Final Working Draft
ISO/TC 194/WG8 N 6
1998-09-01
ISO/WD 10993-10:1998
Revision of Biological evaluation of medical devices
Tests for irritation and delayed type hypersensitivity
ISO/TC 194/WG8 N 6
ISO/ WD 10993-10
ISO/WD 10993-10
Revision of Biological evaluation of medical devices
Tests for irritation and delayed type hypersensitivity
FOREWORD
ISO (the International Organisation for Standardisation) is a world-wide 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 10993-10 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
ISO 10993 consists of the following parts, under the general title Biological evaluation of medical
devices:
- Part 1: Guidance on selection of tests
- Part 2: Animal welfare requirements
- Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity
- Part 4: Selection of tests for interactions with blood
- Part 5: Tests for cytotoxicity: in vitro methods
- Part 6. Tests for local effects after implantation
- Part 7: Ethylene oxide sterilization residuals
- Part 9: Degradation of material related to biological testing [Technical Report]
- Part 10: Tests for irritation and sensitization
- Part 11: Tests for systemic toxicity
- Part 12: Sample preparation and reference material
- Part 13: Identification and quantification of degradation products from polymers
- Part 14: Identif cation and quantifi cation of degradation products from ceramics
- Part 15: Identification and quantification of degradation products from coated and uncoated
metals
and alloys
- Part 16: General guidance on toxicokinetic study design for degradation products and
leachables
- Part 17: Glutaraldebyde andformaldebyde residues in industrially sterilised medical devices
- Part 18: Characterisation of materials.
Future parts will deal with other relevant aspects of biological testing. This part of ISO 10993 is a harmonisation of numerous standards and guidelines, including BS 5736, OECD Guidelines, U.S. Pharmacopoeia and the European Pharmacopoeia. It is intended to be the overall guidance document for the selection and conduct of tests enabling evaluation of irritation and sensitisation responses relevant to materiel and device safety.
Annexes A and B form an integral part of this part of ISO 10993. Annexes C, D and E are for information only.
INTRODUCTION
This part of ISO 10993 assesses possible contact hazards from device-released chemicals that may produce skin and mucosal irritation, eye irritation, and delayed contact sensitisation
Some materials that are included in these devices have been tested, and their skin or mucosal irritation or sensitisation potential has been documented. Other materials and their chemical components have not been tested and may act differently when exposed to biological tissues. It is obligatory upon the manufacturer to evaluate each device for its human toxic potential prior to marketing.
Traditionally, small animal tests are performed prior to human testing to help predict human response. More recently, in vitro tests as well as human tests have been added as alternatives. Despite progress and considerable effort in this direction, a review of findings suggests that currently no satisfactory in vitro test have been devised to eliminate the requirement for in vivo testing. Where appropriate, the preliminary use of in vitro methods is encouraged as screening tests prior to animal testing. In order to reduce the number of animals used, these standards use a step-wise approach with review and analysis of test results at each stage. An animal test is usually required prior to human test.
It is obligatory upon the investigator to conduct these studies using good scientific laboratory practices, complying with regulations related to animal welfare. Statistical analysis of data is recommended. However under special circumstances e.g. pilot studies a low number of individuals in in-vivo tests makes statistical analysis inappropriate.
1 Scope
This part of ISO 10993 describes an approach to the assessment of devices and their constituent materials to produce
a) irritation and
b) delayed type hypersensitivity
This standard includes
a) pretest considerations,
b) the execution of the test, and
c) key factors for the interpretation of the results
These test methods are recommended for most categories of devices and mode of body contact given
in ISO 10093- 1.
Of the tests listed, those appropriate to the end use of the device are to be selected. Guidance is also
given for the preparation of materials specifically in relation to the above tests.
NOTE I It is important to emphasise that pretest considerations may result in the conclusion that testing for irritation and sensitisation is not necessary,
NOTE 2 Guidance on the conduct of supplementary tests which may be required specifically for devices used in the intradermal and in the ocular area, is given in Annex B. and guidance for oral, penile, rectal and vaginal areas are given in Annex C.
2 Normative references
The following standards contain provisions which, through reference in the text, constitute provisions of this part of ISO 10993. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this part of ISO 10993 are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of EC and ISO maintain registers of currently valid International Standards.
ISO 10993- 1: 1992, Biological evaluation of medical devices - Part 1: Guidance on selection of
tests
ISO 10993-2: 1992, Biological evaluation of medical devices - Part 2: Animal welfare requirements
ISO/DIS 10993-9: ----, Biological evaluation of medical devices - Part 9: Framework for identification and quantification of potential degradation of products
ISO 10993-12: 1996, Biological evaluation of medical devices - Part 12: Sample preparation and reference materials
ISO/DIS 10993-13: ----, Biological evaluation of medical devices - Part 13: Identification and quantification of degradation products from polymeric devices
ISO/CD 10993-14: ----, Biological evaluation of medical devices - Part 14: Identification and quantification of degradation products from ceramics *
ISO/DIS 10993-15: ----, Biological evaluation of medical devices - Part 15: Identification and quantification of degradation products from metals and alloys *
ISO/CD 10993-18: ----, Biological evaluation of medical devices - Part 18: Characterisation of materials *
*: Editorial note: May or may not be included, depending on the documents progress.
3 Definitions
For the purpose of this part of ISO 10993, the definitions given in ISO 10993-1, 10993-12 and the following definitions apply.
- Sensitisation (specific hypersensitivity): the induction of specialized immunological memory for an allergen to which an individual is exposed.
- Allergen (sensitizer): a material which is capable of inducing specific hypersensitivity such that on further exposure to the material characteristic side effects are produced.
- irritation: Localised non-specific inflammatory response to single, repeated or continuous application of a material.
- irritant: an agent that produces irritation.
- dose: a quantity to be administered at one time.
- induction: the process that leads to the de novo generation of an altered state of immunological reactivity in an individual to a specific material.
- challenge (elicitation): the process following the induction phase where the immunological effects of subsequent exposures in an individual to the inducing material are examined.
- oedema: Swelling due to abnormal infiltration of fluid into the tissues.
erythema: Reddening of the skin or mucous membrane.
eschar: Scab or discoloured slough of skin.
corrosion: The slow destruction of the texture or material of a tissue, as by the action of a strong irritant.
- ulceration: Open sore representing loss of superficial tissue.
- necrosis: The sum of the morphological changes indicative of cell and/or tissue death and caused by the progressive degradative action of enzymes.
negative control: 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.
- positive control: 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.
- solvent: Material (chemical, vehicle, medium, etc.) used to moisten, dilute, suspend, extract or dissolve the test material.
- reagent control: Solvent used to moisten, dilute, suspend, extract or dissolve the test material, which is evaluated concurrently with the moistened, diluted, suspended, extracted or dissolved test material.
4 General principles, step-wise approach
The available test methods for testing of irritation and sensitisation are developed specifically to detect skin irritation and sensitisation potential. Other types of adverse affects are not predicted by these tests.
This part of ISO 10993 advocates a step-wise approach, which may include any or all of the following:
.
a) literature review;
The first stage is a literature review and shall include an evaluation of chemical and physical
properties, and information on the irritation and sensitisation of the product constituent as well as
structurally related chemicals and materials.
b) characterisation of test material; The second stage involves a chemical characterisation and analysis of the sample according to the general principles described in ISO/DIS 10993-9 Degradation of materials related to biological testing, ISO 10993-12 Sample preparation and reference materials, clause 5-8, ISO/DIS 10993-13 Identification and quantification of degradation products from polymeric devices, ISO/CD 10993-14 Identification and quantification of degradation products from ceramics *, ISO/CD10993-15 Identification and quantification of degradation products from metals and alloys * and ISO/CD 10993- 18 Characterisation of materials *.
*: Editorial note: May or may not be included, depending on the documents progress.
c) in vitro tests
The third stage provides for in vitro assessments.
These should always be considered in preference to in vivo tests and should replace these as new in
vitro methods become available and validated. At the present time there are no validated in vitro tests
(other than simple screens) to detect irritants or sensitizers.
d) in vivo animal tests;
At the fourth stage acute in vivo animal studies are undertaken to test for materials not already classified as severe irritants or strong sensitizers by stages a) or b). Materials that do not demonstrate a potential for toxicity at single exposure may be further evaluated following repeated exposure.
e) non-invasive human tests/clinical trials.
If the material has been demonstrated not to be a sensitizer, studies on skin irritation may be
considered in humans.
f) a positive control should be run at least every 6 months to validate the test system and demonstrate a positive response.
5 Pretest considerations
The general principles presented in 10993-1 Guidance on selection of tests, Clause 5 Testing and the following applies:
5.1 Ceramics, metals and alloys
These materials are normally less complex in terms of the number of chemical constituents and these will be present in significant proportions.
It should be noted that during manufacture and assembly of medical devices additional chemical components may be used as processing aids e.g. lubricants or mould release agents. As well as the chemical components of the starting material and manufacturing process aids, adhesive/solvent residues from assembly and also sterilant residues or reaction products resulting from the sterilisation process may also be present in a finished product. Whether these compounds pose a health hazard/risk depends on the leakage on degradation characteristics of the finished products.
5.2 Determination of compositional information
Full qualitative data for chemical constituents of the material shall be established. Where relevant to biological safety, quantitative data shall also be obtained. When quantitative data is not obtained the rationale shall be documented and justified, validated.
5.3 From existing data sources
Medical device manufacturers should preferably obtain qualitative and quantitative compositional information from the supplier of the starting material. For polymers this often requires access to proprietary information and appropriate formalities may be necessary for transfer and use of such confidential information.
Qualitative information about any additional processing additives (for example, mould release agents) should also be obtained from appropriate members of the manufacturing chain, including converters and component manufacturers.
The composition of ceramics, metals and alloys is likely to be in accordance with ISO materials standard and/or may be specified by the user. However in order to obtain full qualitative and quantitative compositional details it may be necessary to request these from the supplier or manufacturer of the starting material and also from component manufacturers to ensure processing aids are also identified. Material master files held by regulatory authorities are another source of data where they are accessible.
5.4 By analysis
When compositional details are unavailable, only qualitative information is available or new or unknown substances may be expected to develop during the manufacturing process, it may be necessary to undertake analysis of a material.
Analytical methods appropriate for the material under investigation shall be used. All analytical techniques shall be justified, validated and reported and if not already known, the pH and pKa of the material (liquid, solution or extracts of materials) shall be measured prior to any in vivo or in vitro testing. Chemical analysis (qualitative as well as quantitative ) of extracts may give useful information. In this context it should also be emphasised that chemical analysis of the extract may give results that makes testing for irritation and sensitisation unnecessary, as information on irritation and sensitisation potential of the compounds present the extract solution may be available.
In the absence of any initial compositional data a literature study to establish the likely nature of the starting material and any additives is recommended to assist in the selection of the most appropriate methods of analysis for the material concerned.
6 Irritation tests
6.1 In vitro irritation tests
There has been concerted effort over the past 20 years to find alternative in vitro biological tests to replace acute skin and eye irritation tests. Within the past decade national and international organisations have been established to further the development of alternative test methods. While many test methods have been proposed and evaluated, none of the methods has duplicated the physiological responses of the in vivo animal model; consequently, they do not as yet offer a validated alternative test. In parallel with the search for alternative methods, others have been developing methods to quantify the responses of animals and humans in order to better define endpoints using non-invasive techniques. See Annex D. 1.
6.2 Factors to be considered in design and selection of in vivo
tests
Irritation testing of medical devices may be performed with the finished product and/or extracts thereof.
Factors affecting the results of irritation studies include
a) the patch test device;
b) the dose of the test material
c) application of the test material;
d) the degree of occlusion;
e) the application site;
f) the duration and number of exposures; and
g) the techniques used in evaluating the test.
Additional background information is provided in Annex A and ISO 10993-12.
While increased flexibility will allow the investigator to enhance the sensitivity of the test to suit conditions of use and population exposure, consistency in procedure contributes to comparability of test results with different materials and from different laboratories.
Provisions have been included in the test procedures for evaluation of devices and materials that will have repeated and/or prolonged exposure. The investigator, in consultation with the device manufacturer, should design the study to exaggerate the anticipated contact (time and/or concentration) in the clinical situation. While use of an exaggerated concentration or extract of the material is acceptable, this should be borne in mind during interpretation of the results.
For products intended to be used extensively on normal and abnormal skin, no substantial risk is normally accepted; however, many products, in spite of a potential to irritate, are fully acceptable because of their inherent benefit.
If the pH of the test material is less than or equal to 2 or equal to or greater than 1 1,5, the material may be declared an irritant and no further testing is required. However, experimental evidence suggests that acidity and alkalinity of the test material are not the only factors to be considered in relation to the capacity of a material to produce severe injury. The concentration of the test material, its period of contact, and many other physical and chemical properties are also important.
6.3 Animal skin irritation test
6.3.1 Principle
Assessment of the potential of the material under test to produce dermal irritation.
The rabbit is the preferred test animal as evidenced by the large amount of dermal irritation information on this animal in the Registry of Toxic Effects of Chemical Materials (RTECS). Eighty five percent of over 2 000 RTECS entries report test results with the rabbit, 7,5% with man, 4 % with the mouse, and 3 % with the guinea-pig. As a result, rabbits have been used to generate the vast majority of the available data in the open literature.
6.3.2 Test material
If the test material is either a solid or a liquid, it shall be prepared as specified in annex A.
If the test material is to be tested as an extract, it shall be prepared as specified in 10993-12. It is advisable to include a positive and negative control.
6.3.3 Animals and husbandry
Healthy young adult albino rabbits of either sex from a single strain weighing not less than 2 kg shall be used.
The animals shall be acclimatised and cared for as specified in ISO 10993-2.
One animal shall initially be used to evaluate the test material.
A well-defined response in the one animal obviates the need for additional testing.
Unless a well-defined response is observed for solid or liquid materials, a minimum of two further animals shall be used. For extracts, a minimum of two further animals per extract shall be used.
If the response in the test using the minimum of three animals is equivocal or not clear, additional testing shall be considered.
6.3.4 Test procedure
6.3.4.1 Preparation of animals
On the day before the test, closely clip the fur on the backs of the animals a sufficient distance on both sides of the spine for application and observation of all test sites (approximately 10 cm x 15 cm ). Use only animals with healthy intact skin. Abrasion of the test site is not necessary, as evidence indicates similar responses between abraded and non-abraded sites. If repeated exposure is required, follow the procedures in 6.3.4.2, 6.3.4.3 or 6.3.4.4, repeated for a maximum of 21 days.
6.3.4.2 Powder or liquid sample
Apply 0,5 g or 0,5 ml of the test material directly to each test skin site as shown in Figure 1. If the material is a powder, it should be slightly moistened with water or other suitable solvent before application.
Cover the application sites with a 25 mm x 25 mm non-occlusive dressing (such as a gauze patch) and then wrap the application site with a semi-occlusive bandage for a minimum of 4 h . At the end of the contact time, remove the dressings and mark the positions of the sites. Remove residual test material by appropriate means, such as washing with lukewarm water or other suitable, non-irritating solvent, and careful drying.
6.3.4.3 Extracts and extractants
Apply the appropriate extract(s) to the 25 mm x 25 mm four-ply gauze patches ( 0,5 ml per patch), one patch on each side of the animal as shown in Figure 1. Apply a control patch of gauze moistened with the extracting medium to the other side.
Cover the application sites with a semi-occlusive bandage for a minimum of 4 h . At the end of the contact time, remove the dressings and mark the positions of the sites. Remove residual test material by appropriate means, such as washing with lukewarm water or other suitable, non-irritating solvent, and careful drying.
6.3.4.4 Solid sample
Apply the samples of the test material directly to the skin on each side of each rabbit as shown in Figure 1. Similarly, apply the control samples to each rabbit.
Figure 1. Location of skin application sites
When testing solids (which may be pulverised if considered necessary), the test material shall be moistened sufficiently with water or, where necessary, an alternative solvent, to ensure good contact with the skin. When solvents are used, the influence of the solvent on irritation of skin by the test material shall be taken into account.
Cover the application sites with 25 mm x 25 mm non-occlusive dressings (such as a gauze patch) and then wrap the application sites with a semi-occlusive bandage for a minimum of 4 h . At the end of the contact time, remove the dressings and mark the positions of the sites. Remove residual test material by appropriate means, such as washing with lukewarm water or other suitable, non-irritating solvent, and careful drying.
6.3.5 Observation of animals
For acute (single exposure) tests, record the appearance of each application site at 1 h, 24 h, 48 h and 72 h following removal of the patches. Extended observation may be necessary if there are persistent lesions, in order to evaluate the reversibility or irreversibility of the lesions: this need not exceed 14 days.
Repeated exposure shall only be done after completition of the 72 h observation period.
For repeated exposure, record the appearances of the application site at I h after removal of the patches and immediately prior to the next application. After the last exposure, note the appearance of each application site at I h, 24 h, 48 h and 72 h following removal of the patches. Extended observation may be necessary if there are persistent lesions, in order to evaluate the reversibility or irreversibility of the lesions: this need not exceed 14 days.
Describe and grade the skin reactions for erythema and oedema according to the classification system given in Table I for each application site at each time interval and record the results for the test report.
NOTE 3 Histological and non-invasive techniques may assist in certain cases.
6.3.6 Evaluation of results
For acute exposure, determine the Primary Irritation Index (PII) as follows.
For each animal, add together the Primary Irritation Scores for the test material for both erythema and oedema at each time specified and divide by the total number of observations (six: two at each time specified). When vehicle controls are used, calculate the Primary Irritation Score for the vehicle controls and subtract that score from the score for the test material to obtain the Primary Irritation Score.
Only use 24 h, 48 h and 72 h observations for calculations. Observations made prior to dosing or after 72 h, to monitor recovery, are not used in the determination.
ISO/WD
Add the scores for each animal and divide the total by the numbers of animals. This value is the
Primary Irritation Index.
For repeated exposure, determine the Cumulative Irritation Index as follows.
Table I - Classification system for skin reaction
For each animal, add together the Irritation Scores for both erythema and oedema at each time specified. Divide this total by the total number of observations to obtain the Irritation Score per animal.
Add the Irritation Scores of each animal and divide by the total number of animals. This value is the Cumulative Irritation Index.
The Cumulative Irritation Index is compared to the categories of Cumulative Irritation Index defined in Table 2 and the appropriate Category is recorded for the report.
NOTE 4 The categories of Cumulative Irritation Index are based on the data relating the Primary Irritation Index (PII) for chemicals in rabbits to the primary irritation response in humans for a number of chemicals that have been tested in both species.
For any response, determine the Maximum Irritation Response, the time of onset of the response and the time to maximum response.
The Primary or Cumulative Irritation Index is characterised by number and description in Table 2.
Table 2 - Irritation response categories in rabbit
6.3.7 Presentation of results
The test report shall include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of application to the test sites;
f) how the site readings were performed and a record of the observations;
g) assessment of the results.
6.4 Human skin irritation test
6.4. 1 Introduction
To date the prediction of human cutaneous irritation for the purpose of hazard identification relies primarily on the use of experimental animals (See Annex D). There are however problems of extrapolating from animals to humans. For chemicals for which human exposure is high ( e.g., cosmetics and detergents products) risk assessments are frequently performed using human skin patch
tests. Human studies can serve several purposes: I ) direct identification of human hazard by testing chemicals in man rather then in animal species; 2) providing for risk assessment of some chemicals for which human exposure is high and 3) facilitation of extrapolation to man of data obtained previously from animal studies. This Guideline allows skin irritation data to be obtained directly in humans for purposes of hazard identification. Its aim is to determine whether a material presents a significant skin irritation hazard following acute exposure. It is crucial when adopting this approach to observe the safety/ethical standards set out below. Annex D. 1. gives further information on irritation tests.
6.4.2 Safety/ethical standards
In the interests of human safety the following criteria must be met before the study is initiated:
a) the study must meet any local legal requirements and conform fully to the "Helsinki Guidelines" .
b) the study should follow the principles of Good Clinical Practice.
c) the protocol is reviewed before initiation of the study and considered acceptable by an independent ethical review committee.
d) the experiment should be performed in compliance with the national regulations of the testing facility
6.4.3 Initial considerations
Adequate information on the toxicity profile, including percutaneous absorption data, should be
available to indicate that the study does not present any significant health risk.
Materials should not be tested in humans when:
a) they have shown to be irritant in a predictive assay, either in vitro or in vivo;
b) they have been shown to be corrosive in a predictive assay, either in vitro or in vivo.
c) a corrosive potential for human skin can be predicted on the basis of structure-activity relationships and/or physico-chemical properties such as strong acid or alkaline reserve;
d) they present a risk of skin sensitisation or sensitisation of the respiratory tract; e) they present any acute toxicity hazard under test conditions; and/or
f) they present any genotoxic, reproductive or carcinogenic hazard. Further guidance on the selection of human volunteers can be found in 6.4.5.1 and Annex D. 1.
6.4.4
Principle of the test
A single dose of the material to be tested is applied to the occluded skin of human volunteers. Irritation is kept to a minimum by applying the test material for 15 and 30 minute periods and then in hourly increments for up to 4 hours. For testing of relatively weak irritants, a closed patch duration of 24 hours may be necessary. When longer exposure periods are used, this should be done as described in 6.4.5.3.4. Reactions are scored at 24, 48 and 72 hours after treatment, with any reaction regarded as skin irritation being sufficient to terminate treatment in the individuals concerned.
The principal means of evaluation is the proportion of the test panel, which develops an irritant reaction in relation to a concurrent positive control material.
6.4.5 Description of the method
6.4.5.1 Selection of human volunteers -
This Test Guideline is designed for use with healthy human volunteers. It is not necessary specifically to select atopic individuals. The selected human volunteers should be at least 18 years of age, not pregnant and not breast-feeding. In addition, human volunteers with a known sensitivity to the test material or showing any signs of dermatitis should not be selected for the test. The selection of volunteers should be supervised by a dermatologist.
6.4.5.2 Preparation of doses
Liquid test materials are generally used undiluted. When testing solids (which may be pulverised if considered necessary), to insure good contact with the skin, the test material should be moistened with a small amount of water (typically 0.2 ml) or where necessary another suitable vehicle. Care should be taken when using moistened samples to ensure that each subject receives the same dose of the test agent. The amount of water used for moistening should be the same for each individual in the test and should be recorded. When vehicles are used, the influence of the vehicle on irritation of the skin by the test material should be taken into account. Where a vehicle other than water is to be used as the wetting agent for solid compounds, consideration should be given to a vehicle control patch on each subject.
6.4.5.3 Procedure
6.4.5.3.1 Number of
volunteers
At least 30 volunteers are recruited to compose the test panel, with no less than one third belonging to either sex. Without being excessive this number should be sufficient to provide an adequate assessment susceptible to a statistical evaluation.
The test material is applied to a suitable skin site, e.g. the upper outer arm, by means of an occlusive chamber containing a gauze pad. The application site should be an area that is most likely to be exposed to the chemical in a normal use situation. The application site should be the same in all volunteers and should be recorded. Generally the patch should measure at least 18 mm, preferably 25 mm in diameter. The patch should be held in contact with the skin by means of a suitable nonirritating dressing including non-irritating tape for the duration of the exposure period.
6.4.5.3.3 Application of the test material
The patch should deliver an adequate dose per unit area: approximately 50-100 mg/cm2 is considered optimal. When applying liquid test materials, in general 0,2 - 0,4 ml is added onto the gauze pad until it is moistened. When testing solids, in general 0.2 g of the test material are moistened and added onto the gauze pad. Alternatively, the gauze pad could be moistened.
6.4.5.3.4 Duration of exposure
To avoid unacceptably strong reactions, a cautious approach to testing must be adopted. A sequential patch procedure permits the development of a positive but not severe irritant response. The patches are applied progressively starting with a duration of 15 and 30 minutes and up to 1, 2, 3, and 4 hours. The 15 and/or 30 minutes exposure periods may be omitted if there are sufficient indications that excessive reactions will not occur following the 1-hour exposure. Progression to longer exposures including 24 hour closed patch exposure at a new skin site will depend upon the absence of skin irritation (scored at least up to 48 hours) arising from the shorter exposures, in order to ensure that any delayed irritant reaction is adequately assessed.
The application of the material for a longer exposure period is always made to a previously untreated site.
At the end of the exposure period, residual test material should be removed, where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.
6.4.5.3.5 Limited exposure
, . .
In addition to the phased increase in duration of application as described in 6.4.5.3.4, if it is suspected that the material might produce severe irritation, a substantially reduced exposure time should be employed, possibly in a pilot group of volunteers. The progress of the study can then be defined on the basis of the data produced. Subsequent patches are only applied after the 48/72 h readings.
6.4.5.3.6 Clinical observation and grading of skin reactions
Treatment sites are examined for signs of irritation and the responses are scored immediately after patch removal and at 1-2 and 24, 48 and 72 hours after patch removal. If necessary to determine reversibility of the response, the observation period may be extended beyond 72 hours. In addition, the condition of the skin before and after the test should be described exactly (e.g. pigmentation, age, extent of hydration). Skin irritation is scored and recorded according to the grading in Table 3.
Noninvasive bioengineering methods may be applied - See Annex D.
For volunteers who have a response of 1 or greater following an exposure of less than 4 hours, it is assumed that they would present a stronger reaction if exposed for 4 hours to the material. Once a response of I or greater has been obtained, there is no need to subject the reacting volunteer to further treatment with the material. Further observations may be required for proper volunteer care. In addition to the observation of irritation, any other effects should be recorded and fully described. The volunteers should be invited to make comments related to the patch applications (e.g. sensory effects) and assessors trained to note immediate responses (i.e. urticaria) when the patches are removed. Such observations may not indicate an irritant effect but they should be included in the test report if noted. If significant, they should be considered of in the management of the study to ensure proper volunteer care.
The critical data obtained are the number of volunteers who had, or would be expected to have, skin irritation after an exposure up to 4 hour. The time required for an individual to develop a response (if any) does not form part of the results to be evaluated; it relates only to ensuring proper care of the volunteers.
6.4.5.3.7 Selection of a concurrent positive control substance
As humans show variation in their responses to irritants a positive control should be included to determine the suitability of a test panel to detect irritant effects of the test compound. Preferably 20% sodium dodecyl sulphate (SDS) should be used as positive control as its irritants effects are well characterised (See Annex D 1).
By including a routine positive control, there is an opportunity to use it as a benchmark. Skin irritation is not an absolute phenomenon. All materials can give rise to skin irritation; it is simply a matter of dose and the nature and extent of exposure. Thus, skin irritation tests in humans are almost always comparative and should be related to known chemical irritancy.
6.4.6 Data and reporting
6.4.6. I Data
Data should be summarised in tabular form, showing for each individual the irritation scores at 24, 48 and 72 hours after patch removal and any other effects observed.
6.4.6.2 Data evaluation/interpretation
The aim of this test is to determine whether a material presents a significant irritation hazard following acute exposure. Thus if the material produces a frequency of skin irritation in the test panel which is similar to, or greater than, the positive control, it should be regarded as a significant skin irritant. On the other hand, if it produces a frequency of reaction in the test panel, which is substantially and significantly less than the positive control, then it may not be regarded as a significant skin irritant. It is important that interim data generated in the context of volunteer care are not confused with the endpoint data, i.e. the proportion of the panel that exhibit an irritant response. It is also important not to confuse individual variation in the susceptibility to skin irritation with the issue of the general irritation potential of the test material.
6.4.6.3 Test report
The test report must include the following information: a) Test material:
- physical nature and, where relevant, physicochemical properties;
- identification data.
b) Vehicle:
- identification of and justification for the choice of vehicle used to moisten a solid test material.
c) Volunteers:
- number of volunteers who are treated with the test material; age/sex distribution of the volunteers.
d) Results:
- response rate at 0, 1, 2, 24, 48 and 72 hours and at any other times scored.
- tabulation of irritation response data for each individual for each observation time period
(summarised frequency of response rate at e.g. 24, 48 and 72 hours after patch removal);
- description of all irritant reactions observed;
- description of any other effects in addition to irritation observed;
- statistical treatment of the results (comparison with positive control, e.g. using Fisher's exact test);
- description or reference of an in vitro or in vivo animal test, if such is performed before the test in human volunteers, including details of the procedure, and results obtained with test and reference materials.
e) Discussion of the results.
7. Sensitisation tests
7.1 Choice of tests
The two most commonly used methods are the guinea pig maximisation test (GPMT) and the closed
patch test (Buehler test).
The maximisation test is the most sensitive and is the preferred method for single components. It has
also been reported to be useful for the evaluation of extracts. However, the value of this test method is
best documented for single chemicals. A rationale and a list of alternative methods are given in
Annex C.
7.2 Choice of test concentrations
Current guidelines for testing single components for sensitising potential recommend one single test concentration to be used. However, the test result is highly dependent on dose. Therefore qualitative and quantitative analysis of the extract to be tested is highly warranted for the design of the test procedure and for the evaluation of the test results.
7.2.1 Induction
Sensitisation rate is highly dependent on induction dose. It should be moderately toxic or locally irritating, but otherwise not interfere with the health of the animals. The induction dose is chosen based on pilot experiments as described for the individual tests. The use of multiple doses is advised to facilitate the evaluation of the results (see Annex D.2).
7.2.2 Challenge
Challenge concentration is also based on pilot experiments on animals previously not exposed to the test material. A concentration below irritation threshold should be used. The use of multiple doses is advised for the challenge procedure to facilitate the evaluation of the results (see Annex D.2).
7.3 Other important factors determining the outcome of the test
The biochemical and physical characteristics of the test material may influence the choice of test. The maximisation test requires intradermal injections; consequently if the test material cannot be injected intradermally, the closed patch or alternative method shall be used.
A solvent should be selected that optimises exposure by solubilisation and penetration. The concentration of test material should be the highest possible without affecting the ability to interpret results. The bioavailability of the test material is influenced by the choice of vehicle. There is no vehicle that is optimal for all materials.
Most investigators prefer to dissolve the test material because dispersions are prone to form a sediment, making exact dosing difficult. For intradermal induction water, saline, propyleneglycol or a vegetable oil may be used.
The test procedure has several sources of variation between results from different laboratories.
The following factors are important: ambient test conditions, test site on the animal, type of patch design, quantity of test material, quality of occlusion, exposure time and reading of the animals. Animal responsiveness also varies according to genetic factors and husbandry. Comparison of the number of test animals with a positive response at challenge with the appropriate controls is essential for indication of a positive test result, though the severity of reactions will aid in the interpretation.
Borderline reactions at challenge are best clarified by rechallenge. Histopathology has not been shown to be of help in the evaluation of test results.
7.4 Maximisation sensitisation test
7.4. 1 Principle
Assessment of the potential of the material under test to produce skin sensitisation in the guinea pig using the technique applied in the Guinea pig maximisation test for single chemicals.
7.4.2 Test material
If the test material is either a solid or a liquid it shall be prepared as specified in annex A.
If the test material is to be tested as an extract, it shall be prepared as specified in ISO 10993-12.
The concentration of test material shall be the highest possible without affecting the ability to interpret the results (See 7.4.4.2).
7.4.3 Animals and husbandry
Healthy young adult albino guinea-pigs of either sex from a single outbred strain, weighing 300 g to 500 g at the start of the test shall be used. If female animals are used they shall be nulliparous and not pregnant.
The animals shall be acclimatised and cared for as specified in ISO 10993-2. Preliminary tests should be done in one set of animals to determine test concentrations ( 7.4.4.2 ).
For testing powders or liquids, a minimum of ten animals shall be treated with the test material and a minimum of five animals acts as a control group. Additional animals shall be used for the preliminary test.
For testing extracts, a minimum of ten animals shall be treated with each extract and a minimum of five animals acts as a control for each solvent. Additional animals shall be used for the preliminary test.
If no sensitisation is observed it is necessary to use a total of 20 test animals and 10 controls.
7.4.4 Test procedure
7.4.4.1 Preparation
Clip the fur on all treatment sites prior to treatment.
For intradermal injections, inject 0.1 ml per site.
For all topical applications, saturate a patch of filter paper of the appropriate dimensions with the test material and apply the patch to the clipped skin surface under an occlusive dressing wound around the torso of the animal.
7.4.4.2 Preliminary tests
The preliminary tests are intended to determine the concentrations of the test materials to be used in the main test in 7.4.4.3.
Consideration shall be given to the pre-treatment of all animals by injection with Freund's complete adjuvant in order to evaluate the possible excited skin status during the main test and interference with the readings.
Inject a range of concentrations of the test material or extract (in the selected solvent) intradermally into at least two animals.
Select for the intradermal induction phase in the main test the highest concentration that does not cause extensive destruction of the skin and does not otherwise adversely affect the animals.
Topically apply a range of concentrations of the test material or extract to the flanks of at least three additional animals. Remove the occlusive dressings and patches after 24 h, and assess the application sites for erythema and oedema using the grading given in table 4.
Select
a) if possible, for the topical induction phase in the main test, the highest concentration that causes slight erythema but does not otherwise adversely affect the animals;
b) for the topical challenge phase in the main test, the highest concentration that produces no erythema.
7.4.4.3 Main test
7.4.4.3.1 Intradermal induction phase
Make a pair of 0.1 ml intradermal injections of each of the following, into each animal, at the injection sites ( 1, 2 and 3) shown in Figure 2 in the clipped intrascapular region.
a) A 50:50 (V/V) mixture of Freund's complete adjuvant mixed with the chosen solvent. Water for injection or physiological saline (BP, USP or equivalent) for water-soluble materials. For nonaqueous soluble materials, examples of solvents are given in ISO 10993-12. b) The test material or extract at the concentration selected in the preliminary tests; inject the control animals with the solvent alone.
c) The test material or extract at the concentration used in b), emulsified in a 50:50 (V/V) mixture of Freund's complete adjuvant and the solvent; inject the control animals with the solvent mixed/emulsified with adjuvant. 7.4.4.3.2 Topical induction phase
Seven days after completion of the intradermal induction phase, administer the test material or extract by topical application to the intrascapular region of each animal, using 20 mm x 40 mm filter paper, so as to cover the intradermal injection sites. Use the concentration selected in 7.4.4.2 a). Secure with an occlusive dressing. Remove the dressings and patches after 48 h +/- 2 h .
Treat the control animals similarly, using the solvent alone.
Figure 2. location of intradermal injection sites
If the maximum concentration that can be achieved in 7.4.4.2 a) does not produce irritation, pretreat the area with 10 % sodium dodecylsulfate in petrolatum massaged into the skin 24 h +/- 2 h before the topical induction patch is applied. Treat the control groups similarly.
7.4.4.3.3 Challenge phase
At 14 days after completion of the topical induction phase challenge all test and control animals with the test material or extract. Administer the test material or extract and a vehicle control by topical application to the upper flank of each animal using appropriate patches or chambers soaked in the test material or extract at the concentration selected in 7.4.2.2 b). Dilutions of this concentration may also be applied to other untreated sites in a similar manner. Secure with an occlusive dressing. Remove the dressings and patches after 24 h +/- 2 h.
7.4.5 Observation of animals
Observe the appearance of the challenge skin sites of the test and control animals 24 h, 48 h and 72 h after removal of the dressings. Natural or full spectrum lighting is highly recommended to visualise the skin reactions. Describe and grade the skin reactions for erythema and oedema according to the grading given in Table 4 for each challenge site and at each time interval. It is highly recommended that reading be done in a blind manner to counteract bias in the evaluation of the results.
7.4.6 Evaluation of results
Grades of 1 or greater in the test group generally indicate sensitisation, provided grades of less than l are seen on control animals. If grades of 1 or greater are noted on control animals, then the reactions of test animals, which exceed the most severe control reaction, are presumed to be due to sensitisation, If the response is equivocal, rechallenge is recommended to confirm the results from the first challenge. The outcome of the test is presented as the frequency of positive challenge results in test and control animals.
Occasionally, the test group has a greater number of animals showing a response than the controls,
although the intensity of the reaction is not greater than that exhibited by the controls. In these
instances, a rechallenge may be necessary to define the response clearly. A rechallenge shall be
carried out 1 - 2 weeks after the first challenge. The method used shall be as described for the first
challenge, using the other flank of the animal.
7.4.7 Presentation of results
The test report shall include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of application to the test sites;
f) how the site readings were performed and a record of the observations;
g) assessment of the results.
7.5 Closed patch test
7.5.1 Principle
Assessment of the potential of the material under test to produce skin sensitisation in guinea pigs.
7.5.2 Test material
If the test material is either a solid or a liquid it shall be prepared as specified in annex A.
If the test material is to be tested as an extract, it shall be prepared as specified in ISO 10993-12.
7.5.3 Animals and husbandry
Healthy young adult albino guinea-pigs of either sex from a single outbred strain, weighing 300 g to 500 g at the start of the test shall be used. If female animals are used they shall be nulliparous and not pregnant.
The animals shall be acclimatised and cared for as specified in ISO 10993-2. Preliminary tests should be done in one set of animals to determine test concentrations ( 7.5.4.2).
For testing powders or liquids, a minimum of ten animals shall be treated with the test material and a minimum of five animals acts as a control group. Additional animals shall be used for the preliminary test.
For testing extracts, a minimum of ten animals shall be treated with each extract and a minimum of five animals acts as a control for each solvent. Additional animals shall be used for the preliminary test.
If no sensitisation is observed it is necessary to use a total of 20 test animals and 10 controls.
7.5.4 Test procedure
7.5.4.1 Preparation
Clip the fur on all treatment sites prior to treatment.
For all topical applications, saturate a patch (a woven dressing) of the appropriate dimensions with the test material or extract and apply the patch to the clipped area under an occlusive dressing for 6 h . The use of restraint of each animal is highly recommended to ensure occlusion of the test sites. If wrapping is used, its adequacy should be evaluated in every experiment.
7.5.4.2 Preliminary tests
The preliminary tests are intended to determine the concentrations of the test material or extract to be used in the main test described in 7.5.4.3.
Topically apply four concentrations of the test material or extract to the flanks of each of at least three animals using appropriate patches. Remove the occlusive dressings and patches after 6 h . Assess the application sites for erythema and oedema using the grading given in Table 4, 24 h and 48 h after patch removal.
Select
a) for the induction phase in the main test, the highest concentration that causes no more than slight erythema but does not otherwise adversely affect the animals; b) for the challenge phase in the main test, the highest concentration that produces no erythema.
7.5.4.3 Main test
7.5.4.3.1 Induction
phase
Administer the test material or extract by topical application to the clipped left upper back region of each animal using appropriate patches soaked in the test material at the concentration selected in 7.5.4.2 a). Remove the restrainer and occlusive dressings and patches after 6 h. Repeat this procedure at weekly intervals for three weeks. Additional induction application may be warranted. Treat the reagent control animals similarly, using the solvent alone.
7.5.4.3.3 Challenge
phase
Fourteen days after the last application challenge all test and control animals with the test material or
extract. Administer the test material or extract by a single topical application to a clipped untested
area of each
animal using appropriate patches soaked in the test material or extract at the concentration selected in
7.5.4.2 b). Remove the restrainer and occlusive dressings and patches after 6 h.
7.5.5 Observation of animals
At 24 h +/- 2 h after the primary challenge or rechallenge exposure, either
a) depilate all of the animals with a commercial depilatory by placing the material on the test site and surrounding areas according to the manufacturer's instructions; or
b) shave all of the animals on the challenge sites and surrounding areas.
Thoroughly wash the depilated area with warm water and dry the animals with a towel before returning them to their cages. A minimum of 2 h after removal of hair, grade the test sites according to Table 4 . Repeat the grading 48 h +/- 2 h after removal of the challenge patch. Daylight lighting is highly recommended to visualise the skin reactions.
7.5.6 Evaluation of results The grading scale given in Table 4 is applied.
Grades of 1 or greater in the test group generally indicate sensitisation, provided grades of less than 1 are seen on control animals. If grades of 1 or greater are noted on control animals, then the reactions of test animals, which exceed the most severe control reaction, are presumed to be due to sensitisation, Rechallenge is recommended to confirm the results from the first challenge. The outcome of the test is presented as the frequency of positive challenge results in test and control animals.
Occasionally, the test group has a greater number of animals showing a response than the controls,
although the intensity of the reaction is not greater than that exhibited by the controls. In these
instances, a rechallenge may be necessary to define the response clearly. A rechallenge shall be
carried out 1-2 weeks after the first challenge. The method used shall be as described for the first
challenge, using an untested area on the flank of the animal.
7.5.7 Presentation of results
The test report shall include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of application to the test sites;
f) how the site readings were performed and a record of the observations;
g) assessment of the results, including statistical methods.
8. Key factors in the interpretation of test results
A positive test from both methods (7.4 and 7.5) does not necessarily exclude the test material or device from use because the doses of the test material in the test procedure may be exaggerated compared to actual conditions of use. A positive test using any of the validated procedures indicates the need for additional data, either in laboratory animals or humans, that would allow risk assessment of intended human exposure.
Predictive test results generated by the procedures described in the standard can not stand-alone.
Positive tests in any of the assays should be scrutinised by rigorous follow up in order to minimise the likelihood of false positive results. The results should be validated by comparison with other sources of information:
a) industry and consumer complaint data b) experience with devices containing similar components c) diagnostic test results in dermatologic clinics d) retrospective epidemiologic data
The tests included in the standard are important tools for development of safe products provided that these are executed and interpreted by trained personnel.
ANNEX A (Normative)
Preparation of materials for testing
A.1 General
The conduct of the tests and interpretation of the data from irritation/sensitisation tests shall take into account the nature, degree, frequency, duration and conditions of exposure of the device in man. One of the parameters critical to these tests is the preparation of the test material. A general guideline (ISO 10993-12) exists but specific problems are associated with irritation and sensitisation extract preparation.
A.2
Direct contact
A.2.1 Solid materials, which have appropriate physical states (e.g. sheets, films), shall be tested without modification.
A.2.2 Powders (e.g. super-absorbents) shall be tested by direct deposition or by making a paste in an appropriate solvent.
A.2.3 Liquids shall be tested by either direct deposition or in diluted solution made with an appropriate solvent.
A reagent control using the same solvent shall be evaluated in parallel with the moistened, diluted or suspended test material.
A.2.4 If the test material is a solid, prepare samples 25 mm x 25 mm of a thickness that approximates to normal use but is not greater than 5 mm . Prepare suitable negative control samples in the same way. The solid may be pulverised, care being taken to ensure no contamination occurs during this process, or moistened sufficiently with water or a suitable non-irritant solvent to ensure good contact with the tissues. In the case of ceramics where pulverisation is required, it must be remembered that the physico- chemical properties of the ceramic may be altered by reducing the ceramic to a powder with potentially marked effects on biological activity (e.g. when surface area and/or particle size are important such as phagocytosis, which plays an important role in inflammation and the immune response).
The negative control shall physically resemble the test material closely and should be non-irritant. Four-ply gauze may be used as a substitute.
A.2.5 If the test material has to be diluted suspended or moistened, a suitable non-irritant solvent shall be used. Refer to ISO 10993-12 for a list of appropriate solvents.
A.2.6 If the test material is a solid, it may be tested by preparing extracts from the solid. If extracts are tested, extracts shall be prepared as described in ISO 10993-12, using polar, non-polar and/or additional solvents when appropriate.
A reagent control, using the extracting solvent shall be evaluated in parallel with the extract of the test material.
A.2.7 If the final product is sold in a sterile condition, then the test material shall be sterilised using the same process prior to testing. Products sterilised by ethylene oxide present a technical difficulty in that ethylene oxide and its reaction products can produce a biological response in the tests described in this part of ISO 10993. Any adverse biological response shall be evaluated. To enable differentiation to be made between effects produced by the test material and those produced by ethylene oxide residuals when an adverse irritant response is observed, consideration shall be given to evaluation of this response to the device pre- and post-ethylene oxide sterilisation.
ANNEX B (Normative)
Additional irritation tests
The following tests are special evaluation tests and should be considered as additional to the basic tests but not as replacements. They are only relevant for medical devices intended to be applied to these specific areas. Testing of the device according to ISO 10993-6 should be considered.
B.1 Intracutaneous (intradermal) reactivity test
B.1.1 Principle
Assessment of the potential of the material under test to produce irritation following intradermal injection of extracts.
B.1.2 Exclusion from test
Any material shown to be a skin, eye or mucosal tissue irritant or those with a pH of <= 2 or >= 11,5 shall not be tested.
B.1.3 Test material
The test materials shall be extracts and shall be prepared according to the procedures specified in ISO 10993-12.
B.1.4 Animals and husbandry
Healthy young adult albino rabbits of either sex from a single strain weighing not less than 2 kg shall be used. The animals shall be acclimatised and cared for as specified in ISO 10993-2. A minimum of three animals shall be used initially to evaluate the test material. If the response in the initial test is equivocal or not clear, additional testing shall be considered.
B.1.5 Test procedure
On the day before the test, closely clip the fur on the backs of the animals allowing a sufficient distance on both sides of the spine for injection of the extracts. Inject intracutaneously 0.2 ml of the extract obtained with the polar solvent at five sites on one side of each rabbit (see Figure B 1). Use the smallest sized needle, appropriate to the viscosity of the test material, for the intradermal injections.
Figure B I - Arrangement of injection sites
Similarly, inject 0,2 ml of the polar solvent control at five posterior sites on the same side of each rabbit (see Figure B 1).
Repeat the above procedures for the extract obtained with the non-polar solvent and the non-polar solvent control on the other side of each rabbit (see Figure B 1).
If other solvents are used, repeat the above steps for the extract obtained with the other solvents and the solvent controls.
B.1.6 Observation of animals
Note the appearance of each injection site immediately after injection and at 24 h, 48 h and 72 h after injection.
Grade the tissue reaction for erythema and oedema according to the classification system given in Table B I for each injection site and at each time interval observed, and record the results.
NOTE 8 Intradermal injection of oil frequently elicits an inflammatory response.
Injection of an appropriate vital dye such as Trypan blue or Evans blue, intravenously, may be undertaken at the 72 h reading to assist in evaluation of the response by delineating the area of irritation.
Non-invasive techniques may be used to assist in the evaluation if they are available.
B.1.7 Evaluation of results
Determine the Primary Irritation Index as follows.
For each animal, add together the Primary Irritation Scores for the test extract for both erythema and oedema at the three time intervals (24, 48 and 72 hours) and divide by the total number of observations. Add together the Scores for each animal and divide this total by the number of animals. This value is the Primary Irritation Score (PIS). Do the same for the reagent control. Subtract the PIS obtained from the PIS of the test extract - this is the Primary Irritation Indeks (PII).
Table B 2 - Primary irritation response categories in rabbit
B.1.8 Presentation of results
The test report shall include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of injection;
f) how the site readings were performed and a record of the observations;
g) assessment of the results.
B.2 Ocular irritation test
B.2.1 General.
The ocular irritation test should only be considered if safety data can not be obtained by other means,
only for materials that will come in contact with the eye or eye lid (such as ocular implants/intra
ocular lenses).
B.2.2 Principle
Assessment of the potential of the material under test to produce ocular irritation.
Mann and Pullinger described the use of rabbits to predict ocular irritancy in man. These authors advocated the use of pigmented rather than non-pigmented (albino) eyes and relied on description of individual animal responses to assess the irritant effects. Friedenwald et al. reported an albino rabbit model for assessing ocular irritation that provided a scoring system based on the description of individual animal responses. Draize et al. modified Friedenwald's procedure and published a grading system to assist in the evaluation of ocular irritation. Illustrated guides have been published as aids in assessing ocular lesions.
B.2.2 Exclusion from test
Materials and/or final products, which have demonstrated definite corrosion or severe irritation in a dermal study, shall not be further tested for eye irritation. Strongly acidic or alkaline materials (pH <= 2 or > 11,5 ) shall not be tested owing to their predictive corrosive properties. These products shall be considered eye irritants.
B.2.3 Test material
If the test material is a liquid, instil 0.1 ml undiluted into the lower conjunctival sac of one eye.
If the test material is a solid or granular product, grind to a fine dust. When gently compacted, instil that amount which occupies a volume of 0.1 ml and does not weigh more than 100 mg into the lower conjunctival sac of one eye.
NOTE 9 Some products may not be amenable to testing directly in the eye. Mechanical damage can result in making the test useless.
If the test material is contained in a pump spray, expel and instil 0.1 ml as for liquids.
If the test material is contained in an aerosol container, examine by either
a) spraying a single burst of 1 s duration at a distance of 10 cm directed at the open eye; or
b) expelling the aerosol into a cool container and treating as for a liquid.
If the test material is such that it can only be applied as an extract, prepare extracts as described in ISO 10993-12. Instil a 0.1 ml aliquot of the extract into the lower conjunctival sac of one eye.
Under conditions identical with those used above, prepare reagent controls, using both the polar and the non-polar solvent, in the absence of the test material.
B.2.4 Animals and husbandry
Healthy young adult albino rabbits of either sex from a single strain weighing 2 kg to 3 kg shall be used.
The animals shall be acclimatised and cared for as specified in ISO 10993-2.
One animal shall initially be used to evaluate the test material.
A well-defined response in the one animal obviates the need for additional testing.
Unless a well-defined response is observed for solid or liquid materials, a minimum of two further animals shall be used. For extracts, a minimum of two further animals per extract shall be used.
If the response in the test using the minimum of three animals is equivocal or not clear, additional testing shall be considered.
B.2.5 Test procedure
No longer than 24 h before commencement of the test, visually examine both eyes of each rabbit for evidence of ocular abnormality. If either eye shows any abnormality, the rabbit shall be replaced.
When the eyes are examined, sodium fluorescein 2 % BP may be used to visualise any corneal damage. The use of an ophthalmoscope, hand slit-lamp, or other suitable device, is recommended.
Instil the test material as specified in B.2.3.
Following instillation hold the eyelids together for approximately I s .
NOTE 10 The contralateral eye of each animal serves as an untreated control.
If repeated exposure of the material is anticipated and the test material has not demonstrated a significant response in the acute test, a repeat exposure study may be conducted. The duration of the exposure should bear resemblance to the length of use of the test material/device in the clinical situation.
B.2.6 Observation of animals
For animals receiving a single instillation of test material, examine both eyes of each animal approximately I h, 24 h, 48 h and 72 h after instillation.
Extended observation may be necessary if there are persistent lesions in order to determine the progress of the lesions or their reversal; this need not exceed 21 days. Extended observation cannot be justified for animals with severe lesions.
Grade and record any reactions observed in accordance with the scale for grading ocular lesions given in Table B.3.
For animals receiving multiple instillation's of test material, examine both eyes of each animal immediately before and approximately 1 h after each instillation. If there is evidence of irritation after the last treatment, the observations may be extended. Extended observation may be necessary if there is persistent corneal involvement or other ocular irritation in order to determine the progress of the lesions and their reversibility.
Grade and record any reactions observed in accordance with the scale for grading ocular lesions given in Table B.3.
Withdraw an animal immediately from the study and humanely kill it, if at any time it shows
a) very severe ocular damage (e.g. sloughing and ulceration of conjunctival membrane, corneal perforation, blood or pus in the anterior chamber); or
b) blood-stained or purulent discharge; or c) significant corneal ulceration.
Withdraw from the study any animal showing maximum effects on the grading system in Table ? absence of a light reflex (iridial response 2) or corneal opacity (grade 4) without evidence of recovery within 24 h or maximum conjunctival inflammation (chemosis grade 4 together with redness grade 3) without evidence of recovery within 48 h, and kill it humanely.
B.2.7.1 Acute
exposure
If the treated eye in more than one animal shows a positive response (asterisked figures in Table B 3) at any of the observations, then the material is considered an eye irritant and further testing is not required.
If only one of three eyes treated shows a positive reaction or the reactions are equivocal, treat further animals.
When further animals have been treated, the test material is considered to be an eye irritant if more than half of the eyes treated in the test group exhibit a positive reaction (asterisked figures in Table B 3 ) at any stage of the observation.
A severe reaction in only one animal is considered sufficient to label as an irritant.
B.2.7.2 Repeated
exposure
The test material is considered an eye irritant if more than half of the animals in the test group exhibit a positive reaction (asterisked figures in Table B 3 ) at any stage of the observation.
B.2.7.3 Presentation of results
The test report shall include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of instillation;
f) how the ocular readings were performed and a record of the observations;
g) assessment of the results.
ANNEX C (Informative)
Additional irritation tests
C.1 Oral irritation test
C.1.1 General.
The oral irritation test should only be considered for materials with intended contact with oral tissue
and if safety data can not be obtained by other means.
C.1.2 Principle
Assessment of the potential of the material under test to produce irritation of the oral tissue.
C.1.3 Exclusion from test
Any material shown to be a skin or eye irritant or those with a pH of < 2 or >= 1 1,5 should not be tested and should be labelled as a potential oral tissue irritant.
C.1.4 Test material
Prepare test materials according to the procedures specified in annex A .
Test liquid samples by soaking cotton-wool pellets in the test material or by direct flushing of the cheek pouch with the material.
Test solid samples by placing pellets of the test material directly into the cheek pouch or by soaking cotton-wool pellets in an extract prepared according to procedures described in ISO 10993-12.
C.1.5 Animals and husbandry
Syrian hamsters from a single outbred strain should be used. They should be healthy young adults of either sex.
The animals should be acclimatised and cared for as specified in ISO 10993-2.
In addition to the above, fit to each animal a 3 mm to 4 mm wide suitable collar, placed around the neck so that it permits normal feeding and respiration but prevents the animal from removing the cotton- wool pellet. Weigh each animal daily for 7 days. Examine any animal showing a loss of mass during this period and adjust its collar, if necessary. If the animal continues to lose mass, exclude it from the test.
A minimum of three animals should be used initially to evaluate the test material.
NOTE 11 Additional animals treated with a negative control material or control extract may be appropriate.
If the response in the initial test is equivocal or not clear, additional testing should be considered.
C.1.6 Test procedure
Remove the collar from each animal and evert the check pouches. After washing the pouches with physiological saline solution, examine for any abnormality.
For solid materials, place pellets (no larger than 5 mm diameter) of materials directly into the cheek pouch. For liquids or extracts, soak a cotton-wool pellet, recording the volume used, in the test material or extract and place it in one pouch of each animal. Alternatively, an appropriate volume of a liquid may be flushed into the cheek pouch. No sample is placed in the other cheek pouch, which serves as a control. If extracts are tested, appropriate controls should be tested in parallel.
Replace the collar and return the animal to its cage.
The duration of contact should be that expected for actual use of the material, but no shorter than 5
min .
Following the exposure, the collar and cotton-wool pellet are removed and the pouch is washed with
physiological saline, taking care not to contaminate the other pouch.
For acute exposure, repeat the above procedure every hour for 4 h .
For repeated exposure, the number of applications, their duration and their interval should be based on the anticipated contact time in the clinical situation.
C.1.7 Observation of animals
Examine the pouches macroscopically following removal of the pellets and immediately prior to the next dosing (if repeated applications are required).
Describe the appearance of the cheek pouches for each animal and grade the pouch surface reactions for erythema according to the classification system given in Table C 1 for each animal at each time interval. Record the results for the test report.
At 24 h after the final treatment, examine the cheek pouches macroscopically, and humanely kill the hamsters and remove tissue samples from representative areas of the pouches. Place in an appropriate fixative prior to processing for histological examination.
Table C 1 - Classification system for oral and penile reactions
C.1.8 Assessment of
results
C.1.8.1 Macroscopic evaluation
Compare the untreated cheek pouch with the cheek pouch on the contralateral side and, if one is included, with the pouches of animals in the control group.
The scores (Table C I ) for each observation are added and divided by the number of observations to determine the average score per animal.
NOTE 12 These observations may assist in the histological evaluation.
NOTE 13 The initial observations made prior to the first application of the test material are not included in the score average.
C.1.8.2 Histological evaluation
The irritant effects on oral tissue should be evaluated by a pathologist. The pathologist may score each tissue according to the system presented in Table C.2.
The scores for microscopic evaluation for all the animals in the test group are added and divided by the number of observations to obtain a test group average. Repeat for the control group(s). The maximum score is 16.
A total score greater than nine for the microscopic evaluation in the control cheek pouch may indicate underlying pathology or, in a control animal, it may indicate trauma at dosing. Either situation may require a retest if other test or control animals exhibit equivalent high scores.
Subtract the control group average from the test group average to obtain the Irritation Index.
For repeated exposure, Table C.2 may need to be modified to accommodate additional tissue
responses associated with chronic irritation.
C.1.9 Presentation of results
The test report should include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of application;
f) how the site readings were performed and a record of the observations;
g) histological evaluation;
h) assessment of the results.
Table C.2 - Microscopic classification system for oral, penile, rectal and vaginal tissue reaction
C. 2 Penile irritation test
C.2.1 Principle
Assessment of the potential of the material under test to produce irritation of the penile tissue. The penile irritation test should be restricted to materials with intended contact in the penile area.
C.2.2 Exclusion from test
Any material shown to be a skin or eye irritant or those with a pH =< 2 or >= 11,5 should not be tested and should be labelled as a potential penile irritant.
C.2.3 Test material
If the test material is either a solid or a liquid it should be prepared as specified in annex A.
If the test material is to be tested as an extract, it should be prepared as specified in ISO 10993-12.
C.2.4 Animals and husbandry
Male albino rabbits or guinea-pigs should be used. They should be healthy young adults weighing not less than 2 kg for rabbits and 300 g to 500 g for guinea-pigs.
The animals should be acclimatised and cared for as specified in ISO 10993-2.
The length of the penis which can be exposed should be at least 1 cm .
Due to individual pigment variation, animals are observed and scored for erythema prior to the first test application. The classification system given in Table C 1 should be used for scoring any erythema. Animals showing severe discoloration or having an erythema score of two or greater should not be used.
A minimum of three animals should be used initially to evaluate the test material, and three animals as the control group.
If the response in the initial test is equivocal or not clear, additional testing should be considered.
C.2.5 Test procedure
Place the animal in a supine position with the limbs secured by an assistant.
With index and middle finger, gently press the genital area to protrude the penis.
When the penis is protruded, apply enough (approximately 0,2 ml ) of the test material to be sure that the penis is coated.
Allow the penis to retract into the sheath and isolate the area by wrapping the body of the animal between the front and rear legs with loose knit dressing (e.g. nylon hose or roll gauze) and secure the
dressing to the torso. This is to prohibit the animal from licking the test site and confounding the primary irritation by secondary factors.
Alternatively the animal may be secured in an appropriately designed restrainer for 1 h after the last application of the test material and then returned to its own cage.
For acute exposure, repeat the above procedure every hour for 4 h .
For prolonged repeated exposure, the number of applications, their duration and their interval should be based on the anticipated contact time in the clinical situation.
C.2.6 Observation of animals
For acute exposure, note the appearance of the penis 1 h after the initial application (e.g. immediately prior to the next application) and subsequent treatments. Note and record the appearance of the penis at 1 h, 24 h and 48 h after the last application.
For prolonged repeated exposure, note the appearance of the penis at 1 h after the initial application and immediately prior to the next application.
Grade the skin surface reactions for erythema according to the classification system given in Table B. 1 for each animal at each time interval and record the results for the test report.
If any animal exhibits redness prior to the first test application, the score given prior to the first application of the test material is subtracted from the scores for erythema at the timed observations to determine the erythema score due to the test material. The highest possible score for one observation is four.
C.2.7 Assessment of results
C.2.7.1 Macroscopic evaluation
Compare the untreated penis and sheath with the penis of the control animals.
The scores (Table C 1 ) for each observation are added and divided by the number of observations to determine the average score per animal.
NOTE 14 These observations may assist in the histological evaluation.
NOTE 15 The initial observations made prior to the first application of the test material are not included in the score average.
Immediately after the 48 h observation, humanely kill the animals. Dissect free the distal penis and sheath and place into an appropriate fixative prior to processing for histological examination.
C.2.7.2 Histological evaluation
The irritant effects on the penile skin should be evaluated by a pathologist. The pathologist may score each tissue according to the system presented in Table C.2.
The scores for microscopic evaluation for all the animals in the test group are added and divided by the number of observations to obtain a test group average. The maximum score is 16.
Repeat for the control group(s).
A total score greater than nine for the microscopic evaluation in a control animal may indicate trauma at dosing. A retest may be required if other test or control animals exhibit equivalent high scores.
Subtract the control group average from the test group average to obtain the Irritation Index.
For prolonged repeated exposure, Table C.2 may need to be modified to accommodate additional tissue responses associated with chronic irritation.
C.2.8 Presentation of results
The test report should include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of application;
f) how the site readings were performed and a record of the observations;
g) histological evaluation;
h) assessment of the results.
C.3 Rectal irritation test
C.3.1 Principle
Assessment of the potential of the material under test to produce irritation of the rectal tissue.
C.3.2 Exclusion from test
Any material shown to be a skin or eye irritant or those with a pH =< 2 or >= 11,5 should not be tested
and should be labelled as a potential rectal irritant.
C.3.3 Test material
If the test material is either a solid or a liquid, it should be prepared as specified in annex A.
If the test material is to be tested as an extract, it should be prepared as specified in ISO 10993-12.
C.3.4 Animals and husbandry
Healthy young adult albino rabbits of either sex from a single strain weighing not less than 2 kg should be used. If other species are used, the choice should be justified.
The animals should be acclimatised and cared for as specified in ISO 10993-2. A minimum of three animals should be used initially to evaluate the test material, and three animals as the control group.
If the response in the initial test is equivocal or not clear, additional testing should be considered.
The animals should be checked for rectal discharge, swelling and/or other evidence of lower bowel infection, irritation and/or injury prior to each treatment.
C.3.5 Test procedure
Attach a short (6 cm ) soft catheter or blunt-tipped cannula to a syringe with a capacity to deliver more than 1 ml and fill the syringe and catheter such that I ml of the test solution will be dosed. Prepare a separate syringe with attached catheter for each animal.
Secure the animal by placing it in a restraining device which permits access to the perineum, or by an assistant carefully restraining the animal and securing the back legs in such a way to expose the perineum.
Just prior to insertion, moisten the catheter in either the control solution or a suitable lubricant.
Grasp and raise the animal's tail to expose the perineum. Gently insert the moistened catheter deep into the rectum and deposit the entire 1 ml dose from the syringe. Withdraw the catheter and discard it appropriately.
Due to differences in the capacity of the rectum of individual animals, some of the test material may be discharged during or immediately after it is deposited. Gently remove any of the expelled material with a soft tissue.
Repeat the above procedure at 24 h intervals every day for five consecutive days.
For prolonged repeated exposure, the number of applications, their duration and their interval should be based on the anticipated contact time in the clinical situation.
C.3.6 Observation of animals
At 24 h after the initial application and immediately prior to each treatment, note and record the appearance of the perineum for signs of discharge, erythema and irritation.
Animals exhibiting excessive discharge, swelling and/or that are found difficult to dose should be humanely killed and the tissues examined (see C.3.7. 1).
C.3.7 Evaluation of results
C.3.7.1 Macroscopic
evaluation
At 24 h after the last dose, humanely kill the animals. Dissect free the entire lower bowel, open longitudinally and examine for signs of irritation, injury to the epithelial layer of tissue and necrosis.
Place the rectum and distal portion of the large bowel in an appropriate fixative prior to processing for
histological examination.
Compare the rectal tissues of the test rabbits with the rectal tissue of the control rabbits.
Record and describe the macroscopic appearance of the rectal tissue for each animal, noting differences between the test and control sites.
NOTE 16 These observations may assist in the histological evaluation.
C.3.7.2 Histological evaluation
The irritant effects on the rectal tissue should be evaluated by a pathologist. The pathologist may score each tissue according to the system presented in Table C.2.
Add the scores for microscopic evaluation for all the animals in the test group and divide by the number of observations to obtain a test group average. The maximum score is 16. Repeat for the control group(s).
A total score greater than nine for the microscopic evaluation in a control animal may indicate trauma at dosing. A retest may be required if other test or control animals exhibit equivalent high scores. Subtract the control group average from the test group average to obtain the Irritation Index.
For prolonged repeated exposure, Table C.2 may need to be modified to accommodate additional
tissue responses associated with chronic irritation.
C.3.8 Presentation of
results
The test report should include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of application;
f) how the site readings were performed and a record of the observations;
g) histological evaluation;
h) assessment of the results.
C.4 Vaginal irritation test
C.4.1 Principle
Assessment of the potential of the material under test to produce irritation of the vaginal tissue.
C.4.2 Exclusion from test
Any material shown to be a skin or eye irritant or those with a pH of =< 2 or >= 11,5 should not be tested and should be labelled as a potential vaginal irritant .
C.4.3 Test material
If the test material is either a solid or a liquid, it should be prepared as specified in annex A.
If the test material is to be tested as an extract, it should be prepared as specified in ISO 10993-12.
C.4.4 Animals and husbandry
Healthy young adult female albino rabbits from a single strain weighing not less than 2 kg should be used. If other species are used, the choice should be justified.
The animals should be acclimatised and cared for as specified in ISO 10993-2.
A minimum of three animals should be used initially to evaluate the test material, and three animals as the control group.
If the response in the initial test is equivocal or not clear, additional testing should be considered.
The animals should be checked for vaginal discharge, swelling and/or other evidence of vaginal infection, irritation and/or injury prior to each treatment. A check should also be made on the stage in oestrus cycle to ensure a false positive reaction is not given based on physiological changes in the vagina.
C.4.5 Test procedure
Attach a short (6 cm ) soft catheter or blunt-tipped cannula to a syringe with a capacity to deliver more than I ml, and fill the syringe and catheter such that I ml of the test solution will be dosed. Prepare a separate syringe with attached catheter for each animal.
Secure the animal by placing it in a restraining device which permits access to the vagina or by an assistant carefully restraining the animal and securing the back legs in such a way to expose the perineum.
Moisten the catheter in either the control solution or a suitable lubricant.
Grasp and raise the animal's tail to expose the vaginal opening. Gently insert the moistened catheter deep into the vagina and deposit the entire 1 ml dose from the syringe. Withdraw the catheter and discard it appropriately.
Due to differences in the capacity of the vagina of individual animals, some of the test material may be discharged during or immediately after it is deposited. Gently remove any of the expelled material with a soft tissue.
Repeat the above procedure at 24 h intervals every day for a minimum of five consecutive days.
For prolonged repeated exposure, the number of applications, their duration and their interval should be based on the anticipated contact time in the clinical situation.
C.4.6 Observation of animals
At 24 h after the initial application and immediately prior to each treatment, note and record the appearance of the vaginal opening and perineum for signs of discharge, erythema and oedema.
Animals exhibiting excessive discharge, erythema and/or oedema, and found difficult to dose should be humanely killed and the tissues examined (see C.4. 7. 1).
C.4.7 Evaluation of results
C.4.7.1 Macroscopic evaluation
At 24 h after the last dose, humanely kill the animals. Dissect free the entire vagina, open longitudinally and examine for signs of irritation, injury to the epithelial layer of tissue and necrosis.
Place the vagina in an appropriate fixative prior to processing for histological examination. Three sections, to include the cervical, central and caudal portions of each vagina, should be taken.
Compare the vaginas of animals treated with the test material with the vaginas of the control animals.
Record and describe the macroscopic appearance of the vaginal tissue for each animal, noting differences between the test and control groups.
NOTE 17 These observations may assist in the histological evaluation.
C.4.7.2 Histological evaluation
The irritant effects on vaginal tissue should be evaluated by a pathologist. The pathologist may score each tissue according to the system presented in Table C.2.
The scores for microscopic evaluation for all the animals in the test group are added and divided by the number of observations to obtain a test group average. The maximum score is 16. Repeat for the control group(s).
A total score greater than nine for the microscopic evaluation in a control animal may indicate trauma at dosing and may require a retest if other test or control animals exhibit similar high scores. Subtract the control group average from the test group average to obtain the Irritation Index.
For prolonged repeated exposure, Table C.2 may need to be modified to accommodate additional
tissue responses associated with chronic irritation.
C.4.8 Presentation of results
The test report should include
a) a description of the test material(s) or device;
b) the intended use/application of the test material(s) or device;
c) a detailed description of the method employed in preparing the test material or device;
d) the test animals;
e) method of application;
f) how the site readings were performed and a record of the observations;
g) histological evaluation;
h) assessment of the results.
ANNEX D (Informative)
Background information
D.1 Background information on irritation tests
Dermal irritation testing in small animals is performed to help identify materials, which may be potential human skin and/or mucosal tissue irritants. A primary irritant is a material which produces inflammatory changes in the skin as a result of a direct damaging effect characterised by the presence of inflammation, or in the case of severe irritant, vesiculation and/or necrosis. The skin irritation tests in animal and man may give varying results due to variation in a number of test related factors as host, test dose, patch size, degree of occlusion, length of exposure, vehicle, time for reading and quality of reading. Therefore, in skin irritation tests it is important to include a well-known positive and negative control material in order to compare the test results with the control materials making the results relative.
As positive irritant control, sodium dodecyl sulphate (SDS) of purity > 99% is the preferred choice since it is the most widely used control irritant in clinical investigations (York et al.,1996), (Lee et al., 1995) and (Agner,1992). It is also easily and widely available and free from other adverse effects. Nonanoic acid, which has a mode of action different from SDS, may also be used as a positive control (Wahlberg and Maibach, 1980),(Wahlberg et al., 1985). SDS exposure calibrates the panel of human volunteers and acts as a reference point. SDS is classified as a skin irritant according to EU criteria (88/379/EEC Council Directive of 7 june 1988). It is not clear however whether SDS is at, or close to, the threshold level of response at which chemicals should be regarded as skin irritants. Thus rather than using the neat material, it is more appropriate to take as a reference point the minimum level of SDS regarded by at least one regional group (the KU) as a significant acute irritant to skin, which is a 20% w/v aqueous preparation (York et al.,1996).
The use of laboratory animals for skin irritation testing is decreasing due to the development of in vitro models and more frequent use of human volunteers (Simion, 1996; Ponec, 1996). Bioengineering or non-invasive, so-called, objective measuring methods are utilised to quantitate the irritant response and thereby decrease the dependency on the more subjective visual reading scales (Wahlberg, 1983 and 1987) (Serup and Jemec, 1995). However, decades of experience have been obtained with the Draize dermal irritation test on albino rabbits. This method is in the OECD guideline #404. The test material is introduced under gauze patches to intact sites on the clipped dorsum. Applications are made on three rabbits. The patches are secured by adhesive tape and the entire trunk of the animal is wrapped in a semi-occlusive dressing for 4 h . After 4 h, the patches are removed, the test sites cleaned, and any resulting reaction graded for erythema and oedema. The reactions are also scored at 24 h, 48 h and 72 h .
Extensive human data on skin irritation comes from the Research Institute for Fragrance Materials Monographs on essential oils and other aromatics published in Food and Cosmetic Toxicology . An OECD Guideline Draft on Acute dermal irritation study in human volunteers gives additional background information. The chemicals group of the OECD Guideline programme has not yet reached consensus on the need to develop an OECD Guideline for local skin effects in human volunteers.
D.2 Background information on sensitisation tests
Sensitisation in man occurs after single or multiple epicutaneous exposures, and is initiated and elicited by components of the immune system. Most importantly, the hapten (chemical) must be substantive to skin and be able to penetrate. It then reacts with skin proteins to become antigenic. Langerhans cells at the epidermal/dermal border present the antigen to specific lymphocytes which are then activated to initiate the immune responses. A small percentage of these lymphocytes are longlived memory cells and these serve as the primary activators during the challenge phase. Thus, subsequent re-exposures can result in adverse reactions that are mediated by lymphokines released by the activated lymphocytes and other inflammatory cells that are attracted to the area of the lesion. In 1895, Jadassohn employed the patch test to disclose contact allergy to mercury in a clinical patient. This innovative approach provided the scientific basis for subsequent tests aimed at diagnosis and prediction of contact allergy in man and animals. The development of prospective/predictive tests for evaluating the sensitising potential of chemicals followed the pioneer work of Landsteiner and Chase, who firmly substantiated the use of the guinea-pig for studying delayed hypersensitivity. Magnusson and Kligman ( 1969) explored many of the variables of guinea-pig testing and presented a procedure, the guinea pig maximisation test (GPMT) based on intradermal injections (with and without Freund's complete adjuvant, FCA), followed by topical application of the test material to the same area. The original procedure requires pre-treatment of the test site if the test material is nonirritant. By definition, it reputedly detects weak sensitises. because "weak" included a zero incidence of positive reactors. It is a sensitive test and has been extensively used. The use of Freund s complete adjuvant increases the sensitivity of the test method and it may in some cases overestimate the sensitising potential of the compound in question. In 1965, Buehler ( 1965) advocated the use of the closed patch to provide occlusion as a method to exaggerate exposure and to mimic the procedures used in man (Human Repeat Insult Patch Test: HRIPT). It was suggested that the occlusive patch procedure was sensitive and would accurately predict moderate to severe sensitisers and avoid exposing human subjects to the prospect of experiencing adverse reaction during HRIPT's. The data presented demonstrated the superiority of occlusion over intradermal injections and open-type topical application . Stimulation of the immune system by adjuvants was not used. This method is established as a technique that is sufficiently sensitive to detect most weak sensitisers and has been shown to be sufficiently flexible to be used in the Risk Assessment Process. However, the Closed patch test (Buehler test) is less sensitive compared to the GPMT.
These two tests have been the most frequently used for safety assessment, the closed patch test in the United States and the GPMT in Europe. They are also the preferred test methods in current OECD and EU test guidelines. Numerous other tests have been employed and investigated and all of these have their proponents. There are currently several procedures that have been recognised as acceptable for regulatory purposes, provided the procedure is properly documented and validated by the investigator. In all cases the procedures should be performed according to the original references. A list of these other tests is provided in Table D. I.
Table D.1- Alternative delayed contact sensitisation tests
The last assay in Table D.1, the murine local Iymph node assay (LLNA), deserves attention. The
previously described animal assays (a.o. GPMT, Closed patch test ) all identify the potential
allergenicity of a material by evaluation of the skin reaction after challenge. The LLNA differs from
these methods by detecting immune reactivity of potential immunotoxic (allergenic) chemicals by
measuring proliferation of lymphocytes into the draining lymph nodes during the induction phase. In
the assay small groups of inbred mice (n=4) (CBA/CA mice) are painted daily for 3 consecutive days
on the dorsum of the ears with the chemical in question. Several test concentrations and vehicle
controls may be included in order to a obtain dose response relationship (Kimber and Basketter,
1992). To determine if sensitisation has occurred the mice are injected intravenously in the tail vein on
day 4 with radio labelled (3H) thymidine in a buffer. The animals are sacrificed 5 h later and the
draining auricular Iymph nodes are isolated, excised and pooled for each experimental group. A single
cell suspension of Iymph node cells is prepared by mechanical desegregation through a metal gauze
and processed for liquid scintillation counting. The level of incorporated thymidine is measured.
Currently, a chemical is considered allergenic in the LLNA if one or more concentrations of the test
material elicits a three-fold or more increase in isotope incorporation compared with the vehicle
control. The data must not be incompatible with a conventional biological dose response (relationship
(Kimber and Basketter, 1992). The chemicals are classified as "sensitisers" if positive and "not strong
sensitisers" if negative.
An intra and inter-laboratorial evaluation of the LLNA has demonstrated reproducible dose-response
relationship within and between the laboratories (Loveless et al., 1996), (Kimber et al, 1995),
(Basketter et al, 1992) and (Ikarashi et al., 1993). The LLNA has furthermore been able to identify
proven important contact allergens previously identified with the human maximisation test (Basketter
et al., 1994). A close concordance between the LLNA and the GPMT were found for 40 different
moderate to strong sensitising chemicals, however, mild sensitisers responding in the GPMT was not
identified (Basketter and Scholes, 1992). A new experimental design of sensitive mouse Iymph node
assay was developed (Ikarashi, Y., el al., 1993) and validated using thioureas and disperse dyes
(Ikarashi, Y., el al., 1994 og 1996). However, other researchers have found that the LLNA could not
discriminate between allergens and irritants and it is questionable if the LLNA gives any significant
advantages to the guinea pig methods (Edwards et al., 1994, Montelius et al., 1994). The LLNA
method was developed for testing chemicals and no experience with extracts and mixtures has been
published. The ICVAM Immunotoxity working group is currently assessing a proposal to accept the
LLNA as a replacement for the guinea pig maximization test.
The popliteal Iymph node assay (PLNA) using subcutaneous administration in the footpad (De Bakker
et al 1990, Descotes et al 1997, Vial et al 1997) is an alternative Iymph node assay. In the latter assay,
54
besides direct measurement of lymph activation, also so called reporter antigens may be used for further clarification of the immunomodulation caused by the chemical under investigation (Albers et al 1997).
The risk assessment process should not rely on a single model or approach, but should be thoughtfully conducted to provide maximum assurance of safety to the consumer. Generally, this entails both animal and human experimental models. There should be flexibility in the choice of models and approaches as long as the rationale is documented and/or validated. Negative tests in guinea pigs, when they are properly conducted, can generally be definitive if the test concentration has a sufficient safety factor over use conditions. However, one should avoid classifying test materials solely on the basis of incidence and/or severity, without due consideration of eventual product usage.
The risk, e.g., incidence and severity, of the allergic reaction to the product is determined mainly with the following factors: the sensitising potency of the chemical allergen, its amount in the product, bioavailability and the exposure conditions. The relative sensitising potencies of chemicals can be defined in terms 1 of the minimum induction concentration required to induce a given level of sensitisation: the lower this concentration the more potent the sensitiser (Roberts, 1987); (Andersen, el al., 1995), and it was shown that the significant incidence of allergic contact dermatitis was found in users when the residue level of the allergen in the product exceeded its minimum induction concentration obtained by GPMT (Nakamura, et al., 1994).
On the other hand predictive testing of mixtures and products is much less validated and may be performed following testing of product ingredients. However, test design and result interpretation is subject to uncertainty, but several examples document this possibility. In animal experiments with acetone extracts from a sweater that had caused contact dermatitis in man allergens (phosgene chlorophenylhydrazones) were demonstrated (Kojima, 1990). In another case, animal experiments with acetone/chloroform extracts from rubber boots that had caused contact dermatitis in man, mercaptobenzothiazole and dibenzothiazyldisulfide were eventually found to be the causative allergens (Kaniwa, 1992). The importance of adequate extraction liquid was clearly demonstrated. The extracts made with solvent induced hypersensitivity in the guinea pigs while the saline extracts failed to do so.
The Japanese Guidelines for Basic Biological Tests of Medical Materials and Devices (1995) adopts the sample preparation procedure with organic solvents and the risk assessment procedure by comparing the residue yield from the material with the minimum induction concentration of the residue (mixture) obtained from allergenicity testing in animals.
In vitro methods for skin sensitisation: testing is currently not available for routine use (Silva et al.,1996).
ANNEX E (Informative)
Bibliography
General reference for skin and eye irritation and skin sensitisation
tests
World Medical Association. Declaration of Helsinki. recommendation guiding physicians in biomedical research involving human subjects. Adopted by the 18 th. World Medical Assembly, Helsinki June 1964, amended by the 29th. World Medical Assembly, Tokyo, October 1975, the 35 th. World Medical Assembly, Venice, October 1983 and the 415'. World Medical Assembly, Hong Kong, September 1989. Proceeding of the XXVIth. Conference, Geneva, 1993.
Marzalli,F.N. and Maibach,H.I. (eds.) Dermatotoxicology, 3rd ed, Hemisphere Publishing Corporation, 1987.
Organization for Economic Cooperation and Development (OECD) Guideline for the testing of chemical. Acute Dermal Irritation Study in Human Volunteers. Draft document, Nov. 1997.
Organization for Economic Cooperation and Development (OECD) Guidelines for the Testing of Chemicals No. 406 Skin Sensitization, OECD Publications, 1992
Organization for Economic Cooperation and Development (OECD), Guidelines for Testing of Chemicals No.404, Acute Skin Irritation/Corrosion and No. 405 Acute Eye Irritation/Corrosion. OECD Publications, 1992.
Ponec, M. In vitro models to predict skin irritation. In: The Irritant Contact Dermatitis Syndrome. Van der Valk PGM, Maibach HI (eds). Boca Raton, CRC Press, 335-341, 1996.
Serup, J., Jemec, G.B.E. Handbook of non-invasive methods and the skin. CRC Press,
1995
Silva, O. de, Basketter, D.A., Barratt, M.D., Corsini, E. et al. Alternative Methods for Skin Sensitisation Testing. The report and Recommendations of ECVAM Workshop 19. ATLA 24; 683- 705, 1996.
Simion, F.A. In vivo models to predict skin irritation. In: The Irritant Contact Dermatitis Syndrome. Van der Valk PGM, Maibach HI (eds). Boca Raton, CRC Press, 329-334, 1996.
Wahlberg, J.E. Assessment of skin irritancy: measurement of skin fold thickness. Contact Dermatitis 9 (1):21-26, 1983.
Wahlberg, J.E., Wahlberg, E.N. Quantification of skin blood flow at patch test sites. Contact Dermatitis 17 (4):229-233, 1987.
Bibliography for skin and eye irritation tests
Bruner, L.H., Kain, D.J., Roberts, D.A., Parker, R.D. Evaluation of Seven in vitro Alternatives for Ocular Testing Fund. and App. Toxicol.,pp. 136-149, 1991.
Consumer Product Safety Act. 15 U.S.Congress 2041, 1972.
Draize, J.H. Dermal Toxicity. Association of Food and Drug Of ficials of the U.S.,pp. 46-59, FDA, Washington, D.C, 1955.
Draize, J.H. Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics, Austin, Texas, Association of Food and Drug Officials of the United States, Texas State Department of Health, Texas, 1959.
Emtestam, L., Ollmar, S. Electrical Impedance Index in Human Skin: Measurements after Occlusion, in 5 Anatomical Regions and in Mild Contact Dermatitis. Contact Dermatitis, 28; 104, 1993.
European Chemical Industry Ecology and Toxicology Centre. Eye Irritation Testing, Monograph 11, Brussels, Belgium, 1988.
European Chemical Industry Ecology and Toxicology Centre. Skin Irritation, Monograph 15, Brussels, Belgium, 1990.
Grant, M.W. Toxicology of the Eye, 3rd ed, Springfield, III., 1986.
MacMillan, F.S., Rafft, R.R., Cloers, W.B. A Comparison of the Skin Irritation Produced by Cosmetic Ingredients and Formulations in the Rabbit, Guinea Pig, and Beagle Dog to that observed in the Human. Animal Models in Dermatology, Maibach, H.l. (ed.), N.Y., Churchill Livingstone, 1222, 1975.
National Academy of Sciences. Principles and Procedures for Evaluating the Toxicity of Household Substances, Washington, D.C. Comparisons of Skin Irritancy. Toxicol. Appl. Pharmacol., 481-90, 1977.
National Academy of Sciences - National Research Council. In: Principles for Evaluating Chemicals in the Environment. NAS Publication for the Environmental Protection Agency: Eye Irritation, 104105, 1975.
Pesticide Assessment Guidelines, Subdivision F. Hazard Evaluation Human and Domestic Animals. 81 -4 Eye Irritation and 81 -5 Dermal Irritation. Of fice of Pesticide Programs, U.S. Environmental Protection Agency .
Prince, J.H., Diesem, C.D., Eglitis, I., Ruskell, G.L. Anatomy and Histology of the Eye and Orbit in Domestic Animals. Springfield, III., 1980.
Steinberg, M., Akers, W.A., Weeks, M., McCreesh, A.H., Maibach, H.I. A Comparison of Test Techniques Based on Rabbit and Human Skin Responses to Irritants with Recommendations, Regarding the Evaluation of Mildly or Moderately Irritating Compounds. Animal Models in Dermatology. Maibach H.I. (ed.), N.Y., Churchill Livingstone, pp. 1-11, 1975.
Weil, S.C., Scala, R.A. Study of Intra-and Interlaboratory Variability in the Results of Rabbit Eye and Skin Irritation Tests. Toxicol. Appl. Pharmacol. 12; 276-360, 1971.
Wolven, A., Levenstein, I. Techniques for Evaluating Dermal Irritation. J. Soc. Cos. Chem. 18; 199203, 1967.
Bibliography for oral irritation tests
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