oecd 429


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OECD GUIDELINE FOR THE TESTING OF CHEMICALS GUIDELINE 429 Skin Sensitisation : Local Lymph Node Assay K.B.Institute of Pharmaceutical Education and Research, Gandhinagar Roll No – 45

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This is the second guideline to be promulgated for assessing skin sensitisation potential of chemicals in animals. The other guideline (406) utilises guinea pig tests ( The guinea pig maximisation test and the buehler test) A new assay for the determination of skin sensitisation in the mouse, the local lymph node assay (LLNA) Skin Sensitisation : Local Lymph Node Assay

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The LLNA provides certain advantages with regard to both scientific progress and animal welfare. It studies the induction phase of skin sensitisation and provides quantitative data suitable for dose response assessment. In addition, it should be noted that the mild/moderate sensitisers , which are recommended as suitable positive control substances for guinea pig test methods, are also appropriate for use with the LLNA.


INITIAL CONSIDERATIONS An Alternative Method, This does not necessarily imply that in all instances the LLNA should be used in place of guinea pig tests, but rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation.

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The LLNA is an in vivo method and, as a consequence, will not eliminate the use of animals in the assessment of contact sensitising activity . It has, however, the potential to reduce the number of animals required for this purpose Moreover, the LLNA offers a substantial refinement of the way in which animals are used for contact sensitisation testing .

LLNA And Guinea Pig Tests :

The LLNA is based upon consideration of immunological events stimulated by chemicals during the induction phase of sensitisation . Unlike guinea pig tests the LLNA does not require that challenged-induced dermal hypersensitivity reactions be elicited . Furthermore, the LLNA does not require the use of an adjuvant , as is the case for the guinea pig maximisation test. Thus , the LLNA reduces animal distress. LLNA And Guinea Pig Tests

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Despite the advantages of the LLNA over traditional guinea pig tests, it should be recognised that there are certain limitations that may necessitate the use of traditional guinea pigs tests. e.g., false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants LLNA And Guinea Pig Tests

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Figure : Auricular Lymph Node


PRINCIPLE OF THE TEST The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the site of chemical application . This proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective, quantitative measurement of sensitisation . The LLNA assesses this proliferation as a dose-response in which the proliferation in test groups is compared to that in vehicle treated controls . The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index , is determined, and must be at least three before a test substance can be further evaluated as a potential skin sensitiser .


DESCRIPTION OF THE ASSAY Selection of animal species The mouse is the species of choice for this test. Young adult female mice of CBA/Ca or CBA/J strain, which are nulliparous and non-pregnant, are used. At the start of the study, animals should be between 8-12 weeks old, and the weight variation of the animals should be minimal and not exceed 20% of the mean weight.

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HOUSING AND FEEDING CONDITIONS Animals should be individually housed. The temperature of the experimental animal room should be 22ºC (+ 3ºC). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning, the aim should be 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 unlimited supply of drinking water.

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PREPARATION OF ANIMALS The animals are randomly selected, marked to permit individual identification (but not by any form of ear marking), and kept in their cages for at least 5 days prior to the start of dosing to allow for acclimatisation to the laboratory conditions. Prior to the start of treatment all animals are examined to ensure that they have no observable skin lesions.

Reliability check :

Reliability check The positive control should produce a positive LLNA response at an exposure level expected to give an increase in the stimulation index (SI) >3 over the negative control group. The positive control dose should be chosen such that the induction is clear but not excessive. Preferred substances are Hexyl cinnamic aldehyde and Mercaptobenzothiazole


TEST PROCEDURE Number of animals and dose levels A minimum of four animals is used per dose group , with a minimum of three concentrations of the test substance, plus a negative control group treated only with the vehicle for the test substance, and a positive control, as appropriate. In those cases in which individual animal data are to be collected, a minimum of five animals per dose group are used. Doses are selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc. Existing acute toxicity and dermal irritation data should be considered, where available, in selecting the three consecutive concentrations so that the highest concentration maximises exposure whilst avoiding systemic toxicity and excessive local skin irritation.

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The vehicle should be selected on the basis of maximising the test concentrations and solubility whilst producing a solution/suspension suitable for application of the test substance. In order of preference, recommended vehicles are acetone/olive oil (4:1 v/v), dimethylformamide , methyl ethyl ketone , propylene glycol and dimethyl sulphoxide , but others may be used if sufficient scientific rationale is provided. In certain situations it may be necessary to use a clinically relevant solvent or the commercial formulation in which the test substance is marketed as an additional control. Particular care should be taken to ensure that hydrophilic materials are incorporated into a vehicle system , which wets the skin and does not immediately run off. Thus , wholly aqueous vehicles are to be avoided .

Experimental schedule :

Experimental schedule Day 1: Individually identify and record the weight of each animal . Open application of 25μL of the appropriate dilution of the test substance, the vehicle alone, or the positive control (as appropriate), to the dorsum of each ear. Days 2 and 3: Repeat the application procedure carried out on day 1. Days 4 and 5 : No treatment.

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Day 6 : Record the weight of each animal. Inject 250μL of phosphate-buffered saline (PBS) containing 20 μCi (7.4e+5 Bq ) of 3 H-methyl thymidine into all test and control mice via the tail vein . Alternatively inject 250 μ L PBS containing 2 μ Ci (7.4e + 4 Bq ) of iododeoxyuridine and 10 -5 M fluorodeoxyuridine into all mice via the tail vein. Five hours (5 h) later, the animals are killed. The draining auricular lymph nodes from each ear are excised and pooled in PBS for each experimental group (pooled treatment group approach); To further monitor the local skin response in the main study, additional parameters such as scoring of ear erythema or ear thickness measurements (obtained either by using a thickness gauge, or ear punch weight determinations at necropsy) may be included in the study protocol.

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Preparation of cell suspensions A single cell suspension of lymph node cells (LNC) either from pooled treatment groups or bilaterally from individual animals is prepared by gentle mechanical disaggregation through 200 μm -mesh stainless steel gauze. Lymph node cells are washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 o C for 18h. Pellets are either re-suspended in 1 mL TCA and transferred to scintillation vials containing 1.0 mL of scintillation fluid for 3 H-counting, or transferred directly to gamma counting tubes for 125 I-counting.

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Determination of cellular proliferation (incorporated radioactivity) Incorporation of 3 H-methyl thymidine is measured by β-scintillation counting as disintegrations per minute (DPM). Incorporation of 125 I-iododeoxyuridine is measured by 125 I-counting and also is expressed as DPM. Depending on the approach used, the incorporation will be expressed as DPM/treatment group (pooled approach) or DPM/animal (individual approach).

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OBSERVATIONS Clinical observations Animals should be carefully observed once daily for any clinical signs, either of local irritation at the application site or of systemic toxicity. All observations are systematically recorded with individual records being maintained for each animal. Body weights individual animal body weights should be measured at the start of the test and at the scheduled kill of the animals.


CALCULATION OF RESULTS Results are expressed as the Stimulation Index (SI). When using the pooled approach, the SI is obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group; this yields a mean SI. When using the individual approach, the SI is derived by dividing the mean DPM /mouse within each test substance group and the positive control group by the mean DPM/mouse for the solvent/vehicle control group. The average SI for vehicle treated controls is then 1.


DATA AND REPORTING Data Data should be summarised in tabular form showing the mean and individual DPM values and stimulation indexes for each dose (including vehicle control) group. Test report Test substance: identification data ( e.g. CAS number, if available; source; purity; known impurities; lot number ); physical nature and physicochemical properties ( e.g. volatility, stability, solubility); if mixture, composition and relative percentages of components.

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Vehicle: identification data (purity; concentration, where appropriate; volume used); justification for choice of vehicle. Test animals: strain of mice used; microbiological status of the animals, when known; number , age and sex of animals; source of animals, housing conditions, diet, etc. Test conditions: details of test substance preparation and application; justification for dose selection (including results from range finding study, if conducted);vehicle and test substance concentrations used, and total amount of substance applied; details of food and water quality (including diet type/source, water source).

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Reliability check: a summary of results of latest reliability check, including information on substance, concentration and vehicle used; concurrent and/or historical positive and negative control data for testing laboratory. Results : individual weights of animals at start of dosing and at scheduled kill; a table of mean/median (pooled approach) and individual (individual approach) DPM values, as well as the range of values for both approaches, and stimulation indices for each dose (including vehicle control) group; statistical analysis, where appropriate; -time course of onset and signs of toxicity, including dermal irritation at site of administration, if any, for each animal.

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Discussion of results: A brief commentary on the results, the dose-response analysis, and statistical analyses, where appropriate, with a conclusion as to whether the test substance should be considered a skin sensitiser .

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Thank you!

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