Evaluation of sensitisation potential 359 irritation is determined and used for the challenge application. Induction is always performed using the undiluted test substance. METHOD The usual physico-chemical and bacteriological tests are first conducted on the test substances. * The completion of the trial takes 6 weeks and uses twenty animals. ELIMINATION OF ANIMALS SHOWING INDIVIDUAL IRRITATION REACTIONS On day 0, 0.5 g or 0.5 ml of the test substance (or the dilution to be used for the challenge application) is applied to the back of the animal immediately behind the left scapulum. The test substance rests in place beneath an occlusive patch for 48 h. Animals are eliminated if any anomalies are revealed at the site after reading at 1, 7, 24 and 48 h following the removal of the patch. INDUCTION Induction consists of two intradermic injections of Freund's complete adjuvant• and ten applications of the test substance under occlusive patches. The first day of each week the animals are clipped in a region of the back just behind the right scapulum. On days 0 and 9 the guinea-pigs receive an intradermal injection of 0.1 ml of Freund's complete adjuvant diluted to 50•o in sterile isotonic saline solution. The two injection sites should be as close together as possible at the centre of the clipped area. On days 0, 2 and 4 (first week), days 7, 9 and 11 (second week), days 14, 16 and 18 (third week) and day 21, 0.5 g or 0.5 ml of the test substance is applied to the clipped test site of each animal. The test substance is covered by an impermeable occlusive patch 22 mm in diameter. This is held in place by a microporous adhesive border 10 mm wide (Neodermotest). The entire patch is covered between treatment times by an elastic sleeve. The tenth patch is removed on day 23. SUSPENSION OF TREATMENT Treatment is stopped between days 23 and 35, i.e. for a total of 12 days. CHALLENGE APPLICATION On day 35 an area of the abdomen immediately above the left groin is clipped free of fur. At this site 0.5 g or 0.5 ml of test substance (or the chosen dilution) is applied under an occlusive patch for 48 h using the same system as already mentioned A schematic summary of the procedure of the trial is given in Figure 1. * In particular the peroxide value must be determined for fats and essential oils to ensure freshness. t Biom•rieux.

360 M. F. Brulos et al. Lefl side Righl side On 1he back InductJan Checking for absence of individual reactions _T.::.---'- zz •- /.Two mtradermal Ten topicaJ applicationtestof Days 0-1: Topical application of test subst, (oral dilutian) under occlusive patch / injections of substance (0-Sg orO-5 mL) for 48h. / O. ImLofFreund's placed under accJusive Readings at: 1,7' 24 and 48 h after removal of // complete adjuvant patches for 48h dressing. •• Day I (Jstweweeweek)patch(2ndafII18week)andandremoval9JCO(4th7,J4,212:•:DayDaysDays:Days:Dayweek) Animal eliminated if any anomaJy is revealed. Day 9 (2nd •Gu _i•g•• (:•rd inea On the abdomen Challenge a ppli cat ion------__ Co th Week: above) Day 35 : TOplcaJ application of test substance (or non irritant dilution). 0.5 g or 0'.5 mL under patch for 48 h. Day 37 : Removal of occlusive patch. Read•ngs:l and 7h after at days :•8 and From the 2_nd readJng(7h): biopsy if a clear or doubtful macroscopic reaction is noted. Figure 1. Rest period Day 2_.3 to :35: no treatment (End of 4th week, all of 5th week) INTERPRETATION OF RESULTS EVALUATION OF THE REACTION Macroscopic cutaneous examination One, 7, 24 and 48 h after the removal of the occlusive dressing the intensity of the ery- thematous reaction and eschar formation are evaluated using the following scale: no erythema ............................ 0 slight erythema (hardly visible) .......... 1 erythema distinct ...................... 2 erythema moderate to severe ............ 3 erythema severe (red/purple) with the formation of light eschars (profound lesions) 4 N.B. Any other anomaly occurring at the challenge site (eg. papules, vesicles, oedema exfoliation) should also be noted. The induction site must also be unspected in case of any focal reaction (reactivation of induction site). It is extremely important that all readings are performed under the same conditions (particularly the same conditions of lighting). The results are interpreted by calculating the percentage of guinea-pigs sensitised. The animals counted as positive are those which, (a) present at least once in four readings a reaction scoring two or more, or (b) present focal reactions whatever the reaction observed at the challenge site, or (c) present vesicles. Biopsies About 6-7 h after removing the dressing samples of skin for histological examination are taken from the challenge sites of those animals showing distinct macroscopic