EN ISO 7405:1997
(Main)Dentistry - Preclinical evaluation of biocompatability of medical devices used in dentistry - Test methods for dental materials (ISO 7405:1997)
Dentistry - Preclinical evaluation of biocompatability of medical devices used in dentistry - Test methods for dental materials (ISO 7405:1997)
Zahnheilkunde - Präklinische Beurteilung der Biokompatibilität von in der Zahnheilkunde verwendeten Medizinprodukten - Prüfverfahren für zahnärztliche Werkstoffe (ISO 7405:1997)
Art dentaire - Evaluation préclinique de la biocompatibilité des dispositifs médicaux utilisées en art dentaire - Méthodes d'essai des matériaux dentaires (ISO 7405:1997)
Dentistry - Preclinical evaluation of biocompatability of medical devices used in dentistry - Test methods for dental materials (ISO 7405:1997)
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 7405:2000
01-januar-2000
Dentistry - Preclinical evaluation of biocompatability of medical devices used in
dentistry - Test methods for dental materials (ISO 7405:1997)
Dentistry - Preclinical evaluation of biocompatability of medical devices used in dentistry
- Test methods for dental materials (ISO 7405:1997)
Zahnheilkunde - Präklinische Beurteilung der Biokompatibilität von in der Zahnheilkunde
verwendeten Medizinprodukten - Prüfverfahren für zahnärztliche Werkstoffe (ISO
7405:1997)
Art dentaire - Evaluation préclinique de la biocompatibilité des dispositifs médicaux
utilisées en art dentaire - Méthodes d'essai des matériaux dentaires (ISO 7405:1997)
Ta slovenski standard je istoveten z: EN ISO 7405:1997
ICS:
11.060.01 Zobozdravstvo na splošno Dentistry in general
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
SIST EN ISO 7405:2000 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 7405:2000
INTERNATIONAL ISO
STANDARD 7405
First edition
1997-08-l 5
Preclinical evaluation of
Dentistry -
biocompatibility of medical devices used in
dentistry - Test methods for dental
materials
Art dentaire - Évaluation préclinique de la biocompa tibilité des dispositifs
- Méthodes d’essai des matériaux
médicaux utilisés en art dentaire
dentaires
Reference number
ISO 7405: 1997(E)
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SIST EN ISO 7405:2000
ISO 7405: 1997(E)
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.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
International Standard ISO 7405 was prepared by Technical Committee ISO/TC 106, Dentistry, in conjunction with
the World Dental Federation (FDI).
This first edition cancels and replaces ISO/TR 7405:1984, which has been technically revised (see Introduction) and
converted into an International Standard.
Annexes A, B and C of this International Standard are for information only.
0 iso 1997
Ail rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronic or mechanical, including photocopying and
microfilm, without permission in writing from the publisher.
international Organization for Standardization
Case postale 56 l CH-121 1 Genève 20 l Switzerland
Internet central @ iso.ch
x.400 c=ch; a=4OOnet; p=iso; o=isocs; s=central
Printed in Switzerland
ii
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Introduction
This International Standard concerns the preclinical testing of dental materials in medical devices used in dentistry.
It has been developed from and supersedes ISO/TR 7405:1984, Biologica! evaluation of dental materials, and its
supplements, and should be read in conjunction with the ISO 10993, Bio/ogica/ evaluation of medical devices, series
of standards. This International Standard differs from ISO/TR 7405 in several important ways. Firstly, it contains
details of test methods applicable only to dental materials. Many test methods previously included in ISO/TR 7405
are now included in the ISO 10993 series of standards and details of them have therefore been excluded from this
standard. Secondly, only test methods for which the members of the committee considered there was sufficient
published data have been included. Thirdly, in recommending test methods, the need to minimize the use of
animals was given a high priority.
The annexes are informative, to encourage the development of in vitro and in vivo test methods which Will futther
reduce the use of animals in the preclinical evaluation of the biocompatibility of medical devices used in dentistry.
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SIST EN ISO 7405:2000
INTERNATIONAL STANDARD o ISO ISO 7405:19$7(E)
Dentistry - Preclinical evaluation of biocompatibility of medical
Test methods for dental materials
devices used in dentistry -
1 Scope
This International Standard specifies methods for the evaluation of biological effects of dental materials. It includes
testing of pharmacological agents that are an integral part of the device under test.
2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this
International Standard. At the time of publication, the editions indicated were valid. All standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.
ISO 1942-2:1989, Dental vocabulary - Part 2: Dental materials
os0 10993-l:- 11, Biological evaluation of medical devices - Part 1: Evaluation and testing
ISO 10993-2:1992, Bio/ogica/ evaluation of medical devices - Part 2: Animal welfare requirements
- Part 3: Tests for genotoxicity, carcinogenicity and
ISO 10993-3: 1992, Bio/ogica/ evaluation of medical devices
reproductive toxicity
ISO 10993~51992, Biological evaluation of medical devices - Part 5: Tests for cytotoxicity: in vitro methods
Part 6: Tests for local effects after implantation
ISO 10993-6:1994, Bio/ogica/ evaluation of medical devices -
- Part 9: Degradation of materials related to
ISOTTR 10993-9: 1994, Bio/ogica/ evaluation of medical devices
biological testing
Part 10: Tests for irritation and sensitiza tion
ISO 10993-I 0:1995, Bio/ogica/ evaluation of medical devices -
- Part 11: Tests for systemic toxicity
ISO 10993-I I:l 993, Bio/ogica/ evaluation of medical devices
- Part 12: Sample preparation and reference materials
ISO 10993-I 2:-z), Bio/ogica/ evaluation of medical devices
- Part 13: Identification and quantification of
ISO 10993-I 3:-2), Biological evaluation of medical devices
degrada tion products from polymers
ISO 10993-I 4:- 21, Biological evaluation of medical devices - Part 14: Identification and quantifica tion of
degradation products from ceramics
.
ISO 10993-I 5:- 2), Biological evaluation of medical devices - Part 15: Identification and quantification of
degradation products from coated and uncoated metals and alloys
ISO 10993-16:- 2) , Biological evaluation of medical devices - Part 16: Toxicokinetic study design for degrada tion
products and leachables.
1) TO be published. (Revision of ISO 10993~1:1992)
2) TO be published
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3 Definitions
For the purposes of this International Standard, the definitions given in ISO 10993-1, ISO 1942-2 and the following
definitions apply.
3.1 medical device: Any instrument, apparatus, appliance, material or other article, including software, whether
used alone or in combination, intended by the manufacturer to be used for human beings solely or principally for the
pu rpose of
- diagnosis, prevention, monitoring, treatment or alleviation of disease, injury or handicap;
- investigation, replacement or modification of the anatomy or of a physiological process;
- control of conception;
and which does not achieve its principal intended action in or on the human body by pharmacological,
immunological or metabolic means, but which may be assisted in its function by such means.
NOTE 1 Devices are different from drugs, and their biological evaluation requires a different approach.
NOTE 2 In this International Standard, the term “medical device” is understood to include dental devices and dental materials.
3.2 dental material: Substance or combination of substances specially prepared and/or presented for the use of
authorized persons in the practice of dentistry and/or its associated procedures.
3.3 final product: Medical device in its “as-used” state.
NOTE - Many dental materials are used in a freshly mixed state, and evaluation of the materials in both freshly mixed and
set conditions should be considered.
3.4 positive-control material: Material or substance which, when tested by the procedure described,
demonstrates the suitability of the procedure to yield a reproducible, appropriate positive or reactive response in the
test system.
3.5 negative-control material; reference material: Material or substance which, when tested by the procedure
described, demonstrates the suitability of the procedure to yield a reproducible, appropriate negative, nonreactive or
background response in the test system.
4 Categorization of medical devices used in dentistry
4.1 Categorization by nature of contact
For the purposes of this International Standard, the classification of medical devices used in dentistry is derived
from ISO 109934. If a device or material cari be placed in more than one category, the more rigorous testing
requirements shall apply. With multiple exposures the decision into which category a device is placed shall take into
account the potential cumulative effect, bearing in mind the period of time over which these exposures occur.
4.1 .l Noncontact devices
These devices do not contact the patient’s body directly or indirectly, and are not included in ISO 10993.
4.1.2 Surface-contacting devices
These devices include those that contact the surface of intact or breached skin, the surface of intact or breached
oral mucosa, and those that contact the external surfaces of dental hard tissue, including enamel, dentine and
cementum.
NOTE - Dentine and cementum are considered as surfaces; e.g. after gingival recession.
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4.1.3 External communicating devices
These devices include dental devices that penetrate and are in contact with oral mucosa, dental hard tissues, dental
pulp tissue or bone, or any combination of these, and are exposed to the oral environment.
4.1.4 Implant devices (see ISO 10993-l)
These devices include dental devices and implants that are pattially or fully embedded within the soft tissue, bone
or pulpodentinal system of the tooth, or any combination of these, and are not exposed to the oral environment.
4.2 Categorization by duration of contact
For the purposes of this International Standard, medical devices used in dentistry are classified by duration of
contact as described in ISO 10993-1, i.e.:
4.2.1 Limited exposure devices
Devices whose single or multiple use or contact is likely to be up to 24 h;
4.2.2 Prolonged exposure devices
Devices whose single, multiple or long-term use or contact is likely to exceed 24 h but not 30 days;
4.2.3 Permanent contact devices
Devices whose single, multiple or long-term use or contact exceeds 30 days.
5 Selection of biological evaluation test
5.1 The general guidance for the selection of biological evaluation tests stated in ISO 10993-I shall apply. Tests
should be selected from the methods described in the ISO 10993 series of standards or in this International
Standard or in both. If tests not included in these International Standards are selected, a statement shall be made
that indicates that the tests described in the International Standards have been considered and shall include a
justification for the selection of other tests.
5.2 The selection of tests and the overall assessment of the results shall be carried out by an expert who has
appropriate chemical, physical and biological data concerning the device and who is aware of the intended
conditions of use.
5.3 The selection of test methods shall be based upon consideration of:
a) the intended use of the material;
b) the tissue(s) which the material may contact;
c) the duration of the contact.
5.4 According to the categorization of the device, tests shall be considered for use as summarized in table 1. This
table indicates which types of test method shall be considered, but not that they are necessarily required to be
carried out. A decision not to carry out a type of test identified in table 1 shall be justified in the test report on each
device. The types of test listed are regarded as a framework for the preclinical evaluation of the biocompatibility of
dental materials. For most types of test, particular methods are identified, although for some devices it is recognized
that alternative methods not included in the International Standards listed may be more appropriate.
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Table 1 - Types of test for preclinical evaluation of biocompatibility of dental materials
.
Group III
Group II
I
Pulp Endo-
Nature of Duration Cytotoxicrty Cytotoxicity Cytotoxicitj Acute Acute Acute Subchronic Skin irritation Sensitization Subchronic Genotoxicity Local effects Pulp
systemic systemic and ISO 10993- 10, systemic ISO 10993-3, after and dontic
tests systemic systemic CaPPiw
toxicity - intra- 6.2 and 6.3 toxicity - clause 4 implantation dentine test usage
=4lidti ISO 7405, toxicity - toxicity - toxicity -
Application Oral cutaneous Application ISO 10993-6, usage ISO 7405, test
annex A Oral Oral
application reactivity by inhalation clauses 4, 5 test 6.4 ISO 7405
application application
bY
inhalation 30 10993-l 1, ISO 10993-l 0, ISO 10993-l 1, and 6 ISO 7405, 6.5
ISO 10993-l 1 ISO 7405,
6.7.1 5.2 6.7.3 6.3
6.5.1 annex B ISO 10993-l 1
and 5.4
6.5.3
X X
s 24 h
>24hto
Sufface-
X X X X
contacting 30 days
devices ’
> 30 days
Extemal
communi-
cating
devices
X x
NOTE - X indicates test shall be considered for use.
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A justification for the choice of all methods shall be included in the test report of each device. This is of particular
importance when methods not included in this International Standard are used.
For convenience, the types of test have been listed in three groups.
a) Group I
This group comprises in V&O tests of cytotoxicity. General guidance for ~II vitro cytotoxicity tests is presented in
ISO 109935 and shall be followed. Detailed test protocols for the agar diffusion and filter diffusion methods,
appropriate to dental materials, are included in this International Standard. The in vitro cytotoxicity methods include:
1) agar diffusion test (6.1);
2) filter diffusion test (6.2);
3) direct contact or extract tests in accordance with ISO 109935;
4) dentine barrier tests (annex A).
NOTE 1 The order of listing does not indicate any preference for one method over another.
NOTE 2 The use of dentine barrier tests is encouraged and reference to these is presented in annex A.
b) Group II
This group comprises tests in accordance with ISO 10993 and particular tests, where appropriate, are identified:
1) acute systemic toxicity - oral application (ISO 10993-l 1, 6.51 and annex B);
2) acute systemic toxicity - application by inhalation (ISO 10993-l 1, 6.53);
3) subchronic systemic toxicity - oral application (ISO 10993-l 1, 6.7.1);
4) skin irritation and intracutaneous reactivity (ISO 10993-10, 5.2 and 5.4);
5) sensitization (ISO 10993-l 0, 6.2 and 6.3);
6) subchronic systemic toxicity - application by inhalation (ISO 10993-l 1, 6.7.3);
7) genotoxicity (ISO 10993-3, clause 4);
8) local effects after implantation (ISO 10993-6, clauses 4, 5 and 6).
NOTE 1 Alternatives to the LDsO tests are encouraged for acute toxicity testing, and information regarding this point is
presented in annex B.
NOTE 2 In the evaluation of materials following local implantation in accordance with ISO 10993-6, examination of
undemineralized sections, in addition to routine demineralized sections, is recommended.
c) Group Ill
This group comprises tests, specific for dental materials, not referred to in ISO 10993:
1) pulp and dentine usage test (6.3);
2) pulp capping test (6.4);
3) endodontic usage test (6.5).
5.5 A device shall be considered for re-evaluation of its biocompatibility as described in 5.4 when revisions or
modifications to the formula, quality and/or performance specifications are made.
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6 Test procedures specific to dental materials
6.1 Agar diffusion test
6.1 .l Objective
The test is designed to demonstrate the nonspecific cytotoxicity of test materials after diffusion through agar or
agarose.
6.1.2 Cell line
American Type Culture Collection CCL 1 fibroblasts (NCTC clone 929) shall be used; other cell lines may be used if
reproducibility and accuracy of the response cari be demonstrated.
NOTE - Alternative cell lines are presented in annex C.
6.1.3 Culture medium, reagents and equipment
Use Eagle’s Basa1 Medium containing 2,2 g/l sodium bicarbonate, 3,0 g/l HEPES and 50 ml/1 new-born calf serum.
Prepare a double concentration of Eagle’s Basa1 Medium omitting HEPES and reducing sodium bicarbonate to 1 g/l.
Prepare either 3 % agar or 3 % agarose in distilled water.
Sterilize agar by autoclaving and medium by filtration.
Prepare the vital stain by diluting a stock solution of 1 % aqueous neutral red solution (record source) l/lOO with
0,Ol ml/l phosphate-buffered saline solutions immediately before use. Store neutral red solutions protected from the
light. Use Petri dishes of diameter 50 mm to 100 mm suitable for tissue culture.
6.1.4 Sample preparation
For the preparation of test materials, the manufacturer’s recommended instructions shall be followed. The test shall
be performed on either an extract of the material or the material itself, according to the guidance in ISO 10993-5,
clause 4.
For solid materials, prepare circular test specimens of approximately 5 mm diameter, with a flat surface to ensure
adequate contact with the agar overlay.
For setting materials, insert the freshly mixed material into glass or polytetrafluoroethylene rings of internai diameter
5 mm and height 5 mm. When testing materials in the freshly mixed state, place the rings on the agar prior to
inserting the material. When testing after various setting periods, fill the rings SO that the material is flush with the
rim and allow it to set at (37 f 2) “C and a relative humidity of (90 + 10) % until ready for testing.
For fluid specimens or extracts, imbibe 0,l ml of the fluid on a borosilicate microglass filter disc of 5 mm diameter,
placed on the agar.
NOTE - Suitable discs cari be prepared from prefilters3).
6.1.5 Control specimens
Use control specimens as defined in ISO 10993-5, clause 3.
6.1.6 Test procedure
Culture the cells until they reach the end of the log growth phase. Pipette 10 ml of cell suspension
(2,5 x 105 cells/ml) into a sufficient number of Petri dishes and incubate at (37 + 2) “C in a water-saturated
atmosphere with 5 % (I/v) carbon dioxide for 24 h. Heat the sterile agar to 100 “C in a water bath and allow it to
cool to 48 OC. Mix 1 part of agar with 1 part of double-concentration, freshly prepared nutrient medium and heat to
48 OC. Aspirate the liquid nutrient medium from each Petri dish and replace with 10 ml of freshly prepared
agar/nutrient medium mixture.
3) Millipore prefilter AP2502200 is an example of a suitable product available commercially. This information is given for the
convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.
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Allow the agar nutrient medium to solidify at room temperature (approximately 30 min). Add 10 ml neutral red
solution and keep dark for 15 min to 20 min. Aspirate excess neutral red solution.
Protect the culture from light in the presence of neutral red, as the cells cari be damaged.
Apply to each dish two samples of test material, one positive control, one negative control and one extraction
medium control if the last was used. Keep the specimens as far as possible from each other and from the wall of the
Petri dish. Incubate at (37 + 2) OC in a water-saturated atmosphere with 5 % (VIV) carbon dioxide for 24 h. Examine
each test material at least in quadruplicate (i.e. two dishes per test material).
6.1.7 Parameters of assessment
The decolorization zone around the test materials and controls shall be assessed using an inverted microscope with
a calibrated screen, and a Decolorization Index and a Lysis Index determined for each specimen in accordance with
the following criteria:
a) Decolorization Index Description
No decolorization detectable
0
1 Decolorization only under the test substance
2 Decolorization zone not greater than 5,0 mm from the test substance
3 Decolorization zone not greater than 10,O mm from the test substance
4 Decolorization zone greater than 10,O mm from the test substance
5 The total culture is decolorized
Description
b) Lysis Index
No cell lysis detectable
0
1 Less than 20 % cell lysis
2 20 % to 40 % cell lysis
3 > 40 % to < 60 % cell lysis
4 60 % to 80 % cell lysis
5 More than 80 % cell lysis
Calculate the median Decolorization Index and Lysis Index for each test material and present the cell response as
follows:
Cell response = Decolorization Index/Lysis Index
If the index values for the four replicates of the test substance differ by more than 2 units in the range 0 to 3, repeat
the test. With indices of 4 and 5, no repetition is necessary. When extracts are tested, subtract the median index of
the extraction medium alone from the median index of the extraction medium containing test substance to obtain the
index for the test substance alone. If the median index for the extraction medium serving as a control is greater than
1, repeat the test using a different extraction medium.
NOTE - For a valid test, an intact cell layer should be found under the negative control.
6.1.8 Assessment of results
All information gathered in the test shall be taken into account in assessing the test results, particularly any
differences in results between the experimental and control groups. The cell response is based on the median
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Decolorization Index and Lysis Index of at least four replicate tests. The cell response is therefore not necessarily
an integer and the scale used for interpreting the cell response should be continuous. A useful way to grade test
materials is presented below.
Scale Cell response Interpretation
0 o/o Noncytotoxic
1 l/l Mildly cytotoxic
2 2/2/ to 313 Moderately cytotoxic
3 414 to 515 Severely cytotoxic
The results of the assessment shall be included in the test report.
6.1.9 Test report
The results shall be submitted in a test report that includes a complete record of all procedures followed, all results
obtained and any other data necessary for the assessment of results as described in 6.1.8. Details of the
preparation and methods of application of the test material, together with the batch number of the material when
appropriate, shall be included.
6.2 Filter diffusion test
6.2.1 Objective
The test is designed to demonstrate the nonspecific cytotoxicity of test materials after diffusion through a cellulose
acetate filter.
6.2.2 Cell line
American Type Culture Collection CCL1 fibroblasts (NCTC clone 929) shall be used; other cell lines may be used if
reproducibility and accuracy of the response cari be demonstrated.
NOTE - Alternative cell lines are presented in annex C.
6.2.3 Culture medium, reagents and equipment
Prepare culture medium and agar for use as an overlayer as described in 6.1.3. Prepare solutions either for
succinate dehydrogenase staining or for nonspecific hydrolase staining.
For succinate dehydrogenase staining, prepare the following stock solutions:
a) succinate solution, 13,6 g sodium succinate in 100 ml phosphate buffer, pH 7,6;
b) nitro blue tetrazolium chloride solution, 100 mg nitro blue tetrazolium chloride in 100 ml phosphate buffer,
PH 76;
C) phenazine methosulfate solution, 4 mg phenazine methosulfate in 10 ml distilled water, fresh.
Prepare a staining solution of 1 ml succinate solution, 9 ml nitro blue tetrazolium chloride solution and 1 ml
phenazine methosulfate solution.
For nonspecific hydrolase staining, prepare a stock solution of fluorescein diacetate consisting of 5 mg fluorescein
diacetate in 1 ml acetone. For use add 20 ~1 of stock solution to 100 ml phosphate-buffered saline. Use Petri dishes
of 50 mm to 100 mm diameter, suitable for tissue culture.
Use cellulose acetate filters, 47 mm diameter, 0,45 prn pore size4).
4) Millipore HAWD 04753, HA TF 047 SO is an example of a suitable product available commercially. This information is
given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this
product.
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6.2.4 Sample preparation
Prepare the samples in accordance with 6.1.4. All samples, including solid materials, shall be placed on the filter
rather than the agar overlay. The mass of the test specimens shall not exceed 3,5 g. For the preparation of test
materials, the manufacturer’s recommended instructions shall be followed.
6.2.5 Control specimens
Use control specimens as defined in ISO 10993-5, clause 3.
6.2.6 Test procedure
Culture the cells until they reach the end of the log growth phase. Place cellulose acetate filters in the bottom of a
sufficient number of Petri dishes and pipette 6 ml of cell suspension (2,5 x 1 O5 cells/ml) into each. Incubate at
(37 * 2) OC in a water-saturated atmosphere with 5 % (KV’) carbon dioxide for 24 h.
Pipette 5 ml of freshly prepared agar nutrient medium mixture (see 6.1.6) kept at 48 “C into a sufficient number of
Petri dishes and allow it to solidify at room temperature. Aspirate the excess nutrient medium from the dishes
containing cellulose acetate filters, wash the filters with phosphate-buffered saline at (37 * 2) “C and place them on
top of the agar, cell side down. Apply three to five test specimens on top of the filter in each dish and incubate for a
further 2 h at (37 f: 2) OC in a water-saturated atmosphere with 5 % (KV) carbon dioxide. Ensure that the specimens
are in close contact with the surface of the filter. If the test material does not show toxicity after 2 h incubation,
repeat the test with 24 h incubation. In each dish include one positive control and one negative control. In addition,
further controls of a filter with a cell monolayer but without test specimens, and of a filter without cells but with test
specimens shall be used. When extracts are tested, a control using the extraction medium alone shall also be used.
Each test specimen shall be examined at least in quadruplicate.
After incubation, remove the test specimens and gently loosen the filter from the agar. Assess cytochemically the
area of reduced cell enzyme activity by method A or method B.
a)Method A
Demonstrate succinate dehydrogenase (enzyme classification 1.3.99.1) according to the method of Barka and
Anderson [1963]. The incubation period is 3 h at (37 * 2) OC. Wash the filter in distilled water and air-dry prior to
measurement.
b)Method B
Demonstrate nonspecific hydrolase by incubation with fluorescein diacetate solution for 30 min at 4 OC. Examine the
filter under ultraviolet light.
6.2.7 Assessment of cell damage
Cell damage shall be assessed either
a) by measuring the area of decolorization (e.g. by means of an image analysis system) or
b) by using the following scale.
Scale Grading assessment Area of decolorization
0 No difference in staining intensity across the filter .
None
1 A zone of reduced staining intensity, or an unstained zone
< 20 mm2
with a diameter less than that (5 mm) of the test specimen
2 An unstained zone 5 mm to 7 mm in diameter
20 mm2 to 40 mm2
3 An unstained zone greater than 7 mm in diameter
> 40 mm2
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NOTE - The filters beneath negative control specimens and the control filters should be uniformly stained dark blue (if
succinate dehydrogenase is used) or light green (if nonspecific hydrolase is used). The control filters without cells allow
determination of a pos
...
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