EN 1104:2026

• Abstract:

  This document specifies a test method for the determination of the transfer of antimicrobial constituents from paper and board materials and articles intended for food contact.; NOTE The use of this document is determined by legislation pertaining to paper and board intended to come into contact with foodstuffs.

• Status: Not Published
• Publication Date: 15-Dec-2026
• ICS: 67.250 - Materials and articles in contact with foodstuffs
• ICS: 85.060 - Paper and board
• Technical Committee: CEN/TC 172 - Pulp, paper and board
• Drafting Committee: CEN/TC 172/WG 3 - Analytical methods for the assessment of paper and board in contact with foodstuffs
• Current Stage: 5060 - Closure of Vote - Formal Approval
• Start Date: 08-Oct-2026
• Due Date: 16-Sep-2026
• Completion Date: 08-Oct-2026
• Ref Project: kSIST FprEN 1104:2026 - Paper and board intended to come into contact with foodstuffs - Determination of the transfer of antimicrobial constituents

• Revises: EN 1104:2018 - Paper and board intended to come into contact with foodstuffs - Determination of the transfer of antimicrobial constituents
• Effective Date: 07-Aug-2024

EN 1104:2026 is a CEN European Standard for paper and board intended to come into contact with foodstuffs. It specifies a laboratory test method for determining the transfer of antimicrobial constituents from paper and board materials and articles, and it is used where food-contact legislation points to this test.

What does EN 1104:2026 specify?

EN 1104:2026 specifies one test method for checking whether antimicrobial constituents migrate from paper and board intended for food contact. The method is built around two challenge organisms, one bacterium and one fungus, so it can show transfer that affects both bacterial and fungal growth.

The document is organized as a laboratory method standard. Clause 1 gives the scope, Clause 2 lists the normative references, Clause 3 defines the key term used to judge the result, Clauses 4 to 9 describe the principle, equipment, media, test organisms, sampling, procedure and evaluation, and Clause 10 states what the test report must include. Annex A is informative and helps the laboratory interpret the different growth patterns seen on the plates.

The standard is focused on test performance and result interpretation. It is not a general food-contact rule; the note under the scope points readers to the legislation that determines when this method is used.

What are the key requirements of EN 1104:2026?

EN 1104:2026 is a plate diffusion-style microbiological test. In practice, the laboratory prepares agar plates inoculated with test organisms, places paper or board test pieces on the semi-solid medium, incubates them, and then checks whether growth is suppressed around the pieces.

Sampling and test pieces

Clause 7 requires sampling according to EN ISO 186. The samples are handled only by the edges, kept in a sterile vessel or wrapped immediately, and 20 circular test pieces are punched from at least 10 samples per unit. If the material is not homogeneous, the test has to represent each area, and if the food-contact side is unknown, both sides are tested. This matters because the result is judged per side or per area, so the sampling plan has to match the material structure.

Microorganisms and culture media

Clause 6 uses Bacillus subtilis subsp. spizizenii DSM 347 and Aspergillus niger DSM 1957. The document gives preparation media for working cultures and for the actual inhibition test, including medium compositions, pH targets and sterilization conditions, and it allows commercial ready-to-use media when they are comparable. This matters because the test result depends on healthy, standardized growth of the challenge organisms.

Plate preparation and controls

Clause 8 requires at least 3 Petri dishes for each test. For Bacillus subtilis, the test agar is inoculated to a density of 10^4 spores per ml; for Aspergillus niger, the density is 10^5 spores per ml. Three test pieces are placed on each plate, giving 9 test pieces per microorganism and tested area.

Each analysis also needs a negative control and a positive control. The negative control checks that the microorganism grows normally without sample or reference substance, while the positive control checks sensitivity by using penicillin G for Bacillus subtilis and amphotericin B or an isothiazolinon solution for Aspergillus niger. In practice, these controls tell the laboratory whether the run is valid before the sample result is accepted.

Incubation conditions

Clause 8 includes a 2 h prediffusion step at 5 °C ± 3 °C, then incubation for 3 days at 30 °C ± 2 °C for Bacillus subtilis or 5 days at 25 °C ± 2 °C for Aspergillus niger. Plates are stored with the lid underneath to avoid condensation falling onto the sample. The plates may then be read immediately or kept refrigerated for up to 3 days. This matters because timing and condensation can change the visible growth pattern around the test pieces.

How the result is judged

Clause 3 defines an inhibition zone as a growth-free area around the test piece caused by released water-soluble antimicrobial constituents, and it gives a minimum width of 2 mm at the edge for proving its presence. Clause 9 says each of the 9 test pieces is recorded for presence or absence of an inhibition zone, and if at least 2 of the 9 test pieces show an inhibition zone, the tested area or side is reported as positive. That gives users a clear pass/fail style conclusion for each side or area tested.

Reporting

Clause 10 requires the report to identify the standard, the test date and place, the material and test-piece diameter, the result, any deviations from the method, and any unusual observations. This matters for traceability, especially when the result is used in conformity work or supplier discussions.

What terms does EN 1104:2026 define?

Inhibition zone - an area around a test piece where the test microorganism does not grow because antimicrobial constituents have been released from the material.

Test piece - a circular piece cut from the sample and placed on the inoculated agar for the test.

Working culture - the prepared culture used to build the inoculating suspension for Bacillus subtilis or Aspergillus niger.

Inoculating spore suspension - the spore suspension added to the test agar so the plate contains a defined number of challenge spores.

Negative control - a plate without sample or reference substance, used to confirm normal microbial growth.

Positive control - a plate with discs impregnated with the reference antibiotic or antifungal agent, used to confirm that the test organism responds as expected.

Test microorganism - the microorganism used to challenge the sample; here Bacillus subtilis and Aspergillus niger.

Who uses EN 1104:2026?

EN 1104:2026 is used by laboratories that perform food-contact testing on paper and board. It is also relevant to paper and board manufacturers, converters, quality managers, and buyers who need to check whether antimicrobial constituents can transfer from a material or article intended for food contact.

The standard is mainly used for compliance support, product qualification, and supplier verification. In practice, it helps users decide whether a material shows an inhibition effect on the test organisms and how that result should be recorded in a test report.

What changed in EN 1104:2026 from the previous edition?

EN 1104:2026 supersedes EN 1104:2018. The foreword lists these technical changes:

• Clause 3.1 was updated to match current terminology rules.
• Clause 5 and the annex interpretation clauses were revised so measuring the width of the inhibition zone is part of the assessment.
• Clause 5.3 and Clause 7 were clarified for the punch iron and the wording around aseptic or sterile handling.
• Several media clauses were updated to simplify composition wording and storage information.
• The microorganism-preparation clauses were streamlined and clarified.
• The term “conidia” was replaced by “spores” for Aspergillus niger.
• The positive-control wording was updated for amphotericin B and the isothiazolinon solution.
• Clause 7 clarifies the number of test pieces and how the tested sides are handled.
• Clause 8 was rephrased for clearer plate preparation.
• Clause 8.3 allows plate evaluation after refrigerated storage for up to 3 days.
• Clause 9 allows repeating the assay without requiring a new inoculating suspension.
• Annex A corrects and clarifies the control interpretation text.

Which standards are used with EN 1104:2026?

EN 1104:2026 cites three normative references:

• EN ISO 186 - used for sampling paper and board so the test pieces are representative.
• EN ISO 7218 - provides the general microbiological laboratory requirements and guidance used for the equipment and handling conditions.
• EN ISO 11133 - gives the rules for preparing, storing and performance-testing culture media, including the water to use and media quality expectations.

What does the EN 1104:2026 document contain?

EN 1104:2026 contains the laboratory setup for the test, including apparatus, reagents, media formulations and preparation steps. It also gives the strains to use, the way to prepare working cultures and spore suspensions, and the target spore densities for the inoculated agar.

The document includes the sampling plan, test-piece preparation, plate arrangement, incubation conditions and the control plates needed for validation. It also sets out how to read the plates and how many test pieces must show an inhibition zone before the tested area or side is reported as positive.

Annex A is a practical interpretation guide. It uses figures to show normal growth, inhibition zones, modified growth, microbial contamination and, for Aspergillus niger, sporulation activation, so laboratories can distinguish a true inhibition zone from other edge effects.

SLOVENSKI STANDARD
oSIST prEN 1104:2025
01-december-2025
Papir, karton in lepenka v neposrednem stiku z živili - Določanje izločanja
antimikrobnih snovi
Paper and board intended to come into contact with foodstuffs - Determination of the
transfer of antimicrobial constituents
Papier und Pappe vorgesehen für den Kontakt mit Lebensmitteln - Bestimmung des
Übergangs antimikrobieller Bestandteile
Papier et carton destinés à entrer en contact avec les denrées alimentaires -
Détermination du transfert des constituants antimicrobiens
Ta slovenski standard je istoveten z: prEN 1104
ICS:
67.250 Materiali in predmeti v stiku z Materials and articles in
živili contact with foodstuffs
85.060 Papir, karton in lepenka Paper and board
oSIST prEN 1104:2025 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 1104:2025
oSIST prEN 1104:2025
DRAFT
EUROPEAN STANDARD
prEN 1104
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2025
ICS 67.250; 85.060 Will supersede EN 1104:2018
English Version
Paper and board intended to come into contact with
foodstuffs - Determination of the transfer of antimicrobial
constituents
Papier et carton destinés à entrer en contact avec les Papier und Pappe vorgesehen für den Kontakt mit
denrées alimentaires - Détermination du transfert des Lebensmitteln - Bestimmung des Übergangs
constituants antimicrobiens antimikrobieller Bestandteile
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 172.
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, Türkiye 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
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 1104:2025 E
worldwide for CEN national Members.

oSIST prEN 1104:2025
prEN 1104:2025 (E)
Contents Page
European foreword . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Apparatus . 5
6 Reagents and media . 6
6.1 General. 6
6.2 Non-ionic wetting agent. 6
6.3 Nutrient medium for the preparation of Bacillus subtilis spores . 6
6.3.1 Composition . 6
6.3.2 Preparation . 6
6.4 Sabouraud nutrient medium for the preparation of Aspergillus niger spores . 7
6.4.1 Composition . 7
6.4.2 Preparation . 7
6.5 Nutrient medium for the inhibition test with Bacillus subtilis . 7
6.5.1 Composition . 7
6.5.2 Preparation . 7
6.6 Modified Sabouraud nutrient medium for the inhibition test with Aspergillus niger . 7
6.6.1 Composition . 7
6.6.2 Preparation . 8
6.7 Tryptone salt solution . 8
6.7.1 Composition . 8
6.7.2 Preparation . 8
6.8 Test microorganisms . 8
6.8.1 General. 8
6.8.2 Preparation of working cultures . 8
6.8.3 Preparation inoculating spore suspension of Bacillus subtilis . 9
6.8.4 Preparation of inoculating spore suspension of Aspergillus niger . 9
6.8.5 Concentrations of spores of inhibition . 9
6.9 Positive controls . 9
6.9.1 Antibiotic . 9
6.9.2 Antifungal agent . 10
7 Sampling and preparation of test pieces . 10
8 Procedure . 10
8.1 General. 10
8.2 Preparation of plates . 10
8.2.1 General. 10
8.2.2 Test with Bacillus subtilis . 10
8.2.3 Test with Aspergillus niger . 11
8.3 Incubation. 11
9 Evaluation . 11
oSIST prEN 1104:2025
prEN 1104:2025 (E)
10 Test report . 12
Annex A (informative) Guidelines for interpreting results . 13

oSIST prEN 1104:2025
prEN 1104:2025 (E)
European foreword
This document (prEN 1104:2025) has been prepared by Technical Committee CEN/TC 172 “Pulp, paper
and board”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 1104:2018.
This document includes the following significant technical changes with respect to EN 1104:2018:
a) 3.1 has been updated according to current regulations for terms and definitions;
b) Clause 5, A.3.2.3 and A.3.3.2: The statement that the measuring of the width of the inhibition zone is
not compulsory has been removed as the result requires to measure the width of the inhibition zone
in order to check the 2 mm criteria;
c) 5.3 and Clause 7: the precision regarding the punch iron has been updated; usage of the term
“aseptic” or “sterile” which both signifies germ-free;
d) 6.3.1, 6.4.1, 6.5.1, 6.7.1 regarding compositions of media: the term “tryptic digest” for the casein has
been deleted as some media indicate pancreatic digest which is the same thing;
e) 6.3.2, 6.4.2, 6.5.2, 6.6.2: unnecessary details regarding media containers have been deleted;
precision about storage conditions have been updated;
f) 6.8.2, 6.8.3, 6.8.4, 6.8.5: unnecessary details have been deleted and details for a better
understanding of the procedure have been added;
g) 6.8.5: the term “conidia” has been replaced by the term “spores” to harmonize wording since both
terms have the same meaning;
h) 6.9.2: the term “solution” for the amphotericin B has been deleted as it could be bought as pre-
impregnated discs; the note has been deleted as the isothiazolinone solution is not commercially
available;
i) Clause 7: the phrase “for each microorganism” has been deleted as 20 test pieces for both
microorganisms for one tested area is enough (9 test pieces by microorganism and by tested
area/side);
j) Clause 7 and Clause 8: a clarification about the sides tested has been added;
k) 8.2.2 and 8.2.3 have been rephrased for a better understanding;
l) 8.3: evaluation of the plates has been updated since it could be done at least 3 days after the end of
the incubation if the plates are stored at (5 ± 3) °C;
m) Clause 9: the precision “with a new inoculating suspension” has been deleted to allow repeating the
assay with the same inoculating suspension;
n) A.3.2.4: the term “positive” has been replaced by “negative” since the negative control is the nutrient
agar plate without test pieces and inoculated with the test microorganism.
oSIST prEN 1104:2025
prEN 1104:2025 (E)
1 Scope
This document specifies a test method for the determination of the transfer of antimicrobial
constituents from paper and board materials and articles intended for food contact.
NOTE The use of this document is determined by legislation pertaining to paper and board intended to come
into contact with foodstuffs.
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 186, Paper and board — Sampling to determine average quality (ISO 186)
EN ISO 7218, Microbiology of the food chain — General requirements and guidance for microbiological
examinations (ISO 7218)
EN ISO 11133 , Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media (ISO 11133)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology 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
inhibition zone
obvious area in which growth is absent and which forms around test pieces placed on a nutrient
medium inoculated with a preselected test microorganism, due to the release of water-soluble
antimicrobial constituents
Note 1 to entry: The proof of the presence of an inhibition zone is provided by the absence of test microorganism
growth (translucent zone) in a minimum of 2 mm width zone at the edges of the test pieces.
4 Principle
A prepared nutrient medium is mixed with an appropriate inoculum and poured into Petri dishes. The
test pieces are placed on the nutrient medium before its complete solidification and incubated. When
incubation is terminated, the existence of an inhibition zone is an indicator of the release of
antimicrobial constituents.
The test is performed with a bacterium, bacillus subtilis, and with a fungus, aspergillus niger.
5 Apparatus
All laboratory equipment and parts of the equipment, according to EN ISO 7218, shall be used, in
addition to the following.
Under revision.
oSIST prEN 1104:2025
prEN 1104:2025 (E)
5.1 Punch iron, with a diameter = 10 mm to 15 mm, sterilizable or aseptic.
5.2 Pressing device, suitable for pressing the test pieces on the agar plates (e.g. Drigalski spatula).
5.3 Zone reading device, allowing the measurement of the width of the inhibition zone.
6 Reagents and media
6.1 General
When water is mentioned in a formula, distilled or purified water shall be used, according to
EN ISO 11133.
For preparing culture media, general requirements are described in EN ISO 11133. Culture media shall
be prepared as follows, or from commercially available dehydrated culture media according to the
manufacturer's instructions. Ready-to-use medium may be used when its composition and/or growth
performance is comparable to that of formula given in 6.3 and 6.4 (according to EN ISO 11133).
6.2 Non-ionic wetting agent
For example, polyoxyethylenesorbitane monooleate can be used.
6.3 Nutrient medium for the preparation of Bacillus subtilis spores
6.3.1 Composition
— Beef extract: 3,0 g;
— Peptone from casein: 5,0 g;
— Sodium chloride: 5,0 g;
— Agar-agar: 12,0 g;
— Water: 1,0 l.
NOTE The addition of 10 mg/l MnSO4 to the nutrient medium for Bacillus subtilis will support the formation
of spores.
6.3.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Dispense one part of the nutrient medium into flasks (for instance 100 ml or 200 ml flasks) for further
storage and the other part into test tubes for the preparation of working cultures (for instance 10 ml
per tube in 15 to 20 tubes).
Sterilize flasks and test tubes in an autoclave for 15 min at 121 °C. After sterilization, the pH of the
medium shall be equivalent to 7,2 ± 0,2 when measured at 45 °C. Before solidification, position the test
tubes in such a way that the nutrient medium solidifies with a sloping surface.
Medium may be stored according to EN ISO 11133.
oSIST prEN 1104:2025
prEN 1104:2025 (E)
6.4 Sabouraud nutrient medium for the preparation of Aspergillus niger spores
6.4.1 Composition
— Peptone from casein: 5,0 g;
— Meat peptone: 5,0 g;
— D-(+)-glucose C H O ·H O (dextrose): 40,0 g;
6 12 6 2
— Agar-agar: 10,0 g to 15,0 g;
— Water: 1,0 l.
6.4.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Proceed as described in 6.3 by dispensing the medium into flasks and test tubes.
Sterilize in an autoclave for 15 min at 121 °C. After sterilization, the pH of the medium shall be
equivalent to 5,6 ± 0,2, when measured at 25 °C.
Medium may be stored according to EN ISO 11133.
6.5 Nutrient medium for the inhibition test with Bacillus subtilis
6.5.1 Composition
— Peptone from casein: 3,45 g;
— Meat peptone: 3,45 g;
— Sodium chloride: 5,1 g;
— Agar-agar: 13,0 g;
— Water: 1,0 l.
6.5.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Dispense the medium into flasks.
Sterilize in an autoclave for 15 min at 121 °C. After sterilization, the pH medium shall be equivalent to
6,0 ± 0,2, when measured at 45 °C.
Cool the flasks to below 60 °C for the preparation of the inoculation medium (8.2.2) or allow to solidify
if they have to be stored before use.
Medium may be stored according to EN ISO 11133.
6.6 Modified Sabouraud nutrient medium for the inhibition test with Aspergillus niger
6.6.1 Composition
— Peptone from casein: 5,0 g;
— Meat peptone: 5,0 g;
oSIST prEN 1104:2025
prEN 1104:2025 (E)
— D-(+)-glucose C H O ·H O (Dextrose): 10,0 g;
6 12 6 2
— Maltose C H O ·H O: 10,0 g;
12 22 11 2
— Agar-agar: 10,0 g to 15,0 g;
— Water: 1,0 l.
6.6.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Dispense the medium into flasks.
Sterilize in an autoclave for 15 min at 121 °C. After sterilization, the pH of the medium shall be
equivalent to 5,4 ± 0,2, when measured at 45 °C.
Cool the flasks to below 60 °C for the preparation of the inoculation medium (8.2.2) or allow to solidify
if they have to be stored before use.
Medium may be stored according to EN ISO 11133.
6.7 Tryptone salt solution
6.7.1 Composition
— Peptone from casein: 1,0 g;
— Sodium chloride: 5,0 g;
— Water: 1,0 l.
6.7.2 Preparation
Dissolve the components in the water by mixing while heating.
Dispense in
...