November 6, 2014
     Dear Minister,
     I hereby submit the advisory report on the effects of occupational exposure to
     fungal alpha amylase.
     In the advisory report Prevention of work-related airway allergie (report No.
     2008/03), the Health Council proposed a method to derive health-based
     occupational exposure limits, or on risk-based reference values for allergenic
     substances. The present advisory report on fungal alpha-amylase makes use of
     this method. The Health Council has calculated the concentration of fungal
     alpha-amylase in the air, at which occupational exposure leads to an additional
     sensitisation risk of 1%, compared to the background risk in the non-exposed,
     general population.
     The submission letter (in English)                                              71
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<pre>      The conclusions in the advisory report were drawn by the Health Council’s
  Dutch Expert Committee on Occupational Safety (DECOS). The subcommittee
  has taken comments into account from a public review, and included the opinions
  by the Health Council’s Standing Committee on Health and the Environment.
  I have today sent copies of this advisory report to the State Secretary of
  Infrastructure and the Environment and to the Minister of Health, Welfare and
  Sport, for their consideration.
  Yours sincerely,
  (signed)
  Prof. J.L. Severens,
  Vice-President
2 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in 2014 for public review. The
     following persons and organisations have commented on the draft review:
     • W. Eduard, National Institute of Occupational Health, Norway
     • J. Lentz and A. Nayak, National Institute for Occupational Safety and Health,
         the USA.
     Comments on the public review draft                                             73
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<pre> nnex        E
             Prevalence of sensitisation
             to alpha-amylase and of
             respiratory symptoms
             Prospective cohort studies
 tudy design           Exposure information Health information              Results                        Reference
nd population
nformation
87 apprentice bakers, No exposure data      Medical examination and         Specific sensitisation to      Walusiak et al.
xamined in the 1st, presented.              physician-administered          fungal alpha-amylase           200461
nd and 3rd year of                          questionnaire on history of • First year: all negative.
heir training, Poland.                      allergic diseases, atopy,       • Third year: 31/287 persons
                                            smoking, exposure to pet           (10.8%).
                                            allergens, and housing
                                            conditions. The incidence of In the third year 51/287 cases
                                            work-related respiratory        showed sensitisation to
                                            symptoms was calculated         occupational allergens. Of
                                            separately for each follow-up these positive cases:
                                            year and cumulative; skin       • for 16 cases the presence
                                            prick tests to common and          of specific IgE was the
                                            cereal allergens.                  only marker of
                                            In skin prick test positive        occupational sensitisation;
                                            students and in 20 random       • in 27 cases, development
                                            subjects in the 1st and 2nd        of occupational
                                            year, and all students in 3rd      hypersensitivity was
                                            year also a skin prick test was    preceded by skin reactivity
                                            preformed with IgE                 to common allergen at an
                                            antibodies to fungal alpha-        earlier stage.
                                            amylase (cut-off point, ≥0.35
                                            kU/L).                          Background prevalence of
                                                                            sensitisation (new bakery
                                            Nasal provocation tests to      apprentices): 0% (0/287).
             Prevalence of sensitisation to alpha-amylase and of respiratory symptoms                                      75
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<pre>                                               cereal flours. Pulmonary
                                               function tests examination of
                                               persons reporting chest
                                               symptoms.
 30 trainee pastry       No exposure data      Skin prick test to fungal      No positive cases found at the Gautrin et al. 199760,
 akers; Canada;          presented.            alpha-amylase; questionnaire beginning of the study, and 200079, 200280
ollow-up 8 and 16                              on allergy history, lung       after 8 (n = 189), and 16
months after start of                          function tests. Study included months (n = 138) of training.
raining.                                       base-line examination.
                                                                              The low incidence is
                                                                              explained by the finding that
                                                                              trainees rarely handled
                                                                              enzymatic additives and flour
                                                                              containing these additives.
                                                                              The prevalence of atopy (skin
                                                                              prick test to 11 common
                                                                              inhalants) was 54.5%. During
                                                                              the study, 8/186 subjects were
                                                                              positive for flour-deriveD
                                                                              allergen sensitisation.
                                                                              Background prevalence of
                                                                              sensitisation (new bakery
                                                                              apprentices): 0% (0/230).
 25 trainee bakers;      No exposure data      Skin prick tests to fungal     After 30 months:               De Zotti and
taly; follow-up was at presented.              alpha-amylase and to wheat • 3/125 positive for fungal Bovenzi, 200081
 , 18 and 30 months                            flour allergens. Study             alpha-amylase;
 fter start of training.                       included base-line             • 10/125 positive for wheat
                                               examination.                       flour allergens. All
                                                                                  students sensitised for
                                                                                  alpha-amylase were also
                                                                                  sensitised for wheat flour
                                                                                  allergens.
                                                                              • 10/125 showed work-
                                                                                  related respiratory
                                                                                  symptoms.
                Cross-sectional studies
Study design             Exposure information        Health information           Results                    Reference
and population
information
Exposure levels expressed as ng fungal alpha-amylase/m3
517 employees of 31 Full-shift personal airborne Self-administered                Overall prevalence of      Baatjies et al 200936
supermarket bakeries dust was sampled in 18          questionnaire (n=517) on     sensitisation to fungal
in South-Africa.         bakeries on 2 days (n=211). respiratory symptoms,        alpha-amylase.             Exposure measure-
                         Analysis for total mass and employment history and       Specific IgE               ments:
                         flour dust fungal alpha-    job title, degrees of        • all workers: 4% (21/     Baatjies et al. 201030
                         amylase by ELISA-           exposure to flour dust,          513)
                         immunoassay.                baking activities at home    • atopic: 7% (15/213)
                                                     and smoking habits.          • nonatopic: 2% (6/294)
  6             Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>       Inhalable dust levels        Skin prick tests (n=507) to Difference between atopic
       (GM±SD):                     common and work-related     and nonatopic were not
       • bread baker (n=112):       allergens, including fungal significant.
           1.33±2.25 mg/m3;         alpha-amylase.              Skin prick test
       • confectioner (n=38):                                   • all workers: 3% (17/
           0.65±2.08 mg/m3;                                         513)
       • supervisor (n=13):                                     • atopic: 6% (13/213)
           0.56±2.05 mg/m3;         Fungal alpha-amylase        • non-atopic: 1% (4/294)
       • manager (n=13):            specific serum IgE was
           0.51±2.34 mg/m3;         measured by fluorescence    Atopy prevalence among
       • counterhand (serving       enzyme immunoassay          all workers was 42%.
           customers, n=35):        (n=513). A result >0.35
           0.28±1.89 mg/m3.         kU/L was considered         The authors did not present
                                    positive.                   job-title (or exposure)
       Exposure to fungal alpha-                                specific sensitisation
       amylase (GM±SD) (range): Pulmonary function testing prevalence values.
       • bread baker: 0.15±2.32 (spirometry and
           ng/m3 (0.083-19.62); methacholine challenge, Of all workers, 22%
       • confectioner: 0.12±2.14 n=517).                        showed bronchial
           ng/m3 (0.083-6.54);                                  hyperresponsiveness.
       • supervisor: 0.10±1.17
           ng/m3 (0.083-1.17);                                  A relationship between
       • manager: 0.12±1.58 ng/                                 sensitisation to fungal
           m3 (0.083-0.51);                                     alpha-amylase and lung
       • counterhand (serving                                   function was not examined.
           customers): 0.11±1.40
           ng/m3 (0.083-0.64).
       Notes:
       (1) There was no
       association between job-
       title and exposure to fungal
       alpha-amylase (GM) in this
       study, but weighing tasks
       (p=0.057) and use of
       premix products (p=0.010)
       were associated with
       increased exposure to the
       enzyme.
       (2) There was a poor
       correlation between alpha-
       amylase concentration and
       inhalable dust (Pearson
       r=0.33, p<0.001).
       (3) Alpha-amylase
       concentration was below
       detection limit (0.083 ng/
       m3) in 81% of the inhalable
       dust samples, which may
       be explained by the low
       contents of this enzyme in
       premix products: 0.75-100
       ng/mg.
Prevalence of sensitisation to alpha-amylase and of respiratory symptoms                    77
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<pre>225 bakers from 22    Exposure (8-hour TWA) to Interviewer-led                Prevalence of sensitisation Harris-Roberts et al.
bakeries, the UK:     fungal alpha-amylase was questionnaire on work-         to fungal or bacterial alpha-200940
• 40 general bakers   determined for several job related respiratory          amylase (IgE-positive):
• 31 mixers/sievers/  categories. Personal        symptoms, demographic 5.6% (9/160). Prevalence Exposure assess-
   weighers           breathing zone inhalable details, work history and to any enzyme (including ment:
• 7 cleaner jobs      dust samples from 208       smoking habits; lung        enzymes from Aspergillus Elms et al. 200647
• 47 other jobs.      workers in 55 bakeries      function assessment         niger, xylanase, and
                      were analysed for fungal- (n=225).                      hemicellulase): 7.5%
                      amylase (ELISA                                          (12/160).
                      immunoassay). Alpha-        Specific serum IgE (RAST
                      amylase was however not assay) to wheat flour,          Note: the authors did not
                      detectable in a large       enzymes including a         report the prevalence of
                      proportion of the samples. mixture of fungal and        sensitisation to fungal
                      Therefore, below, the       bacterial alpha-amylase, alpha-amylase in each of
                      number enzyme-containing and common allergens           the job categories.
                      samples per total number of (n=160). Workers were
                      samples are listed:         categorized in 4 groups: Abnormal lung function
                      • total (11/171):           • not sensitised;           was more frequent among
                         5.9 ng/m3; <0.78-33.7 • sensitised to wheat flour workers sensitised to
                         (GM and range)              only;                    enzymes but not to wheat
                      • general bakers (5/90), • sensitised to enzymes allergen (odds ratios
                         1.7 ng/m3, <0.78-24.5       only;                    adjusted for job-duration,
                         (GM, range);             • sensitised to wheat flour age, atopy and smoking):
                      • mixers/weighers/sievers and any enzyme.               • abnormal FVC: OR
                         (4/44), <0.78-13.2                                       22.73 (95% CI
                         ng/m3 (range only);      The authors did not             1.91-270.94);
                      • cleaners (1/6), <0.78- separately examine fungal • abnormal FEV1: OR
                         22.0 ng/m3 (range only);alpha-amylase.                   18.92 (95% CI
                      • others (1/31),                                            1.60-223.18).
                         <0.78-33.7 ng/m3 (range
                         only).                                               Atopy was the most
                                                                              important risk factor for
                                                                              sensitisation to workplace
                                                                              allergens (OR 18.4, 5.3-
                                                                              64.3). Correction for atopy
                                                                              was not feasible. Among
                                                                              atopic workers smoking
                                                                              was a strong predictor of
                                                                              sensitisation to wheat or
                                                                              enzymes, corrected for
                                                                              duration of employment
                                                                              and current exposure
                                                                              category (OR 4.7, 1.1-
                                                                              20.8).
239 employees of 20 Full-shift personal           Self-administered           Prevalence of sensitisation Brant et al. 200523
supermarket bakeries, inhalable dust              questionnaire on job-       to fungal alpha-amylase
UK                    concentrations measured classification, job history (specific serum IgE):
                      (IOM sampler; n=89).        and respiratory symptoms • bakers: 8% (5/66)
                                                  and current smoking status • managers: 11% (3/28)
                      Alpha-amylase content in (n=239). Skin prick test to • confectioners: 0%
                      dust samples was analysed common allergens (n=233)          (0/45)
                      by an ELISA                 and RAST to fungal alpha- • assistants: 1% (1/71)
                      immunoassay. Only three amylase specific serum IgE • total: 4% (9/210)
 8           Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>                      samples:                    (n=210).                     The relationship between
                      • one baker (3.3 ng/m3)                                  alpha-amylase sensitisation
                      • one manager (12.4         Atopy was evenly spread and job title was not
                         ng/m3)                   across the working groups. statistically significant
                      • one bakery assistant                                   (p=0.19).
                         (3.9 ng/m3).
                      All reported they                                        Prevalence of atopy was
                      sometimes performed                                      41%, comparable with
                      baking tasks.                                            Baatjies et al 2009.2
20 workers in a       Workers were exposed to     Interview by allergologist,  Prevalence of sensitisation Brisman and Belin
factory producing     fungal alpha-amylase        and spirometry before and    to fungal alpha-amylase 199135
bread mixes and       during mixing and packing   after inhalation of          (skin prick test - IgE - IgG,
dough improvers,      (n=16) or in the laboratory salbutamol/ lactose; skin    respectively):
Sweden                (n=4).                      prick tests to common        • all workers:
                      Nine persons working in     allergens, and to work-          6/20 - 1/20 - 3/20;
                      another part of the factory related allergens including  • with symptoms (rhinitis,
                      served as referent group.   fungal alpha-amylase;            dermatitis):
                                                  RAST assay for specific          4/11 - 0/11 - 0/11;
                      Total dust sampling (1      IgE antibodies to fungal     • without symptoms:
                      personal, 1 stationary      alpha-amylase.                   2/9 - 1/9 - 3/9;
                      sampler, during daily task                               • referents (no
                      duration of 30-60 min).     Workers with positive skin       symptoms):
                                                  prick test underwent nasal       1/9 - 1/9 - 0/9.
                      Enzymatically determined challenge tests with the
                      fungal alpha-amylase        fungal alpha amylase.        Symptoms reported in
                      content at packing station:                              prick test positive workers
                      30 µg/m3 (= peak                                         consisted of rhinitis (n=3)
                      exposure).                                               and dermatitis (n=1).
                                                                               Four employees of the
                                                                               same factory had
                                                                               previously been diagnosed
                                                                               for asthma and/or rhinitis.
                                                                               All were positive to fungal
                                                                               alpha-amylase in the skin
                                                                               prick test.
Exposure levels expressed as mg inhalable dust/m3
197 employees of 6 Breathing zone personal Interview focusing on               No data were presented on Storaas et al. 200567,
bakeries, Norway.     dust samplers (n=58).       occupational rhinitis        the different exposure        200782, 200783
                                                  (n=181) and self-            groups.
                      Four exposure groups:       administered questionnaire
                      • <1.0 mg/m3 (packers, on work tasks, family             Overall prevalence of
                         oven workers,a           history, occupational        sensitisation to fungal
                         administration);         symptoms, smoking habits alpha-amylase:
                      • 1.0-1.9 mg/m3 (mainly and prevalence of allergy • skin prick test:
                         confectionary workers, and atopic dermatitis/             7% (13/183)
                         bread formers);          eczema (n=180).              • specific serum IgE:
                      • 2.0-3.9 mg/m3 (mainly                                      2% (4/183)
                         dough makers);           Allergy tests for            Prevalence of sensitisation
                      • >3.9 mg/m3 (mainly        occupational and common to storage mites was
                         dough makers).           allergens (skin prick, total 20% (37/183).
                                                  and specific serum IgE, and
                                                  histamine release; n=183). Occupational rhinitis, IgE
            Prevalence of sensitisation to alpha-amylase and of respiratory symptoms                                        79
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<pre>                                                    Spirometry, bronchial         and non-IgE mediated,
                                                    provocation test with         preceded lower airway
                                                    metacholine, nasal            symptoms, and was
                                                    challenge and lavage.         associated with asthma
                                                                                  symptoms. Storage mite
                                                    Categorisation of workers sensitisation was related to
                                                    in job titles: dough makers, occupational rhinitis and
                                                    bread formers, oven staff, exposure.
                                                    packers, confectionary
                                                    workers, administration       Bronchial
                                                    and cleaning workers.         hyperresponsiveness
                                                                                  (BHR), determined by
                                                                                  metacholine challenge, was
                                                                                  associated with smoking
                                                                                  and work-related asthma.
                                                                                  BHR, corrected for
                                                                                  baseline lung function, was
                                                                                  not associated with
                                                                                  occupational IgE
                                                                                  sensitisation (defined as
                                                                                  positive to wheat, alpha-
                                                                                  amylase, oats, barley, rye,
                                                                                  soybean, storage mites,
                                                                                  mold or cockroach). The
                                                                                  authors concluded that IgE
                                                                                  sensitisation is not the main
                                                                                  causative factor for airway
                                                                                  hyperresponsiveness and
                                                                                  occupational rhinitis in
                                                                                  bakery workers.
                                                                                  BHR was not associated
                                                                                  with current flour dust
                                                                                  exposure level, with
                                                                                  number of working hours
                                                                                  in a bakery, or with a
                                                                                  history of dough-making.
679 employees of 18 Full-shift personal total       Screening by occupational No data were presented on Smith et al. 200059
flour mills, regularly inhalable dust               physician, using structured the separate job categories.
exposed to flour dust measurements between          interview on type, time of
(workers involved in 1990 and 1998 (n=116)          onset and duration of work-Overall prevalence of
milling, production or                              related respiratory           sensitisation to fungal
packing activities), the Exposure to inhalable dust symptoms (n=679). Skin alpha-amylase (skin prick
UK                       was 8.1 mg/m3 (0.5-217; prick testing to common test positive) was 0.9%
                         median, 8-hour TWA).       allergens, to wheat, soya (6/678). Symptoms in the
                                                    and rice flour, and to fungal positive group included
                         Exposure by job category alpha-amylase (n=678).          allergic rhinitis (n=1), non-
                         (GM and range):                                          specific irritation (n=2),
                         • production (n=78):       Mean duration of              and asymptomatic (n=3).
                           6.2 mg/m3 (0.5-54.7); employment was 12.5              Workers sensitised to
                         • hygiene (n=38):          years (2 months - 47 years) wheat flour and alpha-
                           18.7 mg/m3 (1.1-217).                                  amylase were different.
                                                                                  Overall prevalence of atopy
 0            Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>                                                                               was 37% (248/678).
                                                                               Work-related respiratory
                                                                               symptoms were reported by
                                                                               147/679 workers (22%),
                                                                               mostly occasional and
                                                                               transient, which were
                                                                               classified as non-specific
                                                                               irritation.
                                                                               Allergic respiratory
                                                                               symptoms were reported by
                                                                               8/679 workers (1%, 4
                                                                               rhinitis and 4 asthma).
                                                                               Fungal alpha-amylase was
                                                                               thought to be responsible
                                                                               for one of the 4 cases of
                                                                               asthma, because this
                                                                               worker only had symptoms
                                                                               after handling bread
                                                                               improver.
392 employees from Personal sampling of            Structured interview (three No data were presented of Smith and Wastell
19 bread bakeries and respirable dust (workers occupational physicians         the separate exposure        Smith 199865
77 workers from 3      involved in sieving,        with prior agreed criteria groups.
cake bakeries, UK. weighing and/or mixing/ for diagnosis), and skin
Of the 3 large         dough-making) at various prick tests to common          Overall prevalence of
bakeries, one did not times between 1990 and allergens and work-               sensitisation to fungal
use alpha-amylase and 1996.                        related allergens (wheat, alpha-amylase:
had not done so for at The 1990-1996 dust          soya and rice flour, and    • Bread-bakeries: 16%
least 5 years prior to exposure measurements       fungal alpha-amylase).          (63/392);
the study.             were collated (no local     Workers were allocated to • Cake-bakeries:1% (1/
                       exhaust ventilation, 8-hour four categories:                77; in the bakery not
                       TWA, GM±SD and range): • occupational asthma;               using fungal alpha-
                       Bread bakeries:             • occupational rhinitis;        amylase, employee
                       - sieving (n=35): 11.4±73.1 • respiratory irritation;       previously worked 1
                       mg/m3 (0.9-349.5);          • asymptomatic.                 year in a bread bakery).
                       • weighing (n=26):
                           8.2±146.7 mg/m3                                     Overall prevalence of
                           (1.0-770);                                          work-related respiratory
                       • doughmaking (n=80):                                   symptoms:
                           3.3±19.5 mg/m3                                      • Bread-bakeries: 20.4%
                           (0.1-142.2).                                            (80/392, occupational
                       Cake bakeries:                                              asthma, occupational
                       • sieving (n=12): 35.7±26                                   rhinitis or respiratory
                           mg/m3 (15.9-90);                                        irritation);
                       • weighing (n=8):                                       • Cake-bakeries:10.4%
                           19.2±20.7 mg/m3                                         (8/77, only respiratory
                           (7.4-68.5);                                             irritation).
                       • mixing (n=24): 3.8±4.2
                           mg/m3 (0.5-16.3).                                   This study shows that dust
                                                                               exposure in bakeries poorly
                                                                               correlates with alpha-
                                                                               amylase allergenicity. The
                                                                               authors hypothesize that
             Prevalence of sensitisation to alpha-amylase and of respiratory symptoms                                    81
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<pre>                                                                               the low prevalence of
                                                                               alpha-amylase sensitised
                                                                               workers in the cake
                                                                               bakeries is caused by the
                                                                               lower concentration of
                                                                               alpha-amylase in flour, as
                                                                               compared to bread
                                                                               improvers handled in bread
                                                                               bakeries.
Job- or task-based exposure categories
224 workers in 18     Job categories:             Physician-administered       Overal prevalence of        Jeffrey et al. 199966
small bakeries        A - workers handling flour  questionnaire on work-       sensitisation:
(<50 employees),      directly (dough making,     related symptoms, past       • fungal alpha-amylase:
Scotland.             cleaning, weighing,         medical history, smoking         15% (31/205)
                      moulding, mixing);          status and occupational      • wheat flour: 24%
                      B - workers exposed from    history (n=224). Serum           (49/205)
                      general contamination of    IgE, specific to common • rye flour: 16% (33/205)
                      spaces.                     and bakery allergens,        • common environmental
                                                  including fungal alpha-          allergens: 34% (71/205)
                      Full-shift personal         amylase, was measured
                      sampling of total inhalable using RAST (threshold for No separate data presented
                      and respirable dust (n=144, positive sera was defined as for categories A and B
                      8-hour TWA in mg/m3): mean plus 2.5 standard             regarding sensitisation to
                      • A: 6.7±5.3 (AM±SD) deviations of the                   alpha-amylase.
                      • B: 1.5±1.5 (AM±SD) background level,
                      • A: 4.9±2.3 (GM±SD) established in workers in an Of the sensitised
                      • B: 1.0±2.7 (GM±SD) electronic factory) (n=205).individuals, 18 reported
                                                                               work-related respiratory
                                                                               symptoms, whereas 43.7%
                                                                               of all the workers reported
                                                                               work-related symptoms.
                                                                               There was a statistically
                                                                               significant association
                                                                               between reported work-
                                                                               related symptoms and IgE
                                                                               to alpha-amylase
                                                                               (p<0.001), to wheat flour
                                                                               (p<0.001), and to common
                                                                               allergens (p=0.03).
94 workers in an      Categories:                 Self-administered            Prevalence of sensitisation Vanhanen et al.
enzyme research       • not/rarely (working in questionnaire on work           to fungal alpha-amylase: 199755
laboratory, and 79       laboratories and office history, history of atopy, • not/rarely exposed: 1/59
workers in an enzyme     staff, n=59);            smoking and work-related • occasionally exposed:
producing plant,      • occasionally (handling symptoms; skin prick tests          1/51
Finland.                 mainly liquid enzymes, to common allergens, and • often exposed: 4/63
                         n=51);                   to a series of enzymes,      Most employees sensitised
Enzymes produced: • often (handling mainly among which fungal alpha- to fungal alpha-amylase
cellulase, xylanase,     dry enzymes, n=63).      amylase; RAST with           (5/6) were also atopic.
bacterial alpha-                                  specific IgE antibodies      Only one had symptoms
amylase, and fungal                               against enzymes (used if during work.
alpha-amylase.                                    person was skin prick
                                                  positive to an enzyme). IgG Note 1: The study was
                                                  antibodies to fungal alpha- performed 1.5 yr after the
 2           Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>                                                amylase by ELISA using      appearance of respiratory
                                                conjugated rabbit           symptoms, after which
                                                antihuman IgG.              hygienic measures were
                                                                            taken.
                                                                            Note 2: Workers were
                                                                            exposed to a mixture of
                                                                            enzymes, including
                                                                            bacterial and fungal alpha-
                                                                            amylase.
3,450 workers in a   Categories:                Questionnaire and, in       Prevalence of occupational Smith and Lumley
large food company, • flour milling             selected workers, skin      asthma:                       19967
UK.                     (n=528);                prick tests to workplace    • flour milling:
                     • bread baking             allergens (wheat bran and       0.2% (1/528)
                        (n=1,756);              flour, fungal alpha-        • bread baking:
                     • cake and pastry baking amylase, and hen egg), and        0.6% (10/1756)
                        (n=209);                to common allergens.        • cake baking:
                     • other activities         Differentiation between         0% (0/209)
                        (n=957).                respiratory sensitisation, • other activities:
                                                and non-specific irritation     0.1% (1/957)
                                                based on pattern of
                                                symptoms in relation to     Fungal alpha-amylase was
                                                exposure.                   responsible for 10 cases of
                                                                            sensitisation, all associated
                                                                            with bread baking. Also, all
                                                                            these cases were diagnosed
                                                                            as having occupational
                                                                            asthma (based on work
                                                                            history, and strongly
                                                                            positive prick test).
83 workers in        Categories (based on       Questionnaire on work-      Prevalence of sensitisation Losada et al. 199250
pharmaceutical       questionnaire):            related symptoms and        to fungal alpha-amylase
industry, exposed to • low (intermittent and history of allergy (n=80). (skin prick test):
fungal alpha-amylase    occasional, n=15);      Skin prick tests to common • total: 26/83 (31.3%)
powder, Spain.       • intermediate             allergens, and fungal       • low: 2/15
                        (intermittent and       alpha-amylase (n=83).       • intermediate: 6/27
                        frequent, n=27);        Serum alpha-amylase-        • intense: 18/38
                     • intense (constant,       specific IgE was
                        n=38).                  determined by a reverse     Results questionnaire:
                                                enzyme immunoassay          • nasal symptoms: 47/80
                     No data on exposure levels (positive at ≥150 OD =          (suggestive for allergic
                     presented.                 control + 3xSD).                rhinitis
                                                Bronchial and nasal         • bronchial asthma: 24/47
                                                provocation tests in
                                                symptomatic subjects. Oral Oral challenge of 5
                                                provocation in 5 sensitised sensitised persons
                                                subjects.                   provoked skin and
                                                                            bronchial symptoms in one
                                                Average employment time worker. This person
                                                was 9.5 years.              experienced no symptoms
                                                                            when eating bread.
             Prevalence of sensitisation to alpha-amylase and of respiratory symptoms                                     83
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<pre>No data on exposure levels or job/task categories
89 bakery workers, No exposure data               Skin prick tests to common Prevalence of sensitisation Baur et al. 199864
104 persons with       presented.                 and bakery allergens,         to fungal alpha-amylase.
bakers’ asthma, and                               including fungal alpha-
43 control subjects                               amylase. Specific IgE         Skin prick test positive:
(not working in a                                 antibodies to fungal alpha- • bakery workers:
bakery), Germany.                                 amylase (n=169) were              19% (17/88)
                                                  determined enzymatically • asthmatics: 24%
                                                  (enzyme-allergo-sorbent-          (25/104)
                                                  test). Lung function testing, • control subjects:
                                                  including challenge testing       2% (1/43)
                                                  with methacholine and         • symptomatic bakers:
                                                  alpha-amylase.                    27% (39/142)
                                                                                • asymptomatic bakers:
                                                                                    5% (2/44)
                                                                                Specific IgE positive:
                                                                                • bakery workers:
                                                                                    19% (17/89)
                                                                                • asthmatics:
                                                                                    12% (10/81)
                                                                                • control subjects:
                                                                                    10% (4/41)
                                                                                • symptomatic bakers:
                                                                                    21% (25/119)
                                                                                • asymptomatic bakers:
                                                                                    4% (2/45)
385 workers in 19      No exposure data           Interview on work-related Prevalence of sensitisation Smith et al. 199744
bakeries, currently    presented.                 symptoms by physician, to fungal alpha-amylase:
exposed to dust from                              and skin prick tests to       • all workers:
bread improver, flour                             common and work-related           16% (62/383)
and other ingredients,                            allergens, including fungal • atopics: 31% (40/132)
383 participating in                              alpha-amylase. Based on • non-atopics: 9% (22/
the study, the UK.                                these, workers were               257)
                                                  allocated to the following
                                                  groups:                       Prevalence for diagnostic
                                                  • occupational asthma         categories:
                                                      (alone or in combination • asthma: 100% (2/2)
                                                      with rhinitis);           • rhinitis: 100% (10/10)
                                                  • occupational rhinitis; • respiratory irritation:
                                                  • respiratory irritation          27% (18/66)
                                                      (non-specific);           • asymptomatic:
                                                  • asymptomatic (= no              10% (32/305)
                                                      work-related
                                                      symptoms).                The authors explain the low
                                                                                prevalence of asthmatics
                                                                                among this population by
                                                                                healthy worker effect.
36 workers in an       No exposure data           Self-administered             Results for alpha-amylase Biagini et al. 199669
enzyme producing       presented.                 questionnaires on medical from A. oryzae. Skin prick
factory, the USA.                                 history, and presence of      test positive: 2/36 (6%, non
                                                  symptoms; skin prick test significant increase
 4           Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>Enzymes produced                              and Specific IgE to       compared to controls).
included: alpha-                              measure sensitisation;
amylase from A.                               pulmonary lung function   Authors reported that
oryzae, B.                                    testing.                  specific IgE antibodies to
licheniformis, or B.                                                    alpha-amylase from A.
subtilis formulations;                                                  oryzae was significantly
alkaline proteases;                                                     higher among exposed
amylo-glucosidases.                                                     workers than among non-
                                                                        exposed controls.
                                                                        In general, the most
                                                                        commonly reported
                                                                        symptoms were: itchy,
                                                                        watery eyes (36%),
                                                                        sneezing (33%), chest
                                                                        tightness (31%), muscle
                                                                        aches (31%), cough (28%),
                                                                        flu-like sensation (28%),
                                                                        runny nose (25%).
118 workers in         No exposure data       Measurement of specific   Prevalence:                  Baur et al. 198684
bakeries, and          presented.             IgE to fungal alpha/      • symptomatic: 12 (34%)
confectioners,                                amylase (enzyme           • asymptomatic: 0 (0%)
Germany.                                      immunoassay).             Skin prick test confirmed
                                                                        positive cases.
                                                                        Immediate onset of rhinitis
                                                                        or asthma was observed in
                                                                        4 bakers with symptoms,
                                                                        who were challenged by
                                                                        alpha-amylase by
                                                                        inhalation.
Eight workers in       No exposure data       Skin prick test to fungal Five workers were positive Flindt et al. 197985
enzyme producing       presented.             alpha-amylase.            for fungal alpha-amylase;
factory.                                                                four of them gave also a
                                                                        positive reaction to papain.
Enzymes produced:
papain, alpha-amylase
from A. oryzae,
proteases, trypsin.
AM, average mean; 95% CI, 95% confidence interval; GM, geometric mean; OR, odds ratio; SD, standard deviation; TWA,
time weighed average concentration.
             Prevalence of sensitisation to alpha-amylase and of respiratory symptoms                                   85
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<pre>             Case reports
 tudy design        Exposure information        Health information          Results                           Reference
nd population
nformation
 ive bakers with    Exposure to bread           Tests (skin prick test,     One patient scored positive for Quirce et al.
espiratory allergy, improvers containing fungal specific IgE) for           all sensitisation tests to alpha- 199286
 pain.              alpha-amylase. No exposure sensitisation to fungal      amylase. This person was also
                    data presented.             alpha-amylase and           sensitised to cellulose, but not
                                                bronchial provocation test. to wheat flour allergens.
 aker with asthma, No exposure data presented. Tests (skin prick test,      All tests were positive. Skin Blanco Carmona
 pain.              Alpha-amylase present as specific IgE) for              prick tests with wheat, rye and et al. 199187
                    component in additive for sensitisation to fungal       soya flour extracts were
                    bread making.               alpha-amylase and           negative.
                                                bronchial provocation test.
 aker with asthma, No exposure data presented. Tests (skin prick test,      Positive for sensitisation tests. Birnbaum et al.
 rance.             Alpha-amylase present as specific IgE) for              Provocation test to the enzyme 198888
                    component in additive for sensitisation to fungal       gave a delayed reaction.
                    bread making.               alpha-amylase and
                                                bronchial provocation test.
 6           Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>  nnex        F
              Exposure-response relationships
              Cross-sectional studies as part of a longitudinal prospective cohort
              study by Cullinan et al. 199452
  tudy design and      Exposure information           Health information            Results                       Reference
 opulation
nformation
1) Initial cross-      Exposure assessed by self- Self-administered                 Sensitisation                 Nieuwen-
 ectional phase of a   administered questionnaire questionnaire on work-                                          huijsen et al.
ongitudinal cohort     on job-title and work history, related respiratory symptoms Prevalence, exposure at time 199922
 tudy in 3 bakeries, 1 including exposure to flour in (4-5 years retrospective) and of study:
lour packing factory,  previous employment.           smoking habits. Skin prick • low: 3.1% (7/225)              Exposure
 nd 3 mills where                                     tests to common allergens, • medium: 16.7% (3/18) levels:
wheat was milled,      Full-shift personal inhalable and to wheat flour and fungal • high: 15.4% (2/13)           Nieuwen-
UK.                    dust sampling in 11-15         alpha-amylase (n=256).        Prevalence, highest exposure huijsen et al.
                       workers of each task-based                                   ever:                         199471
 64 workers without exposure group in each            Skin prick tests were         • low: 2.5% (5/203)
 re-employment.        factory (collected 1990/       considered positive if the    • medium: 9.5% (2/21)         Cohort design:
                       1991):                         mean wheal diameter was at • high: 29.4% (5/17)             Cullinan et al.
                       • low: < 1 mg/m3               least 3 mm greater than the Prevalence ratio, highest       199452
                       • medium: 1-5 mg/m3            inert control.                exposure ever:
                       • high: >5 mg/m3                                             • medium:
                                                                                       3.9 (95% CI, 0.8-20.2)
                       Determination of fungal                                      • high:
                       alpha-amylase in dust                                           9.9 (95% CI, 2.8-34.6)
                       samples by sandwich-
                       immunoassay (n=256).                                         Prevalence ratio for
                                                                                    sensitisation relative to
                                                                                    atopy: 2.9 (95% CI, 0.8-9.7).
              Exposure-response relationships                                                                                    87
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<pre>                         Exposure at time of study                              New-work-related symptoms
                        (AM±SD):
                        • low: 0.7±0.8 ng/m3                                    Chest, at time of study:
                        • medium: 10.7±2.2 ng/m3                                • low: 6% (14/225)
                        • high: 46.7±16.6 ng/m3                                 • medium: 0% (0/18)
                        Highest exposure ever                                   • high: 15.4% (4/13)
                        worked in ( AM±SD):                                     Eye and nose, at time of
                        • low: 0.8±0.8 ng/m3                                    study:
                        • medium: 10.5±2.3 ng/m3                                • low: 16.4% (38/225)
                        • high: 48.0±16.6 ng/m3                                 • medium: 21% (4/18)
                                                                                • high: 23.1% (3/13)
                        Mean duration of                                        Skin, at time of study:
                        employment was 28 months                                • low: 5.2% (12/225)
                        (range between 1 month to                               • medium: 5.3% (1/18)
                        maximal 4 years).                                       • high: 30.8% (4/13)
                                                                                Chest, at highest exposure
                                                                                ever:
                                                                                • low: 6.2% (13/203)
                                                                                • medium: 4.6% (1/21)
                                                                                • high: 11.8% (2/17)
                                                                                Eye and nose, at highest
                                                                                exposure ever:
                                                                                • low: 16.2% (34/203)
                                                                                • medium: 22.7% (5/21)
                                                                                • high: 17.7% (3/17)
                                                                                Skin, at highest exposure
                                                                                ever:
                                                                                • low: 5.7% (12/203)
                                                                                • medium: 4.6% (1/21)
                                                                                • high: 23.5% (4/17)
                                                                                None of the 12 workers with
                                                                                positive skin prick test to
                                                                                fungal alpha-amylase had
                                                                                new work-related chest
                                                                                symptoms, one had new
                                                                                work-related eye and nose
                                                                                symptoms and one had new
                                                                                work-related skin symptoms.
                                                                                This might be explained by
                                                                                workers with symptoms
                                                                                moving to a low-exposure
                                                                                environment (healthy worker
                                                                                effect).
2) Cross-sectional      Determination of fungal    Symptoms recorded by self- Prevalence (ratios)           Brisman et al.
 hase of a longitudinal alpha-amylase in dust      administered questionnaire, (Cox regression):            200457
 ohort study in 3       samples by sandwich-       skin sensitisation to fungal
 akeries, 1 flour       immunoassay (n=256).       alpha-amylase by skin prick Prevalence sensitisation:    Exposure levels
 acking factory, and 3 Exposure at time of study   test.                        • low: 8% (18/225)          and categories:
mills where wheat was (AM±SD):                                                  • medium: 9% (2/22)         Nieuwen-
milled, UK.             • low: 0.7±0.8 ng/m3       In this study, a different   • high: 31% (4/13)          huijsen et al.
                        • medium: 10.7±2.2 ng/m3 statistical analysis was       Prevalence ratio,           199471, 199922
                        • high: 46.7±16.6 ng/m3    employed (prevalence ratio) sensitisation:
 8            Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre> 58 workers in        Highest exposure ever         compared to previous studies • medium:                        Cohort design:
 akeries, follow-up 7 worked in ( AM±SD):           on this cohort (odds ratio).      3.1 (95% CI, 0.6-17)        Cullinan et al.
 ears.                • low: 0.8±0.8 ng/m3          Odds ratios are generally     • high: 4.0                     199452
                      • medium: 10.5±2.3 ng/m3      higher than prevalence ratios, (95% CI, 0.9-18)
                      • high: 48.0±16.6 ng/m3       but according to the authors, Prevalence, chest symptoms:
                                                    prevalence ratio is a better • low: 9.7% (22/225)
                      The highest exposure ever estimate of relative risk.        • medium: 22.7% (5/22)
                      worked in was used for                                      • high: 38.5% (5/13)
                      classification. Forty persons                               Prevalence ratio, chest
                      were not classified due to                                  symptoms:
                      missing information.                                        • medium:
                                                                                      1.7 (95% CI, 0.6-4.9)
                      Median duration of                                          • high:
                      employment was (range): 40                                      3.0 (95% CI, 1.1-8.1)
                      (1- 91) months.                                             Prevalence, eye and nose
                                                                                  symptoms:
                                                                                  • low: 24.9% (56/225)
                                                                                  • medium: 45.5% (10/22)
                                                                                  • high: 53.8% (7/13)
                                                                                  Prevalence ratio, eye and
                                                                                  nose symptoms:
                                                                                  • medium:
                                                                                      1.9 (95% CI, 0.9-3.8)
                                                                                  • high:
                                                                                      1.9 (95% CI, 0.9-4.2)
                                                                                  Trends were observed for
                                                                                  eye-nose symptoms, and
                                                                                  sensitisation by increasing
                                                                                  exposure.
                                                                                  Authors noted that precision
                                                                                  of the study is low, because
                                                                                  the distribution of alpha/
                                                                                  amylase exposure was highly
                                                                                  skewed, with 86% of the
                                                                                  workers in the low exposure
                                                                                  category.
3) Nested case-       Categories:                   All cohort members still      Prevalence of sensitisation to Cullinan et al.
 ontrol within a      • low: bread wrappers and employed were surveyed at fungal alpha-amylase                    200153
 rospective cohort       confectioners without      6-monthly intervals for 3     (positive skin prick test): 24/
 tudy. Cohort of 300     direct contact with flour; years. 52 Workers were        300                             Cohort design:
 ew employees from 7 • medium: bread and roll surveyed 7 times. At each • low: 17/58 (cases/                      Cullinan et al.
UK bakeries and flour    makers, cleaners and       survey, self-administered         control), OR 1              199452
mills, not previously    other confectioners;       questionnaire on smoking          (95% CI, -)
 xposed to flour;     • high: workers directly      habits and on work-related • medium: 38/12 (cases/
ollow-up 1-91            handling flour and mixing chest, eye/nose or skin            control), OR 13 (
months (median 40        or braking doughs.         symptoms, that had                95% CI, 0.8-209)
months).                                            commenced after first         • high: 46/31 (cases/
                      Inhalable dust concentrations employment at the study site. control), OR 23
  hose who developed (GM (95% CI)):                 Skin prick tests to common        (95% CI, 0.3-182).
work-related          • low: 0.58 mg/m3 (0.5-0.7 respiratory allergens, to
 ymptoms or with a       mg/m3);                    wheat flour and to fungal al Incidence rates (cases per
              Exposure-response relationships                                                                                   89
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<pre> ositive skin prick test medium: 1.17 mg/m3         alpha-amylase.               100 person-years):
were compared with         (1.0-1.4 mg/m3);         Two controls                 • skin prick test alpha-
 n employment-          • high: 4.37 mg/m3 (3.8-5.1 (nonsymptomatic and              amylase: 2.5
 uration-matched           mg/m3).                  negative skin prick test),   • skin prick test flour: 2.2
 ontrol group.                                      matched for duration of      • eye/nose symptoms: 11.8
                        No exposure data on fungal- employment, were selected    • chest symptoms: 4.1
                        alpha-amylase presented.    for each case (symptomatic
                                                    or positive skin prick test) Approximately 50% of the
                        Median duration of                                       cases developed within 24
                        employment was (range): 40                               months of employment.
                        (1-91) months.
                                                                                 Compared to the controls,
                                                                                 sensitised persons were more
                                                                                 often atopics (OR 4.1, 95%
                                                                                 CI 0.4-47, not significant).
                                                                                 There was no difference in
                                                                                 smoking habits.
                                                                                 Only 25% of workers
                                                                                 reporting chest problems
                                                                                 were sensitised to flour or
                                                                                 fungal alpha-amylase. Even
                                                                                 less workers reporting
                                                                                 eye/nose or skin symptoms
                                                                                 were sensitised to flour of
                                                                                 fungal alpha-amylase (17%
                                                                                 and 18% respectively). This
                                                                                 suggests that a high
                                                                                 proportion of the work-
                                                                                 related symptoms had no
                                                                                 allergic origin but probably
                                                                                 reflect an irritant or
                                                                                 inflammatory response to
                                                                                 airborne dust.
AM, average mean; 95% CI, 95% confidence interval; GM, geometric mean; OR, odds ratio; SD, standard deviation.
 0            Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>              Other cross-sectional studies
 tudy design and           Exposure information       Health information              Results                         Reference
 opulation information
 78 workers from 14        Full-shift personal        Self-administered               Overall prevalence of           Houba et
 akeries, the Netherlands. inhalable dust and fungal questionnaire (n=178) on         sensitisation to fungal alpha- al. 199614,
Maintenance workers        alpha-amylase              work-related symptoms,          amylase:
were excluded.             concentrations measured smoking habits and job             • skin prick test: 9.5%         Doekes et
                           (sandwich-immunoassay). history; lung function                (16/169)                     al. 19986;
                                                      measurements.                   • IgE-positive: 7.7% (13/169)
                           Fungal alpha-amylase                                                                       Houba et
                           exposure levels (job with Skin prick tests to work-related Prevalence, skin prick test:    al. 199670,
                           highest exposure ever      and common allergens, and       • low: 1.4% (1/71)              199789
                           worked in, GM±SD           analysis of specific IgE to     • medium:12.8% (5/39)
                           (range)):                  fungal alpha-amylase in blood • high: 30.4% (7/23)
                           • low: 0.7±4.0 ng/m3       (n=169).                        • indistinct: 8.3% (3/36)
                              (0.2-8.8);                                              • reference
                           • medium: 1.3±3.8          Skin prick tests were              population(laboratory
                              ng/m3 (0.2-33.1) ;      considered positive if at the      animal workers): 1.7%
                           • high: 18.1±4.6 ng/m3 15-min reading, the mean               (7/416)
                              (0.2-221.8);            wheal diameter was at least 3 Prevalence ratio, skin prick
                           • indistinct/variable:     mm greater than the negative test:
                              6.1±8.2 ng/m3           control.                        • medium:8.6 (95% CI,
                              (0.2-150.2).                                               1.01-74)
                                                      Specific IgE was determined • high: 15.9 (95% CI,
                           Job-title categories:      by means of an enzyme              1.95-129)
                           • low (n=71): workers immunoassay using                    • indistinct: 4.6 (95% CI,
                              handling alpha-         monoclonal mouse antihuman         0.48-45)
                              amylase only            IgE. Optical density readings • atopy: 20.8 (95% CI,
                              occasionally;           at 492 nm exceeding the OD of 2.74-158)
                           • medium (n=39):           the reagent blank with more Prevalence among atopic
                              doughmakers and all- than 0.05 was interpreted as a workers (n=49), skin prick test:
                              round staff from        positive reaction.              • low: 4%
                              wheat-bread producing                                   • medium: 35%
                              bakeries and bread and                                  • high: 55%
                              mixed bakers from
                              small bakeries;                                         Prevalence, specific IgE:
                           • high (n=23):                                             • low: 2.5% (2/71)
                              doughmakers in one                                      • medium: 13% (5/39)
                              crispbake factory;                                      • high: 15% (4/23)
                           • indistinct/variable                                      Prevalence ratio, specific IgE:
                              (n=36): all-round staff                                 • medium: 4.6 (95% CI,
                              in the crispbake                                           0.85-22)
                              factory.                                                • high: 3.9 (95% CI, 0.65-24)
                                                                                      Prevalence ratios for IgE were
                           Mean years of                                              not statistically significant
                           employment in bakery                                       except for atopy: 8.3 (95% CI,
                           industry was                                               1.84-38)
                           10.2 ± 8.7 years
                           (range:0.2-43 years).
              Exposure-response relationships                                                                                  91
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<pre>                                                                                    Sensitisation to fungal alpha-
                                                                                    amylase (determined by skin
                                                                                    prick test) was moderately to
                                                                                    strongly associated with self-
                                                                                    reported, work-related rhinitis
                                                                                    and asthmatic complaints
                                                                                    (chest tightness):
                                                                                    Prevalence ratios:
                                                                                    All workers (n=169)
                                                                                    • rhinitis: 4.78 (95% CI,
                                                                                        2.05-11.2)
                                                                                    • chest tightness: 11.95 (95%
                                                                                        CI, 3.21-44.5)
                                                                                    Atopic workers (n=65)
                                                                                    • rhinitis: 3.33 (95% CI,
                                                                                        1.17-9.50)
                                                                                    • chest tightness: 4.44 (95%
                                                                                        CI, 1.00-19.9)
                                                                                    60% of the sensitised workers
                                                                                    reported symptoms, and about
                                                                                    30% of the symptomatic
                                                                                    bakery workers was sensitised
                                                                                    (IgE) to wheat flour or alpha-
                                                                                    amylase. The authors
                                                                                    considered a non-specific
                                                                                    reaction to dust as a possible
                                                                                    explanation.
                                                                                    Smoking habits, age and years
                                                                                    working in the baking industry
                                                                                    were not associated with
                                                                                    sensitisation.
 46 workers from           Personal inhalable dust Self-administered                Overall prevalence of           Droste et al
raditional and industrial concentrations measured questionnaire on respiratory,     sensitisation to fungal alpha- 200356
 akeries (n=74),           (during shift 5-7 hours). asthma and allergy-related     amylase:
 ompared with 251          Alpha-amylase in dust      symptoms, supplemented with   • bakery workers: 7.5% (18/ Droste et
workers from a             samples analysed by        questions on smoking habits.      246)                        al. 200590
 etrochemical plant in the sandwich-immunoassay. Skin prick testing on common       • controls: 0.8% (2/251)
 ame region (controls),                               and bakery-specific antigens  Odds ratio (adjusted for age Data on
  elgium.                  Job categories:            (wheat flour, rye flour, and  and smoking habits) was 11.6 exposure
                           • low: industrial packers;fungal alpha-amylase). Skin    (95% CI, 2.4-55.1).             levels:
                           • medium: industrial       prick tests were considered                                   Bulat et al.
                              bakers and traditional positive if at the 15-min      Overall prevalence of           200420
                              pastry bakers;          reading, the mean wheal       sensitisation for any allergen:
                           • high: traditional bread diameter was at least 2 mm.    • bakery workers: 39.4%
                              bakers, and traditional Lung function tests performed • controls: 42.6%
                              bread + pastry bakers. by spirometry.
                                                                                    Prevalence of sensitisation,
                           Exposure to alpha-                                       alpha-amylase exposure:
                           amylase (GM±SD):                                         • low: 3.0% (1/33)
                           • low (n=34): 0.15 ±                                     • medium: 4.7% (4/86)
  2          Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>                        1.74 ng/m3;                                               • high: 12.6% (13/103)
                     • medium (n=73): 0.47 ±                                      Intergroup difference not
                        7 ng/m3;                                                  statistically significant:
                     • high (n=107): 0.50-                                        medium versus low, odds ratio
                        0.61 ± 4 ng/m3.                                           2.1 (95% CI, 0.2-20.2); high
                     Maximum exposure was                                         versus low, odds ratio 4.4 (95%
                     136 ng/m3 in an industrial                                   CI 0.5-37.2).
                     baker.
                                                                                  Overall work-related
                     Mean duration of                                             symptoms (chronic cough,
                     employment was: 8.9                                          shortness of breath, wheeze):
                     years.                                                       Prevalence:
                                                                                  • low: 5.9% (2/34)
                                                                                  • medium: 18.9% (14/74)
                                                                                  • high: 39.6% (38/96)
                                                                                  Odds ratio (95% CI):
                                                                                  • medium: 3.9 (0.8-18.2)
                                                                                  • high: 10.9 (2.5-48.7)
                                                                                  Spirometry did not reveal
                                                                                  dose/response relationships
                                                                                  with lung function parameters.
86 workers in one US Two exposure categories: Self-administered                   Prevalence of sensitisation to Page et al.
akery.               • low exposure: not         questionnaire on job history fungal alpha-amylase:               200946,
                        handling dough, e.g. and work-related symptoms • low: 4% (2/51)                           201028
                        office, transportation, and smoking habits (n=161). • high: 11% (5/45)
                        oven areas;                                               Prevalence ratio: 2.83 (95%
                     • high exposure:            Specific serum IgE antibodies CI, 0.65-18.84).
                        handling raw materials to fungal alpha-amylase, flour
                        and/or dough: e.g.,      and wheat, and to common         A number of employees
                        bread & bun              allergens to assess atopy        working in lower-exposure
                        production,              (n=96).The method was a          group reported past work in
                        distribution.            highly sensitive enzyme-         higher exposure group;
                     Full-shift air sampling in enhanced chemiluminescent prevalence of sensitisation to
                     personal breathing zone enzyme immunoassay,                  fungal alpha-amylase if these
                     (n=83), and general area Immulite 2000. Traditionally, employees were included in
                     (n=19).                     IgE levels ≥ 0.35 kU/L serum the high-exposure group:
                                                 are considered positive for      • low: 0% (0/33)
                     Inhalable flour dust levels sensitisation. The threshold for • high (current and past):
                     (GM and range):             this assay is 0.10 kU/L.             11% (7/63)
                     • low: 0.24 mg/m3 (ND-
                        1.4)                     There was no difference in       Prevalence of sensitisation to
                     • high: 3.01 mg/m3          prevalence of atopy between fungal alpha-amylase (positive
                        (trace-65)               the two exposure groups.         if IgE ≥ 0.35 kU/L):
                                                                                  • low: 0% (0/33)
                     Fungal alpha-amylase                                         • high (current and past): 6%
                     concentrations (sandwich                                         (4/63)
                     immunoassay; GM and
                     range):                                                      Atopics were significantly
                     • low: 0.12 ng/m3                                            more likely to be sensitised to
                        (0.019-1.2)                                               alpha-amylase at the low cut-
                     • high: 2.10 ng/m3                                           off value (p=0.04).
                        (0.095-11,000)
           Exposure-response relationships                                                                                93
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<pre>                    Inhalable flour dust and                          Prevalence, runny nose:
                    alpha-amylase                                     • low: 6% (6/91-93)
                    concentration were                                • high (16% (10/61-64)
                    positively correlated                             Prevalence ratio:
                    (r=0.64, p<0.01).                                 • 3.81 (95% CI, 1.25-11.61)
                    Mean tenure was 13 years                          Prevalence, stuffy nose:
                    (higher exposure group),                          • low: 6% (6/91-93)
                    and 16 years (lower                               • high: 18% (11/61-64)
                    exposure group).                                  Prevalence ratio:
                                                                      • 2.75 (95% CI, 1.07-7.05)
                                                                      Prevalence, frequent sneezing:
                                                                      • low: 8% (7/91-93)
                                                                      • high: 21% (13/61-64)
                                                                      Prevalence ratio:
                                                                      • 2.68 (95% CI, 1.13-6.34)
                                                                      Note: few symptoms were
                                                                      significantly related to
                                                                      sensitisation. Thus, a
                                                                      preference for the lower or
                                                                      higher cut-off value could not
                                                                      be determined.
M, average mean; 95% CI, 95% confidence interval; GM, geometric mean; SD, standard deviation.
4        Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>nnex G
     Meta-regression analysis
     In deriving a reference value, data by Nieuwenhuijsen et al. (1999) and Houba et al.
     (1996) were combined in a meta-regression analysis as follows.
          First of all the background sensitisation rate was estimated on the basis of
     population studies without occupational exposure. From this data, the committee
     calculated the expected number of cases for each exposure category in the
     Nieuwenhuijsen and Houba study, on the basis of number of persons in the
     exposure group category. Average exposure for each exposure category was
     taken from the respective publications. For each exposure group the ratio of the
     observed and expected number of cases was calculated.
          For the meta-regression analysis, the committee used a linear Poisson model
     RR=α(1 + Kalpha-amylse sensitisation ∗ E), where RR is the relative risk, α is an intercept
     parameter representing the background rate of sensitisation (expected cases), and
     Kalpha-amylse sensitisation is the slope of increase in the AR per unit exposure (E) to
     alpha-amylase (in ng enzyme/m3). Kalpha-amylase sensitisation-values were obtained by
     fitting Poisson regression models using the statistical programme SAS. The
     intercept was fixed (α = 1) to force the model to go through the odds ratio of 1
     (background prevalence of the non-exposed reference groups (zero exposure)). As
     such, the committee derived a Kalpha-amylase sensitisation-value of 0.924 (confidence
     interval 0.344-1.504, p=0.008), resulting in the formula RR=1 + 0.924 ∗ E. With a
     background rate of 1.17 per 100, an extra risk of 1 per 100 (1%) means an additional
     risk of 2.17, which corresponds to a RR of 1.855 (RR=additional risk divided by
     background risk). Using this formula an exposure level of 0.9 ng enzyme/m3 was
     Meta-regression analysis                                                                    95
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<pre>  calculated, which corresponds to an additional risk on sensitisation of 1% compared
  to the background sensitisation rate.
  Exposure group           Exposure level (ng/m3)      Number of persons           Number of persons
                                                       in group                    sensitised
  Nieuwenhuijsen et al. (1999)
     Reference             No data                     No data                     No data
     low                    0.8                        203                         5
     medium                10.5                         21                         2
     high                  48.0                         17                         5
  Houba et al. (1996)
     Reference              0.0                        416                         7
     low                    0.7                         71                         1
     medium                 1.3                         39                         5
     high                  18.1                         23                         7
  Exposure level expressed as geometric mean (highest exposure ever worked in), 8 hour TWA. Reference
  Houba-study: laboratory animal workers not occupationally exposed to fungal alpha amylase.
6 Fungal alpha-amylase (derived from the fungus Aspergillus oryzae)
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<pre>Health Council of the Netherlands
Advisory Reports
The Health Council’s task is to       In addition, the Health Council
advise ministers and parliament on    issues unsolicited advice that
issues in the field of public health. has an ‘alerting’ function. In some
Most of the advisory reports that     cases, such an alerting report
the Council produces every year       leads to a minister requesting
are prepared at the request of one    further advice on the subject.
of the ministers.
Areas of activity
Optimum healthcare                    Prevention                          Healthy nutrition
What is the optimum                   Which forms of                      Which foods promote
result of cure and care               prevention can help                 good health and
in view of the risks and              realise significant                 which carry certain
opportunities?                        health benefits?                    health risks?
Environmental health                  Healthy working                     Innovation and
Which environmental                   conditions                          the knowledge
influences could have                 How can employees                   infrastructure
a positive or negative                be protected against                Before we can harvest
effect on health?                     working conditions                  knowledge in the
                                      that could harm their               field of healthcare,
                                      health?                             we first need to
                                                                          ensure that the right
                                                                          seeds are sown.
www.healthcouncil.nl
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<br><br>