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ISO/TC 194/WG8 N1
ISO/ WD 10993-10

ISO/WD 10993-10
Revision of Biological evaluation of medical devices
Tests for irritation and sensitisation

FOREWORD
INTRODUCTION
1 Scope
2 Normative references
3 Definitions
4 General principles, step-wise approach
5 Pretest considerations
6 Irritation tests
7. Sensitisation tests
8. Key factors in the interpretation of test results
ANNEX

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 organisations, governmental and non-governmental' in liaison with I SO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (EC) on all matters of electrotechnical standardisation.

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.


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 incumbent 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 incumbent 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) sensitisation

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 1 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 use in the ocular. oral rectal, penile and vaginal areas is given in ISO 10993-2.

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 IEC and ISO maintain registers of currently valid International Standards.

*: 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.


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 Identifi cation and quantification of degradation products from polymeric devices, ISO/CD 10993-14 Identifi cation and quantification of degradation products from ceramics *, ISO/CD 10993- 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 sensitises.

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 sensitises by stages a) or b). Materials that do not demonstrate a potential for acute toxicity may be further evaluated following repeated exposure.

e) non-invasive human tests/clinical trials.
If the material has been demonstrated not to be a sensitised, studies on skin irritation may be considered in humans.

f) a positive control should be run periodically 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 teems 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.

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.

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 or only qualitative information is available 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 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 proper chemical analysis of the extract may give results that makes testing for irritation and sensitisation unnecessary.

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 to better define endpoints using non-invasive techniques. See Annex D. 1.

6.2 Factors to be considered in design and selection of tests

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 of exposure; 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 manufacture, 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 11.5, 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). Eightyfive 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 annex B.

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.

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 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.

Describe and grade the skin reactions for erythema and oedema according to the classification system given in Table 1 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 (Pll) 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.

Add the scores for each animal and divide the total by the number 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 deemed 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 (Pll) 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 E). 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: l ) 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. l. 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:


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:


Further guidance on the selection of human volunteers can be found in 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 human volunteers. It is not necessary specifically to select atopic individuals. The selected human volunteers should be generally healthy. at least 18 y ears 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 cm- 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 l-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.


Table 3: Human skin irritation test, grading scale

For volunteers who have a response of l 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

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

6.4.6.1 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 skin 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:

.
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 .
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 D.


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 a 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 generic factors and husbandry.

Comparison of test animals 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 annex B.

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.


Table 4 - Magnusson and Kligman scale

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.


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 7.4.2.2 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 least 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. Daylight 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 3 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

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 I 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 I - 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 annex B.

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-'. 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 3, 24 h and 48 h after patch removal.

Select


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 7.5.4.9 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 resuming them to their cages. A minimum of 2 h after removal of hair, grade the test sites according to Table 3 . 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 I or greater in the test group generally indicate sensitisation, provided grades of less than I are seen on control animals. If grades of I 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.

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.