ISO 21326:2019

INTERNATIONAL ISO
STANDARD 21326
First edition
2019-06
Textiles — Test methods for
determining the efficiency of products
against house dust mite
Textiles — Méthodes d'essai pour déterminer l'efficacité des produits
contre les acariens
Reference number
©
ISO 2019
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 Repelling method by using Petri-dish . 2
4.2 Repelling method by using glass tube, Method A and Method B . 3
4.3 Proliferation method by using Petri-dish Method A and using vial Method B . 3
4.4 Penetration method . 3
5 Preparation of test . 3
5.1 Reagents. 3
5.2 Apparatus . 4
6 Reference sample . 5
7 Preparation of mite medium. 5
8 Test conditions . 6
8.1 Condition for work area . 6
8.2 Testing conditions . 6
9 Test methods . 6
9.1 Repelling method by using Petri dish. 6
9.2 Repelling method by using glass tube . 6
9.3 Proliferation method by using Petri dish (Method A) and using vial (Method B) . 6
9.4 Penetration method . 6
10 Test report . 7
10.1 Overview . 7
10.2 General . 7
10.3 Repelling methods . 7
10.4 Proliferation methods . 7
10.5 Penetration method . 7
Annex A (normative) Preparation of mite medium . 8
Annex B (normative) Counting methods for live mites .10
Annex C (normative) Repelling method by using Petri dish .13
Annex D (normative) Repelling method by using glass tube .17
Annex E (normative) Proliferation method .21
Annex F (normative) Penetration method .26
Bibliography .30
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso
.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 38, Textiles.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved

Introduction
The World Health Organization's (WHO) statement on the correlation of house dust mite to asthma and
other allergic disorders resulted in the increased number of textile products treated against house dust
mite available to the consumer.
However, the testing method to evaluate the efficacy against house dust mite of textiles has not been
standardized to date. This has caused confusion among consumers because of various testing methods
and results.
The purpose of this test method is to standardize the testing method of efficacy of products against
house dust mite in textiles.
INTERNATIONAL STANDARD ISO 21326:2019(E)
Textiles — Test methods for determining the efficiency of
products against house dust mite
1 Scope
This document specifies test methods for efficiency of chemically or physically treated textile products
against house dust mites.
For the products treated by chemicals against house dust mites, the test methods specified in a) to c)
are applied. For the physically treated products, the test method specified in d) is applied.
a) Repelling method by using Petri dish
This method is applied to carpet, bedding surface fabric, bed sheeting, bed covering and blanket.
b) Repelling method by using glass tube (Methods A and B)
This method is applied to wadding (bedding, etc.) with a fibre content of cotton, wool or synthetic
fibre, feathers/down.
c) Proliferation method by using Petri dish (Method A) and using vial (Method B)
Method A is applied to carpet, bedding surface fabric, bed sheeting, bed covering and blanket.
Method B is applied to wadding.
d) Penetration method
This method is applicable to the outer fabric of a futon, bed sheeting and bed covering. However,
this method is not applicable to the multiple component non-woven fabrics and fibre products with
the high stretch properties such as jersey fabrics.
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.
ISO 105-F02, Textiles — Tests for colour fastness — Part F02: Specification for cotton and viscose
adjacent fabrics
ISO 3310-1, Test sieves — Technical requirements and testing — Part 1: Test sieves of metal wire cloth
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 9237, Textiles — Determination of the permeability of fabrics to air
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 http: //www .electropedia .org/
3.1
house dust mite
universal dominant species belonging to Pyroglyphidae, observed in or on floor surfaces, carpets and
bedding with house dust as example
3.2
efficiency of repellency
efficiency of the treatment in repelling house dust mites
3.3
rate of repellency
ratio of the number of live mites in treated material against the number of live mites in untreated
material
Note 1 to entry: The rate of repellency is expressed in percentage (%) and represents the efficacy of repellency of
treated materials.
3.4
rate of suppression of house dust mite reproduction
ratio of the number of live mites in treated material against the number of live mites in untreated
material tested by the proliferation method
Note 1 to entry: The rate of suppression for reproduction of house dust mite is expressed in percentage (%) and
represents the degree of population control of treated materials.
3.5
culture medium
diet for breeding house dust mite
3.6
mite medium
mix of diet and live house dust mite
3.7
live mite
mites which react when stimulated from the outside
Note 1 to entry: It includes all of larva, nymph (protonymph and tritonymph) and adult mites but exclude eggs.
3.8
population density
degree of live mites in a mite medium
Note 1 to entry: The number of live mites in 1 g of the mite medium.
3.9
quiescent period
period in the stage in which activity of mites almost stops and is observed in the latter half of each
development period of a larva, protonymph and tritonymph
4 Principle
4.1 Repelling method by using Petri-dish
The small Petri dish is placed in the centre position of the large Petri dish. The specimen or the reference
fabric is placed in the small dish with the culture medium. The mite medium with 10 000 mites is spread
in the large Petri dish for the mites to migrate to the small dish. After the designated time has elapsed,
the number of live mites that have intruded into the small Petri dish where the test specimen and
culture medium have been placed is counted. The efficiency of repellency is calculated by comparing
the numbers of live mites for the reference test.
2 © ISO 2019 – All rights reserved

4.2 Repelling method by using glass tube, Method A and Method B
The specimen or the reference fabric is placed in one end of the glass tube in the following order:
stuffing, culture medium and the adhesive tape at the end. The mite medium with 10 000 mites is
placed at the opposite end of the glass tube and the mites migrate in the glass tube all the way. After the
designated time has elapsed, the number of live mites in the stuffing, the culture medium and adhesive
tape which are passing through the specimen or reference are counted. The efficiency of repellency is
calculated by comparing the numbers of live mites for the reference test.
Glass tube Method A is a test method for wadding sample. Glass tube Method B is a test method for
down and feather sample. For Method B only, use a stainless steel mesh disc to fix the position of the
specimen in the glass tube.
4.3 Proliferation method by using Petri-dish Method A and using vial Method B
Mite medium with 50 to 80 live mites per 0,1 g is placed on the specimen in a Petri dish or vial. After
the designated time has elapsed, the numbers of live mites on the Petri dish or vial, the specimen and
the mite medium are counted and summed. The suppression effect of house dust mite reproduction is
calculated by comparing the numbers of live mites for the reference test.
The Petri dish Method A is for the carpet samples, etc. and the vial Method B is for wadding samples.
4.4 Penetration method
The specimen or the reference is placed at the upper end of the glass tube and wrapped by plastic wrap
tightly. The mite medium with 10 000 mites are placed at the bottom of the glass tube on the paper filter
which is tightly sealed. After the designated time has elapsed, the number of the mite on the specimen
or the reference which is passing through is counted through the plastic wrap. Check the efficiency for
prevention by comparing the number of house dust mite passing through the fabric in the specimens
and the reference.
5 Preparation of test
5.1 Reagents
The reagents shall be as follows.
5.1.1 Water, grade 3 according to ISO 3696.
5.1.2 Dried yeast, refined beer yeast, dried, mashed and filtered by a sieve.
5.1.3 Saturated sodium chloride solution, 392 g sodium chloride (NaCl) dissolved in 1 000 ml of water.
5.1.4 Nonionic surfactant solution, 0,1 g of nonionic surfactant [Polyoxyethylene sorbitan mono-
oleate (Polysorbate 80) (CAS Number 9005-65-6)] dissolved in 100 ml of water.
5.1.5 Colouring liquid.
Dissolve,
— 6,0 g of crystal violet (C H ClN · 9H O)
25 30 3 2
or
— 0,6 g of methylene blue (C H N S · Cl · 3H O)
16 18 3 2
in ethanol (C H OH) of 100 ml,
2 5
then,
— dilute with water to make 1 000 ml.
5.2 Apparatus
5.2.1 Oven, capable of maintaining a temperature of 70 °C ± 2 °C.
5.2.2 Incubator (or incubation room), capable of maintaining a temperature of 25 °C ± 2 °C in dark
conditions.
5.2.3 Erlenmeyer flask, with a nominal volume of 50 ml.
5.2.4 Beaker, with a nominal volume of 50 ml and 100 ml.
5.2.5 Large Petri dish, made of glass, with an internal diameter of approximately 90 mm and an
internal height of approximately 20 mm.
5.2.6 Small Petri dish, made of glass, with an external diameter of approximately 45 mm and an
internal height of approximately 15 mm.
5.2.7 Glass tube A, hard-coated glass type with an external diameter of 22,0 mm ± 0,6 mm (wall
thickness 1,2 mm ± 0,2 mm) and a length of approximately 100 mm.
5.2.8 Glass tube B, hard-coated glass type with an external diameter of 40,0 mm ± 0,6 mm (wall
thickness 2,0 mm ± 0,2 mm) and a length of approximately 55 mm.
5.2.9 Rubber band, approximately 70,0 mm lay-flat length and approximately 15,0 mm width.
5.2.10 Vial, made of glass with an external diameter of approximately 30 mm, an internal height of
approximately 63 mm and a volume of approximately 30 ml.
5.2.11 Hot-melt adhesive, with appropriate adhesive strength and no effect to mites.
5.2.12 Filter paper, used for counting mites with a diameter of 70 mm or 90 mm and a grid pattern of
5 mm to 10 mm in square.
5.2.13 Adhesive tape, with appropriate adhesive strength and no effect to mites.
5.2.14 Sticky sheet, with an adhesive strength with the ability to anchor mites that may escape.
3 2 3 2
5.2.15 High-density fabric, with air permeability of 1 cm /cm ·s to 10 cm /cm ·s as specified in
ISO 9237 with a fibre content of 100 % cotton.
5.2.16 Standard woven fabric, 100 % cotton fabric used for the reference of the colour fastness test
specified in ISO 105-F02.
5.2.17 Airtight container, made of polypropylene used for food preservation.
5.2.18 Powder diet, for small laboratory animals (mouse, rat, hamster, etc.) and used for breeding mites.
5.2.19 Balance, with a minimum indication of 1 mg with a scale in graduations of 0,1 mg.
4 © ISO 2019 – All rights reserved

5.2.20 Stereoscopic microscope, with an epi-illumination device with 20 × magnification.
5.2.21 Test sieve, as specified in ISO 3310-1.
The sieve opening shall be as follows.
a) The mesh opening size is the range of 500 μm to 700 μm, used in Annex B.
b) The mesh opening size is 300 μm, used in Annex A.
5.2.22 Stainless mesh disk, a circular-shaped wire mesh of approximately 20 mm diameter to fit the
glass tube A (5.2.7), used for both ends of the specimen to keep thickness 20 mm ± 2mm for feather and
down sample in Annex D.
5.2.23 Suction unit, capable of performing suction filtration with an aspirator (or a suction pump) with
a Buechner funnel attached to a vacuum flask. The bypass can be included for adjusting suction force if
necessary.
5.2.24 Counter, capable of counting from 0 to 9 999.
5.2.25 Plastic wrap, as used for food preservation made of polyethylene or polypropylene.
5.2.26 Stuffing, staple fibre such as 100 % polyester with a fineness of 5 dtex to 8 dtex and fibre length
51 mm to 75 mm for counting the number of mites in Annex D.
6 Reference sample
For all test methods, prepare the reference specimens. Reference specimens are untreated products
similar to the test sample. If it is not available, use the same category of the products with the same
structure to the testing sample.
7 Preparation of mite medium
The mite medium used for the test shall be prepared in accordance with Annex A.
After the colonization procedure specified in Annex A, just before the test, prepare the mite medium for
inoculation using the following procedure.
a) Take 0,025 g or 0,050 g of the mite medium which has been well stirred.
b) Count the number of live mites in the mite medium according to B.2.1 or B.2.2. If the amount of mite
medium used is 0,025 g, count the number of each live mites 8 times. If the amount of mite medium
used is 0,05 g, count the number of each live mites 4 times.
c) Calculate the number of live mites in 1 g of the mite medium and the mass of mite medium with
10 000 mites for inoculation according to Formula (1):
10 000
q= (1)
Nm
where
q is the mass of the mite medium (g) with 10 000 live mites;
Nm is the number of live mites in 1 g of the mite medium.
d) Calculate the coefficient of variation values by using Formula (2) for the judgment of test
effectiveness.
n
xx− n−1
() ()
∑ i
i=1
Cv= ×100 (2)
x
where
Cv is the coefficient of variation (C %);
V
x ,x , … x is the counting value of live mites in 0,025 g (or 0,050 g) of mite medium;
1 2 n
is the average of live mites in 0,025 g (or 0,050 g) of mite medium;
x
n is the number of times of counting of mite medium, n = 8 (or n = 4).
e) Prepare the quantity of the mite medium with 10 000 live mites for inoculation in Annexes C, D and F.
f) From the measured value taken using step b), mix the mite medium including live mites with the
culture medium without mites so that the number of live mites is between 50 and 80 in 0,1 g of the
medium. This is used for inoculation in Annex E.
8 Test conditions
8.1 Condition for work area
Temperature 23 °C ± 5 °C and relative humidity (55 ± 15) % is used for the work area.
8.2 Testing conditions
The culture medium for breeding and all testing apparatus assembly are placed in the airtight container
(5.2.17) with the saturated sodium chloride solution (5.1.3) with a concentration of 10 % to control
relative humidity at (75 ± 5) %. The containers are then placed in the incubator at the temperature of
25 °C ± 2 °C.
9 Test methods
9.1 Repelling method by using Petri dish
The test is carried out according to Annex C.
9.2 Repelling method by using glass tube
The tests are carried out according to Annex D.
9.3 Proliferation method by using Petri dish (Method A) and using vial (Method B)
The tests are carried out according to Annex E.
9.4 Penetration method
The test is carried out according to Annex F.
6 © ISO 2019 – All rights reserved

10 Test report
10.1 Overview
The test report shall contain the following information.
10.2 General
a) a reference to this document, i.e. ISO 21326:2019;
b) test method used;
c) name of species of mites used for test (system, source, origin, etc.);
d) type of sample;
e) identification of sample (product name, etc.);
f) measurement result of population density of mite medium;
g) number of live mites in 1 g of mite medium;
h) any deviation from this document.
10.3 Repelling methods
a) Number of invasion mites on the reference assembly (in the case of glass tube method, number of
attractant mites).
b) Number of invasion mites of the testing assembly (in the case of glass tube method, number of
attractant mites).
c) Rate of repellency.
10.4 Proliferation methods
a) Number of live mites in 0,1 g of the mite medium for inoculation (initial population).
b) Number of live mites of the reference assembly at the time of each observation.
c) Number of live mites of the testing assembly at the time of each observation.
d) Rate of suppression of house dust mite reproduction at the time of each observation.
10.5 Penetration method
a) The number of mites which passed through the standard woven fabric (each of the adult, the larva
and the nymph).
b) The number of mites which passed through testing specimens (each of the adult, the larva and
the nymph).
Annex A
(normative)
Preparation of mite medium
A.1 Species of mites
The following species of mites after colonization shall be used for these test methods.
— Dermatophagoides pteronyssinus, or
— Dermatophagoides farinae
A.2 Colonization procedure
A.2.1 Apparatus
A.2.1.1 Breeding container, a Petri dish is used as a glass container with suitable volume.
A.2.1.2 Airtight container for breeding, with the saturated sodium chloride solution (5.1.3) with the
concentration of 10 % to control relative humidity at (75 ± 5) % and the Petri dish used for breeding
container (A.2.1.1) with mite medium is placed in this container (5
...