EN 455-3:2006

SLOVENSKI STANDARD
01-marec-2007
1DGRPHãþD
SIST EN 455-3:2000
Medicinske rokavice za enkratno uporabo - 3. del: Zahteve in preskusi za biološko
ovrednotenje
Medical gloves for single use - Part 3: Requirements and testing for biological evaluation
Medizinische Handschuhe zum einmaligen Gebrauch - Teil 3: Anforderungen und
Prüfung für die biologische Bewertung
Gants médicaux non réutilisables - Partie 3: Exigences et essais pour évaluation
biologique
Ta slovenski standard je istoveten z: EN 455-3:2006
ICS:
11.140 Oprema bolnišnic Hospital equipment
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN 455-3
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2006
ICS 11.140 Supersedes EN 455-3:1999
English Version
Medical gloves for single use - Part 3: Requirements and testing
for biological evaluation
Gants médicaux non réutilisables - Partie 3: Exigences et Medizinische Handschuhe zum einmaligen Gebrauch - Teil
essais pour évaluation biologique 3: Anforderungen und Prüfung für die biologische
Bewertung
This European Standard was approved by CEN on 13 October 2006.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Central Secretariat or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 455-3:2006: E
worldwide for CEN national Members.

Contents Page
Foreword.3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .6
4 Requirements.6
5 Test methods.8
6 Test report .9
Annex A (normative) Method for the determination of aqueous extractable proteins in natural
rubber gloves using the modified Lowry assay .10
Annex B (informative) Immunological methods for the measurement of natural rubber latex
allergens .20
Annex C (informative) Amino acid analysis (AAA) by high pressure liquid chromatography
(HPLC).27
Annex ZA (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC Medical Devices.36

Foreword
This document (EN 455-3:2006) has been prepared by Technical Committee CEN/TC 205 “Non-active
medical devices”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by June 2007, and conflicting national standards shall be withdrawn at the
latest by June 2007.
This document supersedes EN 455-3:1999.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive 93/42/EEC.
For relationship with EU Directive, see informative Annex ZA, which is an integral part of this document.
EN 455 consists of the following parts under the general title "Medical gloves for single use":
 Part 1: Requirements and testing for freedom from holes
 Part 2: Requirements and testing for physical properties
 Part 3: Requirements and testing for biological evaluation
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden,
Switzerland and United Kingdom.
Introduction
Adverse reactions to proteins in latex products have been reported over several years in variable rates of
prevalence. Additionally, adverse reactions due to chemicals, lubricants, sterilization residues, pyrogens or
other residues are described in the scientific literature. Adverse reactions are most often reported due to
gloves made from natural rubber latex, but some of the reactions can also be seen due to gloves made from
synthetic polymers.
EN ISO 10993 specifies requirements and test methods for biological evaluation of medical devices. However
it does not specifically address adverse reactions that can result from the use of medical gloves (e.g,
immediate type allergies). These adverse reactions occur to specific allergens that can be present in gloves.
Several factors contribute to the risk of reaction:
a) the duration and frequency of skin contact with gloves;
b) the exposure to the allergens through direct contact to mucosa and skin (especially when not intact) and
by inhalation of particles;
c) the occlusive nature of the glove/skin interaction during glove use.
This part of EN 455 gives requirements and test methods for evaluation of the biological safety of medical
gloves as part of a risk management process, in accordance with EN ISO 14971 and EN ISO 10993.
1 Scope
This part of EN 455 specifies requirements for the evaluation of biological safety for medical gloves for single use. It
gives requirements for labelling and the disclosure of information relevant to the test methods used.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated references,
only the edition cited applies. For undated references, the latest edition of the referenced document (including any
amendments) applies.
EN 980, Graphical symbols for use in the labelling of medical devices
EN 1041, Information supplied by the manufacturer with medical devices
EN ISO 10993 (all parts), Biological evaluation of medical devices
EN ISO 14971, Medical devices ― Application of risk management to medical devices (ISO 14971:2000)
EN ISO 21171:2006, Medical gloves ― Determination of removable surface powder (ISO 21171:2006)
European Pharmacopoeia, Monograph 2.6.14 Bacterial Endotoxins: publisher EDQM Council of Europe; 226
avenue de Colmar B.P. 907; F-67029 Strasbourg; France http://www.pheur.org
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
chemicals
substances added or formed during any step of the manufacturing process or in storage which may be
available in the final product
NOTE These can include lubricants, chemical coatings and sterilizing agents. Several chemical ingredients are
commonly used during processing of gloves, some of them are known to cause type IV allergic reactions. The type and
amount of residual chemicals added and finally present are variable.
3.2
endotoxins
lipo-polysaccharides originating from the outer cell-membrane of Gram-negative bacteria
NOTE Endotoxins are one type of pyrogen. Sources of endotoxins can include bacterial contamination of the raw
materials, especially the process water used during manufacturing and manual handling of the gloves.
3.3
powder
all water insoluble material on the surface of a glove that is removed by washing under the conditions of the
test
[EN ISO 21171:2006, definition 3.1]
NOTE This includes both deliberately added powder and other processing aids or materials accidentally present
which may be readily detached from the surface of the glove. For the purpose of this European Standard any glove
containing 2 mg or less powder is a powder-free glove and more than 2 mg is a powdered glove (for requirement see 4.4.).
3.4
process limit
highest value likely to be encountered for a validated manufacturing process
3.5
proteins, allergenic
proteins capable of causing a type I allergic reaction
3.6
proteins, leachable
aqueous proteins and peptides extractable from the final product
3.7
pyrogens
substances creating fever in rabbits which can be related to fever and other adverse reactions in humans
4 Requirements
4.1 General
Medical gloves for single use shall be evaluated as described in the EN ISO 10993 series. Part 1 of this series
describes the general principles governing the biological evaluation of medical devices and shall be used to
select the appropriate tests as described in other parts of the series.
A risk management process in accordance with EN ISO 14971 shall be established.
4.2 Chemicals
Gloves shall not be dressed with talcum powder (magnesium silicate).
Chemicals known to be allergenic shall be avoided if technical alternatives exist. Wherever possible allowable
limits for leachable residual chemicals shall be established using EN ISO 10993-17 and these limits shall be
complied with. Wherever this is not possible, the residual chemicals level shall be "As Low As Reasonably
Practicable" (ALARP – see EN ISO 14971).
The manufacturer shall disclose, upon request, a list of chemical ingredients either added during
manufacturing or already known to be present in the product such as accelerators, antioxidants and biocides,
that are known to cause adverse health effects based on current data.
4.3 Endotoxins
The manufacturer shall monitor the endotoxin contamination of sterile gloves using the test method specified
in 5.1 if the gloves are labelled with 'low endotoxin content'. For such labelled gloves the endotoxin content
shall not exceed the limit of 20 endotoxin units per pair of gloves.
4.4 Powder
For powder free gloves the total quantity of powder residues determined according to the test method under
5.2 shall not exceed 2 mg per glove. Any glove containing more than 2 mg powder is a powdered glove.
4.5 Proteins, leachable
The manufacturer shall monitor the process limit of leachable protein in the finished gloves containing natural
rubber latex by the method specified in 5.3 and described in Annex A. The documentation of these results
shall be retained. The results of the test and applied test method shall be made available on request.
The leachable protein level shall be "As Low As Reasonably Practicable" (ALARP).
NOTE Proteins, allergenic: This European Standard specifies a method measuring a broad approximation for the
allergen content, e.g. leachable proteins. There is no direct correlation between leachable proteins and allergen content.
Quantitative methods to measure allergenic proteins are under development, as described in Annex B.
4.6 Labelling
In addition to the labelling specified in EN 1041 and the relevant symbols given in EN 980, the following
requirements apply:
a) medical gloves containing natural rubber latex shall be labelled at least on the packaging, of the smallest
packaging unit with the following symbol (EN 980 general requirements for symbols apply);
Figure 1 — Symbol for products containing natural rubber latex

b) the labelling shall include the following or equivalent warning statement together with the symbol:
‘(Product) contains natural rubber latex which may cause allergic reactions, including anaphylactic
responses;
c) the labelling shall include a prominent indication of whether the glove is powdered or powder-free;
d) sterile powdered gloves shall be labelled with the following or equivalent:
'CAUTION: Surface powder shall be removed aseptically prior to undertaking operative procedures in
order to minimize the risk of adverse tissue reactions;
NOTE 2 This caution statement can be given on the inner wrapping.
e) for any medical glove containing natural rubber latex the product labelling shall not include:
 any term suggesting relative safety, such as low allergenicity, hypoallergenicity or low protein;
 any unjustified indication of the presence of allergens;
f) if the manufacturer labels the gloves with the protein content, the process limit, measured as specified in
5.3 shall be given.
NOTE 3 This does not allow a protein labelling claim below 50 µg/g. Lower claims are not considered to be reliable
given the expected process variation in manufacture and inter-laboratory testing.
5 Test methods
5.1 Endotoxins
Except where non-removable interferences in the Limulus Amoebocyte Lysate (LAL) procedures are present,
selection, validation and use of technique shall be as described in the European Pharmacopoeia, Monograph
2.6.14, "Bacterial Endotoxins". The results shall be expressed in endotoxin units (E.U.) per pair of gloves.
NOTE 1 Where non-removable interferences in the LAL procedure are present, the bacterial endotoxin level cannot be
accurately measured.
The minimum number of pairs of gloves recommended to be tested in relation to the number of items in the
batch are two pairs of gloves for a batch size under thirty, three pairs of gloves for a batch size thirty to one
hundred, and 3 % of a batch above size one hundred, up to a maximum of ten pairs of gloves per batch.
The outside surface of a pair of gloves is extracted with 40 ml of endotoxin-free water (Water LAL, European
Pharmacopoeia, for not less than 40 min and not more than 60 min at a temperature between 37 °C and
40 °C in a way to ensure that all surfaces come into contact with the extraction medium. The extract is
centrifuged, if necessary, for 15 min at 2000 g to remove particles after which the liquid component is
decanted and tested for endotoxin immediately afterwards.
NOTE 2 Other methods for the analysis of endotoxins exist and these can be used for routine quality control purposes
provided they have been validated and a correlation established against the reference method specified in this European
Standard.
5.2 Powder
The test method for the determination of powder residues described in EN ISO 21171 shall be used.
5.3 Proteins, leachable
The test method for the analytical determination of leachable protein shall be the modified Lowry method
given in Annex A or a suitably validated method which has been correlated against the modified Lowry
method.
NOTE 1 An example of a validated analytical method is given in Annex C.
NOTE 2 The immunological methods in Annex B are currently not validated against the modified Lowry method but
may be correlated to clinical response data.
6 Test report
The test report shall include at least the following information:
 a reference to this European Standard, i.e. EN 455-3;
 the type of gloves and manufacturing batch code;
 the name and address of the manufacturer or distributor and of the test laboratory if different;
 the date of the test;
 the description of the test method applied;
 the test results.
Annex A
(normative)
Method for the determination of aqueous extractable proteins in natural
rubber gloves using the modified Lowry assay
A.1 Scope
This method is for the determination of the amount of aqueous extractable proteins in gloves for medical use
made from natural rubber (NR). It has been validated during inter-laboratory round-robin tests. The lower
quantification limit is approximately 10 µg protein per g of glove (i.e. 2 µg protein per ml of extract) depending
on the glove weight.
Chemicals such as surfactants, accelerators and antioxidants added to the NR latex during the manufacture of
the gloves can interfere with the colour development during the determination, some materials may reduce
colour development while others can increase it. If the test method yields results that appear erroneous due to
interferants, then any validated amino acid analysis method can be used (as an example see the method
given in Annex C).
NOTE Persons using this method should be familiar with normal laboratory practice. This method does not purport to
address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate
safety and health practices and to ensure compliance with any national regulatory conditions.
A.2 Principle
Water soluble proteins are extracted into a buffer solution and then precipitated with acids in the presence of
sodium deoxycholate to concentrate them and to separate them from water soluble substances which may
interfere with the determination. The precipitated proteins are redissolved in alkali and quantified
colorimetrically by a modified Lowry method. The assay is based on the reaction of proteins with copper and
Folin reagent in an alkaline medium to give a characteristic blue colour. Spectrophotometric measurements
are performed at a fixed wavelength in the range 600 nm to 750 nm.
A.3 Reagents
A.3.1 General
Wherever water is called for, double distilled water or water of equivalent quality should be used. All other
reagents should be of analytical quality.
A.3.2 Extractant
A.3.2.1 N-tris-[Hydroxymethyl]-methyl-2-aminoethanesulfonic acid (TES), hemisodium salt.
A.3.2.2 Extraction buffer, 0,1 M, prepared by dissolving 24 g TES (A.3.2.1) in 1 l water. Any equivalent
buffering system can be used provided the solution has sufficient buffering capacity to hold a pH of 7,4 ± 0,2
in the glove extracts.
NOTE Prepare a sufficient quantity for the glove extraction (A.6.2), the preparation of the protein standard solutions
(A.6.3.2) and the blank.
A.3.2.3 Dye solution, Bromophenol blue, sodium salt solution, prepared by dissolving 100 mg
bromophenol blue in 1 l of water. Prepare a fresh solution every four weeks.
A.3.3 Lowry protein assay reagents
NOTE Reagents can either be prepared from off-shelf chemicals [1] or be purchased as commercial kits. The method
1)
for this European Standard was validated with a commercial kit.
A.3.3.1 Reagent A, Copper reagent (alkaline copper tartrate or copper citrate solution).
A.3.3.2 Reagent B, Diluted Folin reagent.
A.3.4 Sodium hydroxide, 0,1 M aqueous solution.
A.3.5 Sodium deoxycholate (DOC), 3,47 mM, prepared by dissolving 0,15 g sodium deoxycholate in water
and diluting with water to 100 ml. Do not use this solution more than four weeks after it has been prepared.
A.3.6 Trichloroacetic acid (TCA), 4,4 mM in water, prepared by dissolving 72 g TCA in water and diluting
with water to 100 ml.
A.3.7 Phosphotungstic acid (PTA), prepared by dissolving 72 g PTA in water and diluting with water to
100 ml. Do not use this solution more than four weeks after it has been prepared.
2)
A.3.8 Ovalbumin, from chicken egg lyophilized, salt-free.
A.4 Apparatus
A.4.1 Synthetic gloves, powder-free.
A.4.2 Centrifuge, capable of reaching at least 6000 g
A.4.3 Centrifuge tubes, 30 ml or 50 ml polypropylene tubes with a low protein binding capacity of 10 µg per
tube or less. Do not use glass equipment because of surface absorption of proteins.
NOTE A method for the determination of protein binding capacity is described in A.5.
A.4.4 Filter units, single use, with 0,22 µm pore size and a low protein binding capacity of 10 µg per filter or
less.
NOTE A method for the determination of protein binding capacity is described in A.5.
A.4.5 Syringes, disposable, 20 ml, made of polyethylene or polypropylene.
A.4.6 Micro tubes, 2 ml, made of polypropylene.
A.4.7 Quartz cuvette, of 1 cm path length.
A.4.8 Microtitre plate, with 96 wells, flat bottomed, made of polystyrene, or disposable cuvettes (A.4.9).

1)
Lowry Micro DC Protein Assay Kit (catalogue number 500-0116), available from BioRad Laboratories, 2000 Alfred
Nobel Drive, Hercules, CA 9456547, USA. This information is given for the convenience of users of this European
Standard and does not constitute an endorsement by CEN of the product named.
2)
This ovalbumin is prepared from fresh chicken egg whites by ammonium sulfate fractionation and repeated
crystallisation at pH 4,5; for example Sigma A 5503, chicken egg albumin, Grade V, available from Sigma Chemical Co.
P.0. Box 14506, St Louis, MO 63178, USA is suitable. This information is given for the convenience of users of this
European Standard and does not constitute an endorsement by CEN of the product named.
A.4.9 Disposable cuvettes, 1,5 ml semi-micro, 1 cm path length, made of polystyrene
A.4.10 Microplate reader, operating at a wavelength in the range 600 nm to 750 nm
A.4.11 Spectrophotometer, operating in the wavelength range 230 nm to 750 nm.
A.4.12 Vortex mixer.
A.4.13 Micropipettes, with disposable polypropylene tips.
A.4.14 Clamps, for sealing gloves watertight during extraction. Pairs of aluminium bars lined with foam
rubber and which can be screwed together (see Figure A.1) or 170 mm long plastic clips for haemodialysis are
suggested.
A.4.15 Shaker.
A.5 Measurement of protein binding capacity
A.5.1 General
Single use polypropylene equipment (which is known to have a low protein binding capacity) is recommended
for use throughout. The protein binding capacity shall be checked by the following methods before using
centrifuge tubes or filter units from a new batch. The test shall be carried out within one day.
A.5.2 Protein binding capacity of centrifuge tubes
A.5.2.1 Prepare in a centrifuge tube (A.4.3) 30 ml of a reference solution containing 10 µg/ml ovalbumin
by dilution of the protein stock solution (A.6.3.1) with the extraction buffer (A.3.2.2).
A.5.2.2 Transfer 10 ml test portions of the ovalbumin solution (A.5.2.1) to each of two fresh centrifuge
tubes and shake the tubes on a shaker (A.4.15) ensuring that the whole surface is wetted by the solution.
After 30 min transfer the solutions to a further two tubes and shake them. Repeat the procedure until each of
the 10 ml portions has been exposed to five tubes. Store the remaining test solutions.
A.5.2.3 Determine the concentration of the protein in the reference solution and two test solutions in
triplicate using the method given in A.6.4 to A.6.6.
A.5.2.4 Calculate the average absorbed ovalbumin from the expression:
10(R−T )
O =
= 2 (R-T)
where
O is the absorbed ovalbumin in µg/tube;
R is the mean of three determinations of the ovalbumin content of the reference solution in µg/ml;
T is the mean ovalbumin content of the test solution after passage through the tubes
(i.e. the mean of six values) in µg/tube.
The value for the absorbed albumin (O) shall be less than 10 µg per tube, otherwise the tubes are unsuitable
for the determination.
A.5.3 Protein binding capacity of filter units
A.5.3.1 Prepare in a centrifuge tube (A.4.3) 30 ml of a reference solution containing 10 µg/ml ovalbumin
by dilution of the protein stock solution (A.6.3.1) with the extraction buffer (A.3.2.2).
A.5.3.2 Prepare two stacks of five filter units (A.4.4). Filter 10 ml of the reference solution through each
filter stack into a centrifuge tube (A.4.3).
A.5.3.3 Determine the protein content of the reference solution and the two test solutions in triplicate
using the method given in A.6.4 to A.6.6.
A.5.3.4 Calculate the average absorbed ovalbumin from the expression:
10(R−T )
O =
= 2 (R-T)
where
O is the absorbed ovalbumin in µg/tube;
R is the mean of three determinations of the ovalbumin content of the reference solution in µg/ml;
T is the mean ovalbumin content of the test solution after passage through the filter units
(i.e. the mean of six values) in µg/tube.
The value for the absorbed albumin (O) shall be less than 10 µg per filter, otherwise the filters are unsuitable
for the determination.
A.6 Procedure
A.6.1 General
The procedure involves the extraction of the gloves followed by purification and concentration of the extract by
a factor of five. The determination on the extract is performed by reference to a calibration curve prepared
using standard protein solutions which have been concentrated in the same manner.
The extraction procedure employed is one in which the inside of one glove and the outside of a second glove
are extracted simultaneously. It allows the extraction volume to be minimized at 25 ml and avoids any loss of
proteins to container surfaces because the extraction buffer is only exposed to the gloves.
NOTE Alternative extraction procedures may be used if they are validated against this method. A round robin test by
selected laboratories in Europe and in the USA revealed equivalent results with the ASTM Standard D5712:1995 [2] when
extracting cut pieces of gloves for 2 h at 25 °C in TES buffer pH 7,4.
A.6.2 Extraction procedure
A.6.2.1 Use synthetic gloves (A.4.1) to handle the glove samples used for the extraction.
Take eight glove specimens of the same size and the same lot and separate them into four pairs. In the case
of hand-specific gloves, choose four right-handed and four left-handed samples and separate them into two
right-handed and two left-handed pairs.
Mark the cuff of one glove of each pair at a point (200 ± 10) mm from the tip of the middle finger and weigh the
glove (m ) to the nearest 0,1 g. For each pair, insert the second glove inside the marked one so that they fit
together as shown in Figure A.1 a).
NOTE The method for introducing one glove into the other is not of critical importance provided the gloves are
manipulated as little as possible. One way it can be achieved is by sliding a rod in the thumb and the little finger of the
inner glove to help to introduce them into the corresponding fingers of the second glove. Use the rods to insert the three
other fingers.
A.6.2.2 Pour into the inner glove a sufficient quantity of dye solution (A.3.2.3) to fill all five fingers.
Introduce 25 ml extraction buffer (A.3.2.2) at (25 ± 5) °C between inner and outer gloves. For larger gloves
this volume may be increased to a maximum of 50 ml. Remove most air bubbles and seal the gloves with the
clamp (A.4.14) at the 20 cm mark so as to produce a watertight seal as shown in Figure A.1 b).
A.6.2.3 Fix the gloves to the shaker (A.4.15) and shake for (120 ± 5) min at (25 ± 5) °C.
A.6.2.4 Remove the clamp and separate the gloves carefully. Take care not to contaminate the extract
with the dye solution. If the extract is coloured blue, it shall be discarded and the extraction repeated with
fresh gloves.
A.6.2.5 Carefully transfer the extract into a centrifuge tube (A.4.3) and clarify the extract either by
centrifugation at not less than 2000 g for 15 min or filtration through a single use filter unit (A.4.4), or a
combination of both as appropriate. Either store the resulting clear solution refrigerated at 2 °C to 8 °C and
carry out the determination within 48 h or alternatively freeze aliquots of the solution at -18 °C or lower for a
period not exceeding 2 months before analysis.
A.6.2.6 Cut the section of the cuff above the 20 cm mark from the extracted outer glove, wipe the liquid
off the surface with tissue, allow to dry at room temperature and weigh it to the nearest 0,1 g (m ). Calculate
the mass (m) of the extracted part of the glove as follows:
m = m − m
1 2
A.6.3 Protein standard
A.6.3.1 Stock protein solution
Prepare a solution of ovalbumin (A.3.8) with a nominal concentration of 1 mg/ml by dissolving 25 mg
ovalbumin in 25 ml extraction buffer (A.3.2.2). Filter the solution through a 0,22 µm filter (A.4.4) and determine
the true concentration of ovalbumin by using an UV spectrophotometer to measure the absorbance at 280 nm
3)
using a quartz cuvette (A.4.7). If the absorbance is divided by 0,715 it will give the exact concentration in
mg/ml. The solution is stable for 2 days under refrigeration or for 2 months frozen at -18 °C. Thawing requires
heating to 45 °C for 15 min.
A.6.3.2 Protein standard solutions
Prepare serial dilutions of the protein stock solution (A.6.3.1) using the extraction buffer (A.3.2.2), to make
solutions with nominal concentrations of about 100 µg/ml, 50 µg/ml, 20 µg/ml, 10 µg/ml, 5 µg/ml and 2 µg/ml.
Use extraction buffer as a blank. The solutions are stable for 2 days refrigerated or for 2 months frozen at -
18 °C. Thawing requires heating to 45 °C for 15 min.
A.6.4 Precipitation and concentration of protein
A.6.4.1 Carry out the procedure in duplicate at (25 ± 5) °C.

3)
Assuming a molecular weight of 43000 D and a molar extinction of 30745 at 280 nm and pH 7,4, the extinction of 1
mg/ml ovalbumin in 0,1 M TES buffer pH 7,4 is 0,715 using a light path of 1 cm [3].
A.6.4.2 Accurately transfer 1 ml each of the blank, protein standard solutions (A.6.3.2) and the four glove
extracts (A.6.2.5) into micro tubes (A.4.6). Add 0,1 ml of DOC (A.3.5), mix by vortexing and allow to stand for
10 min. Add 0,1 ml of TCA (A.3.6) and 0,1 ml PTA (A.3.7), mix by vortexing and allow to stand for a further
30 min.
A.6.4.3 Centrifuge at 6000 g for 15 min. Decant the supernatant liquid and drain for 5 min by inverting
each centrifuge tube on an absorbent paper.
A.6.4.4 Add 0,2 ml of 0,1 M sodium hydroxide solution (A.3.4) to each tube including the blank. Mix on a
vortex mixer to re-dissolve the precipitated proteins. Ensure that the proteins are completely re-dissolved to a
clear solution. Depending on the glove this sometimes needs an overnight standing refrigerated at (5 ± 3) °C.
If any precipitate remains, add a further, measured quantity of the sodium hydroxide solution by 0,2 ml
increments up to a total of 1 ml and use a 0,2 ml aliquot for subsequent steps. It can be useful to dilute the
extract of such samples prior to precipitation.
NOTE The process of concentrating the protein by precipitation and re-dissolving is intended to purify the protein and
to rid it of interferants. It is inevitable that during this process a certain amount of protein is lost and it is assumed for
purposes of the test that the same percentage will be lost from the protein standard solutions as from the test sample
extracts. However, the loss should be kept to the minimum as gross losses would not be reproducible.
A.6.5 Colour development
A.6.5.1 The method described here is adapted to the commercial kit used for validation. Other kits or
reagents prepared from off-shelf chemicals can require other volumes and incubation times.
A.6.5.2 Add 0,125 ml Reagent A (A.3.3.1) into each micro tube containing the re-dissolved protein
solutions including the blank. Mix well. Add 1 ml Reagent B (A.3.3.2), cap the tube, vortex and allow the colour
to develop fully for 30 min. Should precipitation occur at this stage, centrifuge or filter to remove the precipitate
before measuring the absorbance.
A.6.6 Measurement
A.6.6.1 Micro-plate reader
Pipette a consistent volume of the solution (A.6.5.2) to the well of a microtitre plate (A.4.8) so that the well is
almost full, e.g. 490 µl in a 500 µl well. Measure the absorbance versus the blank at a specific wavelength in
the range of 600 nm to 750 nm.
NOTE It is important for uniform results that the standard solutions, together with the glove extracts, be analysed
together within 1 h after a stable colour has developed.
A.6.6.2 Spectrophotometer
Transfer the solution (A.6.5.2) to a cuvette (A.4.9) and measure the absorbance against the blank at a specific
wavelength in the range 600 nm to 760 nm.
NOTE It is important for uniform results that the standard solutions, together with the glove extracts, be analysed
together within 1 h after a stable colour has developed.
A.7 Expression of results
A.7.1 Calculation
A.7.1.1 Calibration curve
Calculate the average absorbance of the duplicate determinations. If the individual values differ by more than
20 % repeat the determination. Prepare the calibration curve by plotting average absorbance measurements
against the true concentration of the original protein standard solutions as shown in Figure A.2. The calibration
curve should be linear over the range of 0 µg to 100 µg protein/ml in the original protein standard solutions.
NOTE Some protein is lost during the concentration process. It is assumed that the same percentage of protein is
lost from the standards as from the test samples during the concentration process.
A.7.1.2 Concentration of extract
For each of the four extracts, calculate the average absorbance of the duplicate determinations (see A.6.4.1).
If the individual values differ by more than 20 % repeat the determination. Determine the concentrations of the
extracted samples (C) in µg/ml extract by reading them directly from the linear portion of the curve.
NOTE In the event that the calibration curve is non-linear, the value can be calculated by quadratic regression. It is
suggested that commercial computer software for curve fitting and calculation of unknown concentrations is more practical.
A.7.2 Results
The protein content of each sample is given by
(V ⋅C⋅F)
P =
m
where
P is extractable protein in µg/g of glove;
V is the volume of extraction medium used in ml;
C is the protein concentration of the extract in µg/ml;
F is the dilution factor;
NOTE F is the real volume of NaOH solution in ml used to re-dissolve the protein divided by 0,2.
m is the mass of glove extracted in g (A.6.2.6).
Report the mean protein content of the four determinations of glove extracts.
Dimensions in millimetres
Key
1 outer glove (glove 1)
2 inner glove (glove 2)
3 extraction buffer
4 dye solution
5 glove clamp
Figure A.1 — Extraction of gloves (cross section)

Y absorbance at 750 nm    1 absorbance
X ovalbumin concentration (µg/ml)   2 computer generated polynoma of best fitting
Y = -4E - 0,5 x² + 0,013 x + 0,024 7
Concentration Absorbance
2,1 0,036
5,2 0,099
10,4 0,159
20,8 0,291
52,0 0,583
104,0 0,945
Figure A.2 — A typical standard curve measured in a spectrophotometer at 750 nm with 1 cm path
length
A.7.3 Statistical information
Nine laboratories participated in an inter-laboratory exercise as part of a scientific study supported by the EU
in 1996 to 1998 and published in the final report MAT 1 – CT 940060 European Commission Directorate
General XII. In this experiment both were tested the precision of the Lowry method and the precision of the
whole procedure including the extraction. The whole method includes additionally the variation of the protein
content from glove to glove, which is in some cases much higher than the variation of the method. The results
are summarized in Table A.1.
Table A.1 — Statistical information
Number of Number of Number Mean Repeatability Reproducibility
measurements extracts of days coeffiecient of coefficient of
in µg/ml
variation in % variation in %
(within (between
laboratories) laboratories)
Glove 8 triplicates 1 used by all 1 63,9 4,9 9,6
extract participants
Glove 15 triplicates 5 61,7 6,8 6,3
extract
Glove A 5 triplicates 5 1 88,8 7,9 22,5
Glove A 5 triplicates 5 5 84,5 6,1 20,3
Glove B 3 triplicates 3 1 109 20,2 23,3
Glove C 3 triplicates 3 1 727 8,3 23,0
Glove D 3 triplicates 3 1 46,5 10,1 31,8
Mean extract without extraction procedure 5,0 8,0
Mean whole procedure (glove A to D) 10,5 24,2

The limit of quantification was set to 10 µg/g because it is dependent on the thickness (weight) of the gloves. It
was found to be between 1 µg/g and 5 µg/g.

A.8 References
[1] Lowry OH, Rosebrough, NJ, Farr AL, Randall RJ, Protein measurement with Folin Phenol reagent. J Biol
Chem 1951 : 193 : 265-275
[2] ASTM D 5712:1995, Standard test method for analysis of protein in natural rubber and its products
[3] Kidwai SA, Ansari AA, Salahuddin, Effect of succinylation (3-carboxypropionylation) on the conformation
and immunological activity of ovalbumin. Biochem J 1976 : 155 : 171-180

Annex B
(informative)
Immunological methods for the measurement of natural rubber latex
allergens
B.1 Introduction
Immediate allergic reactions to natural rubber latex (NRL) proteins are recognized as an important medical
and occupational health problem. A major source of sensitisation has been considered to be proteins or
peptides eluting from protective NRL gloves [1].
Although the amount of extractable total protein usually correlates reasonably well with the allergen content of
NRL gloves measured by skin prick test (SPT) or human IgE-based assays [2], [3], [4], [5], the total protein
methods also measure non-allergenic proteins that are unikely to be relevant in NLR allergy. Therefore, there
has been an increasing need for methods capable of specifically and accurately measuring allergens in NRL
goods. It is agreed that allergen-specific assays would provide much more accurate and reliable information
both for regulatory purposes and for monitoring manufacturing processes. The availability of specific assays
has, however, been scanty. Moreover, the still incomplete knowledge on the overall significance of the wide
spectrum of NRL allergens has made it difficult to decide which of the numerous allergens present in the NRL
source material should be measured.
Semiquantitative methods, such as RAST-inhibition and IgE ELISA inhibition, based on the use of human IgE
antibodies, have been available for several years in research laboratories. Drawbacks of these methods are
that they are difficult to standardise and they suffer from limited availability of human sera containing clinically
relevant latex-specific IgE antibodies. In addition, it should be noted that the standards used do not equate to
glove proteins. The principle that an ideal test for assessing allergenic potential of NRL products should be
based on specific allergen quantification has recently been adopted and endorsed in the ongoing
standardisation work both in Europe [6], [7] and the US [8].
Substantial progress has recently been made in the development of specific and quantitative assays for
individual NRL allergen quantification [9], [10]. These new assays, based on the capture-enzyme
immunoassay (EIA)-principle and on the use of monoclonal antibodies and purified or recombinant allergens,
are specific; they can be properly standardized and are of sufficient sensitivity and reproducibility. In this
informative annex current methods for NRL allergen measurement are reviewed.
B.2 Natural rubber latex allergens in manufactured rubber products
Of the some 250 different proteins or polypeptides demonstrated in the NRL source material, the liquid latex of
the rubber tree, Hevea brasiliensis, about one fourth to one fifth have been shown to bind with IgE and
represent allergens [11], [12]. The mixture of plant proteins in source material reflects the stress response of
the rubber tree to wounding (the tapping procedure). Several of these proteins are defense proteins that have
been well preserved in plants during evolution. The structural homologies shared with these proteins provide
the molecular basis for the common cross-reactions of latex-allergic patients’ IgE towards various plant
proteins. It is likely that all of the significant allergens probably are present in the liquid NRL but, as referred to
above, the majority of proteins and polypeptides present in the NRL source material are likely to be irrelevant
in the assessment of allergenic properties of manufactured NRL products. The WHO/IUIS Allergen
Nomenclature Committee lists (March 2004) 13 NRL allergens characterized t the molecular level
(www.allergen.org.), most of which have been cloned and produced by recombinant DNA techniques.
An optimal test should be designed to accurately measure all allergens that can be present in manufactured
rubber products. This could include epitopes present on the natural proteins, as well as new epitopes on the
break down products resulting from the harsh rubber manufacturing processes. So far a limited number of
allergens has been demonstrated in NRL products. The current literature supports the contention that at least
Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02, and/or fragments or polymers of them carrying IgE-binding
epitopes, can be present in manufactured products [13], [14], [15], [16], [17], [18]. Whether additional
allergens, such as Hev b2, Hev b7 or Hev b13 [19], turn out to be important rubber product-specific allergens,
still awaits confirmation.
B.3 Methods for measuring natural rubber latex allergens
B.3.1 Qualitative methods
Immunoelectrophoretic methods and immunoblotting techniques, used extensively in the early 1990s,
demonstrated and tentatively characterised several NRL proteins to which IgE from sera of NRL-allergic
patients bind. It is however today agreed that these methods alone cannot be used for reliable identification of
allergens [11], [12], [20], [21].
B.3.2 Semiquantitative methods
B.3.2.1 Skin prick testing in voluntary latex-allergic subjects
Allergenicity of NRL extracts can be semiquantitatively assessed by skin prick testing (SPT) in a statistically relevant
number of NRL-allergic patients. The size of the reaction is dependent on and proportional to the quantity of the
allergens t
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