OECD GUIDELINES 404

404: OECD GUIDELINE FOR THE TESTING OF CHEMICALS: 

VIDHI DHAGIA M.Pharm (Pharmacology) Roll Number 23 K.B. INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH 404 : OECD GUIDELINE FOR THE TESTING OF CHEMICALS

Acute Dermal Irritation/Corrosion: 

Acute Dermal Irritation/Corrosion

DEFINITIONS: 

DEFINITIONS DERMAL IRRITATION : Dermal irritation is the production of reversible damage of the skin following the application of a test substance for up to 4 hours. DERMAL CORROSION : Dermal corrosion is the production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions .

PowerPoint Presentation: 

IN VIVO testing should not be undertaken until all available data relevant to the potential dermal corrosivity /irritation of the substance have been evaluated in a weight-of-the-evidence analysis This analysis should decrease the need for in vivo testing for dermal corrosivity /irritation of substances. A preferred sequential testing strategy, which includes the performance of validated and accepted in vitro or ex vivo tests for corrosion/irritation, is included as a Supplement to this Guideline . Major deviation - should be justified . If a determination of corrosivity or irritation cannot be made using a weight-of-the-evidence analysis , consistent with the sequential testing strategy, an in vivo test should be considered ( see Supplement).

PRINCIPLE OF THE IN VIVO TEST: 

PRINCIPLE OF THE IN VIVO TEST The substance to be tested is applied in a single dose to the skin of an experimental animal and untreated skin areas of the test animal serve as the control. The degree of irritation/corrosion is read and scored at specified intervals. The duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects observed. Animals showing continuing signs of severe distress and/or pain at any stage of the test should be humanely killed, and the substance assessed accordingly.

PREPARATIONS FOR THE IN VIVO TEST: 

PREPARATIONS FOR THE IN VIVO TEST SELECTION OF ANIMAL SPECIES The healthy young adult albino rabbits are used. A rationale for using other species should be provided. PREPARATION OF THE ANIMALS Approximately 24 hours before the test, fur should be removed by closely clipping the dorsal area of the trunk of the animals. Care should be taken to avoid abrading the skin, and only animals with healthy, intact skin should be used. Some strains of rabbit have dense patches of hair that are more prominent at certain times of the year. Such areas of dense hair growth should not be used as test sites.

PowerPoint Presentation: 

HOUSING AND FEEDING CONDITIONS Animals should be individually housed. The temperature of the experimental animal room should be 20°C (+/- 30°C) for rabbits . Relative humidity should be between 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unrestricted supply of drinking water.

PowerPoint Presentation: 

TEST PROCEDURE

Application of the test substance: 

Application of the test substance The test substance should be applied to a small area (approximately 6 cm) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In cases in which direct application is not possible (e.g., liquids or some pastes), the test substance should first be applied to the gauze patch, which is then applied to the skin. The patch should be loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period. Access by the animal to the patch and ingestion or inhalation of the test substance should be prevented.

PowerPoint Presentation: 

Liquid test substances are generally used undiluted. When testing solids (which may be pulverised , if considered necessary), the test substance should be moistened with the smallest amount of water (or, where necessary, of another suitable vehicle) sufficient to ensure good skin contact. When vehicles other than water are used, the potential influence of the vehicle on irritation of the skin by the test substance should be minimal, if any. At the end of the exposure period (4 hours) residual test substance should be removed , where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis . DOSE LEVEL: A dose of 0.5 mL of liquid or 0.5 g of solid or paste is applied to the test site.

Initial test (in vivo dermal irritation/corrosion test using one animal): 

Initial test ( in vivo dermal irritation/corrosion test using one animal)

PowerPoint Presentation: 

It is strongly recommended that the in vivo test be performed initially using one animal , especially when the substance is suspected to have corrosion potential . When a substance has been judged to be corrosive no further animal testing is needed. However , in cases of insufficient evidence , limited animal testing may be carried out using the following approach: Up to three test patches are applied sequentially to the animal. The first patch is removed after three minutes . If no serious skin reaction is observed, a second patch is applied at a different site and removed after one hour. If the observations at this stage indicate that exposure can humanely be allowed to extend to four hours, a third patch is applied and removed after four hours, and the response is graded .

PowerPoint Presentation: 

If a corrosive effect is observed after any of the three sequential exposures, the test is immediately terminated . If a corrosive effect is not observed after the last patch is removed, the animal is observed for 14 days, unless corrosion develops at an earlier time point. In those cases in which the test substance is not expected to produce corrosion but may be irritating, a single patch should be applied to one animal for four hours.

Confirmatory test (in vivo dermal irritation test with additional animals): 

Confirmatory test ( in vivo dermal irritation test with additional animals)

PowerPoint Presentation: 

If a corrosive effect is not observed – confirm using up to two additional animals, each with one patch, for an exposure period of four hours. If an irritant effect is observed - the confirmatory test may be conducted in a sequential manner, or by exposing two additional animals simultaneously. In the exceptional case , in which the initial test is not conducted, two or three animals may be treated with a single patch, which is removed after four hours. When two animals are used, if both exhibit the same response, no further testing is needed. Otherwise, the third animal is also tested. Equivocal responses may need to be evaluated using additional animals.

Observation period: 

Observation period The duration of the observation period should be sufficient to evaluate fully the reversibility of the effects observed. However, the experiment should be terminated at any time that the animal shows continuing signs of severe pain or distress. To determine the reversibility of effects, the animals should be observed up to 14 days after removal of the patches. If reversibility is seen before 14 days, the experiment should be terminated at that time.

Clinical observations and grading of skin reactions: 

Clinical observations and grading of skin reactions All animals should be examined for signs of erythema and oedema , and the responses scored at 60 minutes, and then at 24 , 48 and 72 hours after patch removal. For the initial test in one animal, the test site is also examined immediately after the patch has been removed. Dermal reactions are graded and recorded according to the grades in the Table below. If there is damage to skin which cannot be identified as irritation or corrosion at 72 hours, observations may be needed until day 14 to determine the reversibility of the effects. In addition to the observation of irritation, all local toxic effects, such as defatting of the skin, and any systemic adverse effects (e.g., effects on clinical signs of toxicity and body weight), should be fully described and recorded. Histopathological examination - to clarify equivocal responses.

PowerPoint Presentation: 

The grading of skin responses is necessarily SUBJECTIVE. To promote harmonisation, the personnel performing the observations need to be adequately trained in the SCORING SYSTEM used (see Table below). A guide for grading skin irritation and other lesions is as follows:

TABLE: GRADING OF SKIN REACTIONS: 

TABLE: GRADING OF SKIN REACTIONS Erythema and Eschar Formation CLINICAL SIGNS GRADE No erythema 0 Very slight erythema (barely perceptible) 1 Well defined erythema 2 Moderate to severe eryhtema 3 Severe erythema (beef redness) to eschar formation preventing grading of erythema 4

Oedema Formation: 

Oedema Formation CLINICAL SIGNS GRADE No oedema 0 Very slight oedema (barely perceptible) 1 Slight oedema (edges of area well defined by definite raising) 2 Moderate oedema (raised approximately 1 mm) 3 Severe oedema (raised more than 1 mm and extending beyond area of exposure) 4

DATA AND REPORTING: 

DATA AND REPORTING Study results should be summarised in tabular form in the final test report and should cover all items listed in following teat report.

Evaluation of results: 

Evaluation of results The dermal irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard for the irritant properties of a material. Instead, individual scores should be viewed as reference values, which need to be evaluated in combination with all other observations from the study. Reversibility of dermal lesions should be considered in evaluating irritant responses. When responses such as alopecia (limited area), hyperkeratosis, hyperplasia and scaling, persist to the end of the 14-day observation period, the test substance should be considered an irritant.

Test report: 

Test report The test report must include the following information: Rationale for in vivo testing: W eight-of-evidence analysis of pre-existing test data, including results from sequential testing strategy. Description of relevant data available from prior testing. Data derived at each stage of testing strategy. Description of in vitro tests performed, including details of procedures, results obtained with test/reference substances Weight-of-the-evidence analysis for performing in vivo study.

PowerPoint Presentation: 

Test substance : Identification data ( eg . CAS number; source; purity; known impurities; lot number); Physical nature and physicochemical properties ( eg . pH, volatility, solubility, stability) If mixture, composition and relative percentages of components. Vehicle: Identification, concentration (where appropriate), volume used Justification for choice of vehicle.

PowerPoint Presentation: 

Test animals: Species/strain used, rationale for using animals other than albino rabbit; Number of animals of each sex; Individual animal weights at start and conclusion of test; Age at start of study; Source of animals, housing conditions, diet, etc. Test conditions: Technique of patch site preparation; Details of patch materials used and patching technique; Details of test substance preparation, application, and removal.

PowerPoint Presentation: 

Results: Tabulation of irritation / corrosion response scores for each animal at all time points measured; Descriptions of all lesions observed; Narrative description of nature and degree of irritation or corrosion observed, and any histopathological findings; Description of other adverse local (e.g. Defatting of skin) and systemic effects in addition to dermal irritation or corrosion.

PowerPoint Presentation: 

SUPPLEMENT TO TEST GUIDELINE 404

A Sequential Testing Strategy For Dermal Irritation And Corrosion: 

A Sequential Testing Strategy For Dermal Irritation And Corrosion Although this sequential testing strategy is not an integral part of Test Guideline 404, it expresses the recommended approach for the determination of skin irritation/corrosion characteristics. The Guideline provides guidance for the conduct of the in vivo test and summarises the factors that should be addressed before initiating such a test. It also recommends the performance of validated and accepted in vitro or ex vivo tests for skin corrosion/irritation under specific circumstances .

STEP1: 

ACTIVITY FINDINGS CONCLUSIONS Existing human and/or animal data showing effects on skin or mucous membranes Corrosive Apical endpoint; considered corrosive. No testing is needed. Irritating Apical endpoint; considered irritating. No testing is needed. Not corrosive or irritating Apical endpoint; not considered irritating or corrosive. No testing is needed. If No information available, or available information is not conclusive then go for step 2. STEP 1

STEP2: 

Perform SAR evaluation for skin corrosion/irritation Predict severe damage to skin Considered corrosive. No testing is needed. Predict irritation to skin Considered irritating. No testing is needed. If No predictions can be made, or predictions are not conclusive or Negative, go for step 3 STEP 2

Step3: 

Step 3 Measure pH (consider buffering capacity, if relevant) pH ≤ 2 or ≥ 11.5 (with high buffering capacity, if relevant) Assume corrosivity . No testing is needed. 2<pH < 11.5, or pH ≤ 2.0 or ≥ 11.5 with low/no buffering capacity, do step 4

Step4: 

Step 4 Evaluate systemic toxicity data via dermal route Highly toxic No further testing is needed. Not corrosive or irritating when tested to limit dose of 2000 mg/kg body weight or higher, using rabbits Assume not corrosive or irritating. No further testing is needed. Such information is not available or is non-conclusive, do step 5

Step5: 

Step 5 Perform validated and accepted in vitro or ex vivo test for skin corrosion Corrosive response Assume corrosivity in vivo. No further testing is needed. Substance is not corrosive, or internationally validated in vitro/ ex vivo testing methods for skin corrosion are not yet available, do step 6

Step6: 

Step 6 Perform validated and accepted in vitro or ex vivo test for skin irritation Irritant response Assume irritancy in vivo. No further testing is needed. Substance is not an irritant, or internationally validated in vitro or ex vivo testing methods for skin irritation are not yet available, do step 7

Step7: 

Step 7 Perform initial in vivo rabbit test using one animal Severe damage to skin Considered corrosive. No further testing is needed. No severe damage, then do step 8

Step8: 

Step 8 Perform confirmatory test using one or two additional animals Corrosive or irritating Considered corrosive or irritating. No further testing is needed Not corrosive or irritating Considered not corrosive or irritating. No further testing is needed

PowerPoint Presentation: 

Thank you…..