prEN 455-3

Medical gloves for single use - Part 3: Requirements and testing for biological evaluation

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.

Medizinische Handschuhe zum einmaligen Gebrauch - Teil 3: Anforderungen und Prüfung für die biologische Bewertung

Dieser Teil von EN 455 legt Anforderungen für die Bewertung der biologischen Sicherheit von medizinischen Handschuhen zum einmaligen Gebrauch fest. Er enthält Anforderungen an die Kennzeichnung und die Informationsangaben entsprechend den angewendeten Prüfverfahren.
ANMERKUNG   Medizinische Handschuhe, die für den einmaligen Gebrauch gekennzeichnet sind, sind Medizinprodukte für den einmaligen Gebrauch nach der Verordnung (EU) 2017/745. Ein Medizinprodukt zum einmaligen Gebrauch bezieht sich auf ein Produkt, das für die Verwendung an einer einzigen Person an einem einzigen Tag vorgesehen ist.

Gants médicaux non réutilisables - Partie 3 : Exigences et essais pour évaluation biologique

La présente partie de l’EN 455 spécifie les exigences permettant d’évaluer la sécurité biologique des gants médicaux non réutilisables. Elle mentionne les exigences d’étiquetage des gants et de diffusion des informations concernant les méthodes d’essai utilisées.
NOTE   Conformément au règlement (UE) 2017/745, les gants médicaux étiquetés non réutilisables sont des dispositifs médicaux à usage unique. Un dispositif médical à usage unique désigne un dispositif conçu pour être utilisé sur une personne physique au cours d’une procédure unique.

Medicinske rokavice za enkratno uporabo - 3. del: Zahteve in preskušanje za biološko ovrednotenje

General Information

• Status: Not Published
• Publication Date: 27-Feb-2024
• ICS: 11.140 - Hospital equipment
• Technical Committee: CEN/TC 205 - Non-active medical devices
• Drafting Committee: CEN/TC 205/WG 3 - Medical gloves
• Current Stage: 4599 - Dispatch of FV draft to CMC - Finalization for Vote
• Start Date: 19-Jan-2023
• Directive: 2017/745 - Regulation (EU) 2017/745 of the European Parliament and of the Council of 5 April 2017 on medical devices, amending Directive 2001/83/EC, Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009 and repealing Council Directives 90/385/EEC and 93/42/EEC
• Mandate: M/565 - Medical devices and in vitro diagnostic medical devices in support of Regulation (EU) 2017/745 of the European Parliament and of the Council and Regulation (EU) 2017/746
• Mandate: M/575 - Medical devices in support of Regulation (EU) 2017/745 and in vitro diagnostic medical devices in support of Regulation (EU) 2017/746
• Ref Project: oSIST prEN 455-3:2022 - Medical gloves for single use - Part 3: Requirements and testing for biological evaluation

Relations

• Revises: EN 455-3:2015 - Medical gloves for single use - Part 3: Requirements and testing for biological evaluation
• Effective Date: 18-Dec-2019

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN 455-3:2022
01-maj-2022
Medicinske rokavice za enkratno uporabo - 3. del: Zahteve in preskušanje 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: prEN 455-3
ICS:
11.140 Oprema bolnišnic Hospital equipment
oSIST prEN 455-3:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 455-3:2022
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oSIST prEN 455-3:2022
DRAFT
EUROPEAN STANDARD
prEN 455-3
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2022
ICS 11.140 Will supersede EN 455-3:2015
English Version
Medical gloves for single use - Part 3: Requirements and
testing for biological evaluation

Gants médicaux non réutilisables - Partie 3: Exigences Medizinische Handschuhe zum einmaligen Gebrauch -

et essais pour évaluation biologique Teil 3: Anforderungen und Prüfung für die biologische

Bewertung

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 205.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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 CEN-CENELEC

Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 455-3:2022 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 5

Introduction .................................................................................................................................................................... 6

1 Scope .................................................................................................................................................................... 7

2 Normative references .................................................................................................................................... 7

3 Terms and definitions ................................................................................................................................... 7

4 Requirements ................................................................................................................................................... 8

4.1 General ................................................................................................................................................................ 8

4.2 Chemicals ........................................................................................................................................................... 8

4.3 Endotoxins ......................................................................................................................................................... 8

4.4 Powder-free gloves ......................................................................................................................................... 9

4.5 Proteins, leachable ......................................................................................................................................... 9

4.6 Labelling ............................................................................................................................................................. 9

5 Test methods ................................................................................................................................................. 10

5.1 Endotoxins ...................................................................................................................................................... 10

5.2 Powder ............................................................................................................................................................. 10

5.3 Proteins, leachable ...................................................................................................................................... 10

6 Test report ...................................................................................................................................................... 11

Annex A (normative) Method for the determination of aqueous extractable proteins in

natural rubber gloves using the modified Lowry assay ................................................................. 12

A.1 Scope ................................................................................................................................................................. 12

A.2 Principle .......................................................................................................................................................... 12

A.3 Reagents .......................................................................................................................................................... 12

A.4 Apparatus ........................................................................................................................................................ 13

A.5 Measurement of protein binding capacity .......................................................................................... 14

A.5.1 General ............................................................................................................................................................. 14

A.5.2 Protein binding capacity of centrifuge tubes ..................................................................................... 14

A.5.3 Protein binding capacity of filter units ................................................................................................ 15

A.6 Procedure........................................................................................................................................................ 15

A.6.1 General ............................................................................................................................................................. 15

A.6.2 Extraction procedure .................................................................................................................................. 16

A.6.3 Protein standard .......................................................................................................................................... 16

A.6.3.1 Stock protein solution ................................................................................................................................ 16

A.6.3.2 Protein standard solutions ....................................................................................................................... 17

A.6.4 Precipitation and concentration of protein ........................................................................................ 17

A.6.5 Colour development .................................................................................................................................... 17

A.6.6 Measurement ................................................................................................................................................. 17

A.6.6.1 Micro-plate reader ....................................................................................................................................... 17

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A.6.6.2 Spectrophotometer ...................................................................................................................................... 18

A.7 Expression of results ................................................................................................................................... 18

A.7.1 Calculation....................................................................................................................................................... 18

A.7.1.1 Calibration curve .......................................................................................................................................... 18

A.7.1.2 Concentration of extract ............................................................................................................................ 18

A.7.2 Results .............................................................................................................................................................. 18

A.7.3 Statistical information ................................................................................................................................ 20

A.8 References ....................................................................................................................................................... 21

Annex B (informative) Immunological methods for the measurements of natural rubber

latex allergens ................................................................................................................................................ 22

B.1 Introduction.................................................................................................................................................... 22

B.2 Natural rubber latex allergens in manufactured rubber products ............................................ 22

B.3 Methods for measuring natural rubber latex allergens ................................................................. 23

B.3.1 Qualitative methods .................................................................................................................................... 23

B.3.2 Semiquantitative methods ........................................................................................................................ 23

B.3.2.1 Skin prick testing in voluntary latex-allergic subjects .................................................................... 23

B.3.2.2 IgE-ELISA inhibition (also known as RAST-Inhibition) ................................................................... 23

B.3.3 Specific quantitative methods .................................................................................................................. 24

B.3.3.1 Capture enzyme immunoassays (EIA) for NRL allergen quantification ................................... 24

B.3.3.2 Background ..................................................................................................................................................... 24

B.3.3.3 Description of capture EIA methods ...................................................................................................... 24

B.3.3.4 Performance of the capture EIAs in comparison with IgE-based allergen assays ................ 24

B.4 Conclusion ....................................................................................................................................................... 25

B.5 References ....................................................................................................................................................... 25

Annex C (informative) Amino acid analysis (AAA) by high pressure liquid chromatography

(HPLC) ............................................................................................................................................................... 28

C.1 Background ..................................................................................................................................................... 28

C.2 Principles of the determination of proteins by HPLC ...................................................................... 28

C.3 Material ............................................................................................................................................................ 28

C.4 Buffers and solutions .................................................................................................................................. 29

C.4.1 Norvalin-100 .................................................................................................................................................. 29

C.4.2 Norvalin-1 ........................................................................................................................................................ 29

C.4.3 o-Phthaldialdehyde (OPA)......................................................................................................................... 29

C.4.4 Boratebuffer ................................................................................................................................................... 29

C.4.5 Stop-solution .................................................................................................................................................. 29

C.4.6 Phosphate buffer ........................................................................................................................................... 30

C.4.7 Solvent 1 ........................................................................................................................................................... 30

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C.4.8 Solvent 2 .......................................................................................................................................................... 30

C.4.9 Sodium carbonate solution (0,1 M) ....................................................................................................... 30

C.5 Hydrolysis ....................................................................................................................................................... 30

C.5.1 Samples ............................................................................................................................................................ 30

C.5.2 Standards ........................................................................................................................................................ 30

C.5.3 Incubation (hydrolysis) ............................................................................................................................. 30

C.5.4 Free amino acids ........................................................................................................................................... 30

C.6 Analysis (HPLC) ............................................................................................................................................ 30

C.6.1 Sample preparation ..................................................................................................................................... 30

C.6.2 Derivatisation ................................................................................................................................................ 31

C.6.3 HPLC .................................................................................................................................................................. 31

C.6.4 Calculation ...................................................................................................................................................... 31

C.7 Examples ......................................................................................................................................................... 31

C.7.1 Standard .......................................................................................................................................................... 31

C.7.2 Glove extract .................................................................................................................................................. 31

C.8 Advantages and disadvantages of the HPLC method ....................................................................... 32

C.8.1 Advantages ..................................................................................................................................................... 32

C.8.2 Disadvantages ............................................................................................................................................... 32

C.9 References ...................................................................................................................................................... 35

Annex ZA (informative) Relationship between this European Standard and General Safety

and Performance Requirements of Regulation (EU) 2017/745 [OJ L 117] aimed to be

covered............................................................................................................................................................. 37

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European foreword

This document (prEN 455-3:2022) has been prepared by Technical Committee CEN/TC 205 “Non-active

medical devices”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 455-3:2015.

Compared to the previous edition the following main changes have been introduced:

a) addition of a note in the Scope on the single-use;
b) addition of a new listing a) in 4.6, Labelling, on the single-use.

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;
— Part 4: Requirements and testing for shelf life determination.
The following part is under development:
— Part 5: Extractable chemical residues.
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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 10993.

Users and choosers who are looking for guidance for selection, storage and use of medical gloves for

single use are referred to CEN/TR 16953:2017.
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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.

NOTE Medical gloves labelled as single use are medical devices for single use only according to the Regulation

(EU) 2017/745. A single use medical device means a device that is intended to be used on one individual during a

single procedure.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 ISO 10993-1:2020, Biological evaluation of medical devices — Part 1: Evaluation and testing within a

risk management process (ISO 10993-1:2018, including corrected version 2018-10)

EN ISO 10993-5:2009, Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity

(ISO 10993-5:2009)

EN ISO 10993-10:2013, Biological evaluation of medical devices — Part 10: Tests for irritation and skin

sensitization (ISO 10993-10:2010)

EN ISO 15223-1:2021, Medical devices — Symbols to be used with information to be supplied by the

manufacturer — Part 1: General requirements (ISO 15223-1:2021)

EN ISO 21171:2006, Medical gloves — Determination of removable surface powder (ISO 21171:2006)

European Pharmacopoeia, General chapter 2.6.14 Bacterial Endotoxins: publisher EDQM - Council of

Europe; 7 allée Kastner, CS 30026, F-67081 Strasbourg; France http://www.edqm.eu/

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
endotoxins

lipo-polysaccharides originating from the outer cell-membrane of Gram-negative bacteria

Note 1 to entry: 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.

3.2
powder

all water insoluble material on the surface of a glove that is removed by washing under the conditions of

the test
[SOURCE: EN ISO 21171:2006, 3.1]
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Note 1 to entry: 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 document

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.).
3.3
process limit
highest value likely to be encountered for a validated manufacturing process
3.4
proteins, allergenic
proteins capable of causing a type I allergic reaction
3.5
proteins, leachable
aqueous proteins and peptides extractable from the final product
3.6
pyrogens

substances creating fever in rabbits which can be related to fever and other adverse reactions in humans

4 Requirements
4.1 General

Based on EN ISO 10993-1:2020 medical gloves are classified as limited contact duration surface devices

and require compliance to EN ISO 10993-5:2009 and EN ISO 10993-10:2013.
4.2 Chemicals

Chemicals which are relevant in accordance with the scope of this document are substances added or

formed during any step of the manufacturing process or in storage which may be available in the final

product.

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.

Gloves shall not be dressed with talcum powder (magnesium silicate).

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 which are known to cause adverse health effects based on current data.

Upon request the manufacturer shall provide evidence of the steps taken to reduce the risk to the end-

user of exposure to chemicals used in the manufacturing process which, based on current data, are known

to cause adverse health effects.

Manufacturers may only declare the absence of a substance if the substance is not used in any part of the

manufacturing process. No compounds shall be used in the manufacture of the product, which are known

to form a substance that is subject of such a declaration.
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.

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4.4 Powder-free gloves

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 strive to minimize the leachable protein level.

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.

NOTE This document 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 described in Annex B.
4.6 Labelling

In addition to the relevant symbols given in EN ISO 15223-1:2021, the following requirements apply:

a) Medical gloves shall be labelled for single use on one individual during a single procedure;

NOTE 1 This is in accordance with the Regulation (EU) 2017/745.

For any medical glove the product labelling shall not include any term suggesting disinfection,

reprocessing or re-use.

b) For any medical glove containing chemical ingredients such as accelerators, antioxidants and

biocides either added during manufacturing or already known to be present in the product, which

are known to cause Type IV allergy, they shall be labelled on the packaging of at least the smallest

packaging unit as follows.

The labelling shall include the following or equivalent warning statement “(Product) contains

chemicals which may cause Type IV allergic reactions.”;

c) For any medical glove containing chemical ingredients such as accelerators, antioxidants and

biocides either added during manufacturing or already known to be present in the product, which

are known to cause Type IV allergy, the product labelling shall not include:

— any term suggesting relative safety, such as low allergenicity, hypoallergenicity or low or

reduced content of Type IV allergens;

— any unjustified indication or misleading claims of the absence or presence of allergens;

d) medical gloves containing natural rubber latex shall be labelled on the packaging of at least the

smallest packaging unit with the EN ISO 15223-1:2021 symbol for latex (reference number 5.4.5).

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.”;

e) the labelling shall include a prominent indication of whether the glove is powdered or powder-free;

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f) 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.
g) for an
...