Document rBb7GgQpmO5Nmkemaa9KK5YE0

AR226-3158 CONFIDENTIAL SPONSOR ElfAtochemSA Cours Michelet La Defense 10 92091 Paris-la-Defense CEDEX France TEST SUBSTANCE STUDY TITLE SKIN SENSmZATION TEST IN GUINEA-PIGS (Maximization method ofMagnusson and Kligman) STUDY DIRECTOR Xavier Manciaux STUDY COMPLETION DATE 28 August 2000 TEST FACILITY err Centre International de Toxicologie BP 563 - 27005 Evreux - France IFM Recherche wrai SM.C.W oe s M am f SIECC SOCMl :HKStir 27003 EWEUX Company Sanitized. Does not contain TSCA'CBI CENTRE INTERNATIONAL DE TOXICOLOGIE B.P. 563 2700'; Fvro.^ r^ :--- 2 CONTENTS STATEMENT OF THE STUDY DIRECTOR 4 OTHER SCIENTIST INVOLVED IN THIS STUDY 4 STATEMENT OF QUALITY ASSURANCE UNIT 5 SUMMARY 6 RESUME 8 1JNTRODUCTION 10 2. MATERIALS AND METHODS 10 2.1 TEST SUBSTANCE AND OTHER SUBSTANCES 10 2.1.1 Identification of the test substance 10 2.1.2 Vehicle 10 2.1.3 Dosage form preparation 10 2.1.4 Other substances 11 2.2 TEST SYSTEM 11 2.2.1 Animals 11 2.2.2 Environmental conditions 11 2.2.3 Food and water 12 2.3 TREATMENT 12 2.3.1 Preliminary test 12 2.3.2 Main study 13 2.3.2.1 Preparation of the animals 13 2.3.2.2 Induction phase by intradermal and cutaneous routes 13 2.3.2.2.1 Intradermal route 13 2.3.2.2.2 Cutaneous route 13 2.3.2.3 Challenge phase 14 2.4 SUMMARY DIAGRAM 15 Figure 1: Treatment sites 15 2.5 SCORING OF CUTANEOUS REACTIONS 16 2.6 CLINICAL EXAMINATIONS 16 2.7 BODY WEIGHT 16 2.8 PATHOLOGY < 16 2.8.1 Necropsy 16 2.8.2 Skin samples 16 2.9 DETERMINATION OF THE ALLERGENICITY LEVEL 16 -,' company Sanitized. Does not contain TSCA CB1 2.10 CHRONOLOGY OF THE STUDY 2.11 PROTOCOL ADHERENCE 2.12 ARCHIVING 3. RESULTS 3.1 CHOICE OF THE VEHICLE 3.2 PRELIMINARY STUDY 3.2.1 Administration by intradermal route 3.2.2 Application by cutaneous route 3.3. MAIN STUDY 3.3.1 Clinical examinations 3.3.2 Body weight 3.3.3 Challenge phase - Scoring of cutaneous reactions 4. CONCLUSION APPENDICES 1. Test article description and analytical certificate 2. Diet formula 3. Individual body weight values 4. Positive control to check the sensitivity of Dunkin-Harfley guinea-pigs 17 18 18 19 19 19 19 20 20 20 20 21 22 23 24 27 29 31 and 32 .------,,S.md.Do^n^c^nTSCACB. 4 STATEMENT OF THE STUDY DIRECTOR The study was performed in compliance with the principles of Good Laboratory Practice as described in: . OECD Principles on Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98)17. . Decret N 98-1312 du 31 decembre 1998 concemant les Bonnes Pratiques de Laboratoire (Journal Officiel du ler janvier 1999), Ministere de 1'Economie, des Finances et de 1'mdustrie. . Commission Directive 1999/1 I/EC of 8 March 1999 adapting to technical progress the Principles of Good Laboratory Practice as specified in Council Directive 87/18/EEC on the hannonization of laws, regulations and administrative provisions relating to the application of the Principles of Good Laboratory Practice and the verification of their applications for tests <-on chemical substances (OJ No. L 77 of 23.3.1999). I declare that this report constitutes a true and faithful record of the procedures undertaken and the results obtained during the performance of the study. This study was performed at CIT, Centre International de Toxicologie, BP 563, 27005 Evreux, France. Toxicology X. Manciaux Study Director Doctor of Pharmacy Date: 28 August 2000 OTHER SCIENTIST INVOLVED IN THIS STUDY For Pharmacy: P.O. Guillaumat Doctor of Pharmacy 'Company Sanitised. Does no? contain TSCA CB' STATEMENT OF QUALITY ASSURANCE UNIT Type of inspections Protocol Report Inspections 14 December 1999 24 July 2000 Dates Reported to Study Director (*) 15 December 1999 17 August 2000 Reported to Management (*) 13 December 1999 17 August 2000 In addition to the above-mentioned inspections, at about the same time as the study described in the present report, "process-based" and routine facility inspections of critical procedures relevant to this study type were also made by the Quality Assurance Unit. The findings of these inspections were reported to the Study Director and to QT Management. The inspections were performed in compliance with CIT Quality Assurance Unit procedures and the Good Laboratory Practice. The reported methods and procedures were found to describe those used and the results to constitute an accurate and complete reflection of the study raw data. 'OoA^e~^\^' L. Valette-TaIbi Date: 28 August 2000 Doctor of Biochemistry Head of Quality Assurance Unit and Scientific Archives (*) The dates indicated correspond to the dates of signature of audit reports by Study Director and Management. Company Sanitized. Does nol contain r^^f^t SUMMARY ^I^U^U^U^m AUh^tt^^^^l^lQchen^A, Paris-la-Defense, France, the potential of the test substance to induce delayed contact hyper-sensitivity was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406,17th July 1992) and EC (96/54/EEC, B.6, 30 July 1996) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Methods Thirty guinea-pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals: . Freund's complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCI (both groups), . test substance at the chosen concentration in the chosen vehicle (treated group) or vehicle alone (control group), . test substance at the chosen concentration in a mixture FCA/0.9% NaCI 50/50 (treated group) or vehicle at the concentration of 50% in a mixture FCA/0.9% NaCI 50/50 (control group). On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On day 8, the test substance (treated group) or the vehicle (control group) was applied topically to the same test site which was then covered by an occlusive dressing for 48 hours. On day 22, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. Test substance concentrations were as follows: ^^^^^^^^ Induction (treated group) . intradermal injections (day ^.^ss^^^^SSA ^t the concentration of 25% (w/w) in sterile isotonic saline solution (0.9%^^^^^^^^ . topical application (day 8):^^^HHBHundiIuted. Challenge (all groups) H^^^^^^^^^U^^^in^diluted. . topical application (day 22); At the end of the study, animals were killed without examination of internal organs. No skin samples were taken from the challenge application sites. Company "Sanitizacr. Ooesnb! contain TSCA CB7 ! Results No clinical signs and no deaths were noted during the study. No well-defined cutaneous reactions were observed after the challenge application. Conclusion Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test substanceM|BHHHIHBHIHI does not induce delayed contact hypersensitivity in guinea-pigs. According to the classification criteria laid down in Directive 93/2 I/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance should not be considered as a skin sensitizer. _gy2_g,_jai,^c--g--s,^^^_^--^w."-^.>,----1-;;-.--^."--~..^t,-.,--.^,?i>..,;i- .b^At-a' RESUME B^^^^I^UUI^Il1 AhuleinandedeEl^tochem SA, Paris-la-Defense, France, Ie potentiel du produit induire une hypersensibilisation cutanee retardee est evalue chez Ie Cobayeseionlametiliodede maximisation de Magnusson et Kligman et conformement aux lignes directrices de 1'OCDE (n0 406, 17 juillet 1992) et de la CEE (96/54/EEC, B.6, 30 juiUet 1996). L'etude est realisee conformement aux regles de Bonnes Pratiques de Laboratoire. Methodes Trente cobayes sont repartis en 2 groupes : un groupe temoin de 5 males et 5 femelles et un groupe traite de 10 males et 10 femelles. Au jour 1, trois paires d'injections intradermiques sont effectuees au niveau de la region interscapulaire de tous les animaux : . Adjuvant complet de Freund (FCA) dilue a 50 % (v/v) dans du NaCI a 0,9 % (groupe traite et groupe temoin), . produit a tester a la concentration choisie dans Ie vehicule (groupe traite) ou vehicule seui (groupe temoin), . produit a tester a la concentration choisie dans une mixture FCA/ NaCI a 0,9 % 50/50 (groupe traite) ou vehicule a la concentration de 50 % dans une mixture FCA/ NaCI a 0,9 % 50/50 (groupe temoin). Au jour 7, une application cutanee de laurylsulfate sodique a 10 % (p/p) dans de la vaseline est effectuee sur la meme zone dans Ie but d'induire une irritation locale. Au jour 8, Ie produit (groupe traite) ou Ie vehicule (groupe temoin) sont appliques sur Ie meme site, qui est ensuite recouvert d'un pansement occlusif pendant 48 heures. Au jour 22, tous les animaux des groupes traite et temoin recoivent une application cutanee declenchante de produit sur Ie flanc droit Le flanc gauche sert de temoin et recoit Ie vehicule seul. Le produit et le vehicule sont maintenus sous pansement occlusif pendant 24 heures. devaluation des reactions cutanees est effectuee environ 24 et 48 heures apres 1'enlevement du pansement. Les concentrations de produit sont les suivantes: Induction (groupe traite) _ NaCI a 0,9%, application cutanee (jour 8): __ . Application declenchante (tous les groupes^^^^ .application cutanee (jour22):^^^^^^^^HEion dilue. ^BBB^^^^^^^^^^^^^^^^^^- A la fin de 1'etude, les animaux sont sacrifies sans examen des organes internes. Aucun prelevement cutane n'est effectue au niveau des sites d'application declenchante. 'Company Sanitized. Does not contain TSCA CB( Resulfats Aucun signe clinique ni aucune mortalite ne sont notes pendant 1'etude. Aucune reaction cutanee bien definie n'est observee apres 1'application declenchante. Conclusion ^----JC^B^^ Dans nos conditions_expenmgntales et selon la methode de maxunisation de Magnusson et Kligman, Ie produit n'induit pas de reaction cutanee attribuable a une hypersensibilisation cutaneeretaroee^De^leCobaye. Selon les criteres de classification decrits dans la Directive 93/21/CEE (27 avril 1993) portant du-huitieme adaptation au progres technique de la Directive 67/548/CEE, Ie produit est considere non sensibilisant par contact avec la peau. company Sanitizea. Does hoi contain TSCA CBl 10 1. INTRODUCTION The objective of this study, performed according to the maximizationmethodo^Magnusson and Kligman (1), was to evaluate the potential of the test substance^HIBHU| to induce delayed contact hypersensitivity in guinea-pigs. The results of the study are of value in predicting the contact sensitization potential of the test material in humans. The study was conducted in compliance with: . OECD guideline No. 406,17th July 1992, . EC Directive No. 96/54/EEC, B.6, 30 July 1996. 2/- MATERIALS AND METHODS 2.1 TEST SUBSTANCE AND OTHER SUBSTANCES 2.1.1 Identification of the test substance The test substance I^HUlB use<^ m ^le ^^Y was supplied by the Sponsor. It was identified as follows: . name: . batch number ^^^^^^^^* E - l f pr A ot to o c co he l a m nd fili Ia ng bneullminbge:|TMB^H^ ^^^^^^^II^| . . description: yellow liquid ^^^^^^^^ . container one plastic flask . date of receipt 16 December 1999 . storage conditions: at room temperature . expiry date: November 2000. Data relating to the characterization of the test substance are documented in a test article description and an analytical certificate (presented in appendix 1) provided by the Sponsor. 2.1.2 Vehicle The choice of the vehicle was based on tests to check the homogeneity (visual check) of the preparation (for cutaneous application and intradermal injections) and its free passage through a needle (for intradermal injections). The highest concentrations which satisfied these criteria were called the maximal practicable concentrations. The vehicle used was 0.9% NaCI, batch No. LR92011 (Laboratoire Fresenius, 92316 Sevres, France). 2.1.3 Dosage form preparation ,A11 dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day. (1) Magnusson B. and Kligman A.M.: The identification of contact allergens by animal assay. The guinea-pig maximization test. J. Invest. Derm., 52: 268-276 (1969). ~c=om--p--an-y.-S_an.rtizea-,;.D--oes--ho-i c,o-n-t-a,in ,.IS.,c"Aa C.,,B-.I Company Sanitized. Does not contain TSC 11 2.1.4 Other substances The other substances used were Freund's complete adjuvant, batch No. 39H8926 (Sigma, 38297 Saint-Quentin-Fallavier, France); sodium lauryl sulfate, batch No. 107H0006 (Sigma, 38297 Saint-Quentm-Fallavier, France) and vaseline, batch No. 1144 (Cooperative Pharmaceutique Fran9aise, 77000 Melun, France). 13. TEST SYSTEM 2.2.1 Animals , Species and sex-: male and female guinea-pigs. Strain and sanitary status: Hartley Cri: (HA) BR, Caesarian obtained. Barrier sustained - Virus Antibody Free (COBS - VAF). Reason for this choice: species generally accepted by regulatory authorities for this type of study. The strain used has been shown to produce a satisfactory sensitization response using known sensitizers. Breeder Charles River France, 76410 Saint-Aubin-les-Elbeuf, France. Number . one male and one female for the preliminary test, . 30 animals (15 males and 15 females) for the main test. Females were nulliparous and non-pregnant. Allocation of the animals to the groups: on day -1, the animals were weighed and randomly allocated to two groups: a control group of ten animals (five males and five females) and a treated group of 20 animals (ten males and ten females). Age/weight: on day 1, the animals of the main test were approximately 3 months old and had a mean body weight standard deviation of 364 14 g for the males and 341 16 g for the females. Acclimatization: at least 5 days before the beginning of the study. Identification of the animals: ear-tattoo. 2.2.2 Environmental conditions The conditions in the animal room were set as follows: . temperature: 21 2C . relative humidity: 30 to 70% . light/dark cycle: 12 h/12 h . ventilation: approximately 12 cycles/hour of filtered, non-recycled air. The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. During the acclimatization period and throughout the study, the animals were housed individually in polycarbonate cages (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle. Dust-free sawdust was provided as litter (SICSA, 94142 Alfortville, France). Bacteriological and chemical analyses of the sawdust, including the detection of possible contaminants (pesticides, heavy metals), are performed regularly by external laboratories. The results of these analyses are archived at CIT. (Company Sanitized. Does not contain T8CA C^< 12 2.2.3 Food and water During the study, the animals had free access to "106 pelleted diet" (UAR, 91360 Villemoissonsur-Orge, France). Food is analysed regularly by the supplier for composition and contaminant levels. The diet formula is presented in appendix 2. Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analyses of the water and diet, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines), are performed regularly by external laboratories. The results of these analyses are archived at CIT. No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced the outcome of theTstudy. 2.3 TREATMENT 2.3.1 Preliminary test A preliminary test was conducted in order to determine the concentrations to be tested in the main study. By intradermal route (tested concentrations: 75%, 50% and 25% (w/w)): . 24 hours before treatment, the dorsal region of the animals was clipped, . intradermal administrations of the dosage form preparations (0.1 ml) were performed in the interscapular region, . cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections. By cutaneous route (tested concentrations: 100% and 50% fw/w)): . 24 hours before treatment, both flank regions of the animals were clipped, . the filter paper of a chamber (Finn Chamber) was fully-loaded with the dosage form preparations. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours, . cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings. Criteria for selection of concentrations The following criteria were used: . the concentrations should be well-tolerated systemically and locally, . intradermal injections should cause moderate irritant effects (no necrosis or ulceration of the skin), . cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, . cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects. ,,. company Sanitized. Does not contain TSCA C6t 13 6 2.3.2 Main study 2.3.2.1 Preparation of the animals For all animals, the application sites were: . clipped on days -1 and 7 (interscapular region 4 cm x 2 cm), . clipped and shaved on day 21 (each flank 2 cm x 2 cm). 2.3.2.2 Induction phase by intradermal and cutaneous routes 2.3.2.2.1 Intradermal route On day 1, six injections were made deep into the dermis of a 4 cm x 2 cm clipped interscapular area, using a needle (diameter 0.50 x 16mm) mounted on a 1 ml plastic syringe (0.01 ml graduations). Three injections of 0.1 ml were made into each side of this interscapular region (i.e. three pairs of sites), as follows: Injection Site Anterior Treated group FCA at 50% (v/v) in 0.9% NaCI Control group FCA at 50% (v/v) in 0.9% NaCI Middle test substance at 25% (w/w) in 0.9% NaCI 0.9% NaCI 3 Posterior* test substance at 25% (w/w) in a vehicle at 50% (w/v) in a mixture mixture FCA /0.9% NaCI 50/50 FCA /0.9% NaCI 50/50 FCA: * : Freund's complete adjuvant The test substance was first dissolved in the aqueous phase prior to mixing with FCA. The final concentration of the test substance was equal to that used in injection 2. The anterior and middle pairs of injections were performed close to each other and nearest the head, while the posterior pair was performed towards the caudal part of the test area. 2.3.2.2.2 Cutaneous route On day 7, the interscapular area was clipped. As the test substance was shown to be non-irritant during the preliminary test, the animals were treated with 0.5 ml of sodium lauryl sulfate at the concentration of 10% (w/w) in vaseline, in order to induce local irritation. On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the undiluted test substance and was then applied to the interscapular region of the animals of the treated group. The animals of the control group received an application of the vehicle alone under the same experimental conditions. The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster. , any Sanitize'd. Does no! contain TSCA CBI 14 2.3.2.3 Challenge phase On day 22, the animals of treated and control groups received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chamber)was fully-loaded with the undiluted test substance and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. The chambers were held in contact with the skin for 24 hours by means of an adhesive anallergenic waterproof plaster. Company SanIHzea. Dsesnai contain TSCA CBf 15 2.4 SUMMARY DIAGRAM Figure 1: Treatment sites Induction site Intradermal injections (day 1)* Cutaneous application (day 7): Sodium lauryl sulfate 10% in vaseline Cutaneous application (day 8): vehicle (control group) or test substance at the chosen concentration (treated group) Challenge application sites Cutaneous application (day 22) * Intradermal injections: 50% Freund's complete adjuvant and sterile isotonic solution (0.9% NaCI) Q vehicle (control group) or test substance at the chosen concentration in the vehicle (treated group) v (D vehicle at 50% (control group) or test substance at the chosen concentration (treated group) in a mixture of Freund's complete adjuvant/ 0.9% NaCI 50/50 '^s mi eontain TsGA CB? 16 2.5 SCORING OF CUTANEOUS REACTIONS Twenty-four and 48 hours after removal of the dressing of the challenge application, both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions, according to the following scale: . no visible change.................................................................................................................. 0 . discrete or patchy erythema.................................................................................................. 1 . moderate and confluent erythema......................................................................................... 2 . intense erythema................................................................................................................... 3 Any observed oedema was recorded. Any other lesions were noted. 2.6 CLINICAL EXAMINATIONS The animals were observed at least once a day during the study in order to check for clinical signs and mortality. 2.7 BODY WEIGHT The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1) and on the last day of the study (day 25). 2.8 PATHOLOGY 2.8.1 Necropsy At the end of the study, all the animals were killed by carbon dioxide asphyxiation. No necropsy was performed. 2.8.2 Skin samples No skin samples were taken. 2.9 DETERMINATION OF THE ALLERGENICITY LEVEL The animals of the treated group show a positive reaction if macroscopic cutaneous reactions are clearly visible (score > 1) and are of greater intensity and/or duration of response than the maximum reaction seen in control animals, or if macroscopic reactions are confirmed at microscopic examination as being due to the sensitization process. company Sanitized. Does not contain TSCA CB? 17 Determination of the allergenicitv level The allergenicity level of the test substance is calculated by comparing the number of animals showing positive reactions with the number of surviving treated animals at the end of the study. % of animals showing a reaction 0- 8 9 29 65 81 - 28 64 80 100 Allergenicity level I n m IV v Classification weak mild moderate strong extreme According to the Commission Directive 93/2 I/EEC, when the reactions are positive in at least 30% of the treated animals, the test substance has sensitization properties and the symbol Xi, the indication of danger "Irritant" and the sentence "R 43: May cause sensitization by skin contact" must be applied. The sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer, MERCAPTOBENZOTHIAZOLE. In a recent study performed under CIT experimental conditions, the strain of guinea-pigs used showed, a satisfactory sensitization response in 80% animals (see appendix 4). 2.10 CHRONOLOGY OF THE STUDY The chronology of the main test is summarized as follows: Procedure Arrival of the animals Weighing and allocation of the animals into groups Weighing, induction by intradennal injection Sodium lauryl sulfate application Induction by cutaneous route Removal ofocclusive dressings Challenge cutaneous application Removal of occlusive dressings Scoring of cutaneous reactions after . 24 hours . 48 hours Weighing,-sacrifice of the animals Date 27 January 2000 31 January 2000 1 February 2000 7 February 2000 8 February 2000 10 February 2000 22 February 2000 23 February 2000 24 February 2000 25 February 2000 25 February 2000 Day -5 -1 1 7 8 10 22 23 24 25 25 'RSswpany^an^ed. T?OBP no? conta^ TS^-A CW 18 2.11 PROTOCOL ADHERENCE The study was performed in accordance with the Study Protocol No.^lH^Hkhd subsequent amendments. There were no deviations from the agreed Study Protocol. 2.12 ARCHIVING The study documentation and specimens generated during the course of the study are archived at CIT, 27005 Evreux, France, for 10 years after the end of the in vivo phase of the study. The archived study materials include: . protocol and possible amendments, . raw data, . correspondence, . final report and possible amendments. On completion of this period, the archived study materials will be returned to the Sponsor, or may be archived at CTT for a further period. In addition, raw data not specific to the study including, but not limited to, certificates of analyses for food, water and bedding (if applicable) and records of environmental data and equipment calibration, are also archived at CIT and retained for at least 30 years. Cempany Sanitized. Does no! contain TSftA cm 19 __ 3. RESULTS 3.1 CHOICE OF THE VEHICLE The vehicle chosen was 0.9% NaCI: a homogeneous dosage form preparation was obtained whatever the proportion. The dosage form preparation at the concentration of 75% (w/w) passed freely through a needle and into the dermis. 3.2 PRELIMINARY STUDY 3.2.1 Administration by intradermal route Results were as follows: Animal number Concentration <afthe test substance % (w/w) 24 hours Scoring after treatment 48 hours 6 days male 301 75 + FCA I 75 I N A N A 50 + FCA I N A 50 I 25 + FCA I I A I A 25 I U LI female 302 75 + FCA I 75 I N A N A 50 + FCA I N A 50 I I A 25 + FCA I I A 25 I U U FCA: mixture Freund^ Complete AdjuvaiH/0.9% NaCI 50/50 (v/v) N : necrosis I : irritation U : slight irritation A : crusts In order to respect the criteria for the selection of concentrations (the concentration should be well-tolerated systemically and locally, intradermal injections should cause moderate irritant effect but no necrosis or ulceration of the skin), concentration chosen for the main study was 25% (w/w). Company Sanitized. Does not contain TSCA CBi 20 3.2.2 Application by cutaneous route Results were as follows: Animal number male 301 Cconcentration of the test substance % 100 RF 50 (w/w) LF female 302 100 RF 50 (w/w) LF RF: right flank LF: left flank Scoring after remo^fal of the dressing 24 hours 48 hours 0 0 0 0 0 0 0 0 Off removal of the dressing, no residual test substance was observed. h order to respect the criteria for the selection of concentrations (the concentrations should be well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effect), concentration chosen for the topical application of the induction phase (day 8) and for the challenge application (day 22) was 100%. 3.3 MAIN STUDY 3.3.1 Clinical examinations No clinical signs and no deaths were observed during the study. 3.3.2 Body weight The body weight gain of the treated animals was similar to that of the control animals (appendix 3). .Company Sanitized. Does not conta'n TRCA CRF 21 3.3.3 Challenge phase - Scoring of cutaneous reactions On removal of the dressing, no residual test substance was observed. Scoring of skin reactions was as follows: Sex Male Female Animal number 121 122 123 124 125 136 137 138 139 140 24 hours LF 0 0 0 0 0 0 0 0 0 0 Sex Male Animal number 126 127 128 129 130 131 132 133 134 135 24 hours LF 0 0 0 0 0 0 0 0 0 0 Female 141 142 143 144 145 146 147 148 149 150 0 0 0 0 0 0 0 0 0 0 . LF : left flank (vehicle) RF : right flank (undiluted test substance) S : drynessofthe sidn Control group RF LF 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Treated group RF LF 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/S 0 0 0 0 1 0 0 0 0 0 0 0 48 hours RP 0 0 0 0 0 0 0 0 0 0 48 hours RF 0 0 0 0 0/S 0 0 0 0 0 0 0 0 0 0 0 0/S 0 0 0 p&CTpany Sanitized, Does not contain TSO& f^i 22 No cutaneous reactions were observed in the animals of the control group. In the treated group, only a discrete erythema (grade 1) was noted in 1/20 animals at the 24-hour reading. Dryness of the skin was recorded in 3/20 animals at the 48-hour reading. No other cutaneous reactions were observed. 4. CONCLUSION Under our experimental condition and Kligman, the test substance hypersensitivity in guinea-pigs. ion method of Magnusson does not induce delayed contact According to the classification criteria laid down in Directive 93/2 I/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance should not be considered as a skin sensitizer. Company Sanitized. Does no! contain TSftA rfll 23 APPENDICES company Sanitized. Decs not contain TSCA Cei 24 1. Test article description and analytical certificate Company Sanitized. Does not contain T8CA CBt 25 TOXICOLOGY DEPARTMENT CONFIDENTIAL elf atochem s.a. La defense 10, Cours Michelet DTI421 2nd December 1999 92091 Paris-Ia-Defense cedex, France TEST ARTICLE DESCRIPTION J IDENTITY Test article name Purity Origin Batch Elf Atochem filing number Atochem Villers-Saint-Paul PHYSICAL AND CHEMICAL PROPERTIES Appearance Specific gravity Boiling point Flash point Solubility Liquid 1070 kg/m3 at 20C (liquid) 80C 25C (closed cup) Partially soluble in water (20C) Soluble in : Methanol, Ethanol (400 g/l approximately) Insoluble in : Hydrocarbons, DMSO TOXICOLOGICAL INFORMATION AND USE SAFETY See Material and Safety Data Sheet . STORAGE AND DISPOSAL Storage : Expiry date : Disposal _________ in dark and at room'temperature November 2000 incineration 'ipany Sahiiizea. iDoes not contain TSCA C^i 26 elf atochem BSfo) ViDers Saint Paul, Ie 18 Novembre 1999 CERTIFICAT D'ANALYSE LotN ^ Extrait Sec Unit^ % pH Pouvoir moussant ml Tension superficidle Taux de 1145 H202 Mn/m % % . Pouvoir jRImogeoe Sur cycio hexane sur eau de ville Pouvoir HItDOgene Sur cycio hexane sur eau de mer Sec. Sec. Resultat 39,5 Specification de fabrication 39,5 ^ 40,5 Methode d'analyse LCU622 7.3 7a8 LCU642 80 60 a 100 LCU664 15,2 Oal6 LCU663 6,4 Oa7 LCU639 <0,01 Oa0,2 LCL1637 11 Oa20 LCU668 14 Oa60 LCU 668 Le Chef de Service du Laboratoire SIGNATURE: NOM : Robert Cntppo ^,,^"eswlc'"""TSCACBt 27 2. Diet formula IjSlBlpariy Saii'iliz.ed. Does ncrt coM^" i-e^ll'" 28 Ref: 106 COMPLETE DIET GUINEA-PIG MAINTENANCE DIET Appearance: 4.5 mm diameter granules Conditioning: bags of 25 kgs Daily portion: Guinea-pigs 35-50 g, water ad libitum. FORMULA % Cereals ..................................... Grain byproducts and legumes. Vegetable protein (soya bean meal, yeast) ............................. Vitamin and mineral mixture..., AVERAGE ANALYSIS Calorific value (Kcal/kg).......... Moisture.................................... Proteins..................................... Lipids........................................ Carbohydrates (N.F.E.) Fibre.......................................... Minerals (ash)........................... AMINO ACID VALUES (calculated in mg/kg) Arginine.................................... Cystine...................................... Lysine........................................ Methionine................................ Tryptophan................................ Glycine...................................... FATTY ACID VALUES (calculated in mg/kg) Palmitic acid............................. Palmitoleic acid........................ Stearicacid................................ Oleic acid.................................. Linoleic acid............................. Linolenic acid........................... 42 46 2600 10 17 3 49 13 8 8500 2500 7200 2100 2000 6000 3600 0 700 5900 11200 3000 P. Ca K. Na Mg Mn Fe.. Cu Zn , Co. I...., Cl., MINERALS (calculated in nig/kg) Nat CMV val. 7400 val. 1400 5400 5600 12000 0 Total 8800 11000 12000 1300 3270 60 170 1950 130 40 150 3250 3400 100 320 10 15 25 40 45 85 0.1 1.5 1.6 0 0 0 0 0 0 __ ____ V_IT_A_MINS (calculated per kg) CMV Vitamin A Vitamin D3 Vitamin Bl Vitamin B2 Vitamin B3 Vitamin B6 Vitamin B12 Vitamin C Vitamin E Vitamin K3 Vitamin PP Folic acid P.A.B. acid Biotin Choline Meso-mositol val. 3500 IU 30 IU 6mg 5mg 22 mg 0.7 mg ).003 mg Omg 15 mg 5mg 97 mg 2.2 mg Omg 0.02 mg 1010 mg Omg val. 7500 IU 2000 IU 6.4 mg 6.4 mg 26 mg 2.7 mg 0.012 mg 400 mg 60 mg 12.6 mg 14.5 mg 1.3mg 2.5 mg 0.06 mg 60 mg 62.5 mg Total 11000IU 2030 IU 12.4mg 11.4 mg 48 mg 3.4 mg 0.015 mg 400 mg 75 mg 17.6mg 11L5mg 3.5 mg 2.5 mg 0.08 mg 1070mg 62.5 mg This food is supplemented with stabilized coated vitamin C, avoiding the need of other food substances (greenery, ascorbic acid) if used within 4 months of date of manufacture. UAR, 7 rue Gallieni, 91360 Villemoisson - Tel: 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. UAR : 1992) iCompany ganffize'd. Does not contain TSCA CBI 29 3. Individual body weight values ^mpafy ticsnit'sseci, (Joes ffoi cor^n '''PC & I'R' INDIVIDUAL BODY WEIGHT VALUES (g) Groups Sex Animals -1 Days 1 (1) 1 Male 121 344 349 143 122 376 383 136 123 362 355 84 124 377 384 107 125 360 364 113 M 364 367 117 SD 14 16 24 '" Female 136 354 355 111 137 333 330 105 138 353 347 72 139 331 339 111 140 330 352 112 M 340 345 102 SD ^ 10 17 2 Male 126 337 346 156 127 350 349 138 128 366 371 158 129 393 387 105 130 360 372 97 131 341 346 193 132 360 358 103 133 350 357 163 134 375 376 143 135 353 361 118 M 359 362 137 SD 17 14 31 Female 141 142 . 143 144 145 146 147 148 149 150 333 340 340 373 347 343 325 332 368 340 330 346 337 366 350 319 314 328 371 331 142 169 95 40 131 59 99 132 134 99 M 344 339 110 SD 15 19 39 25 492 519 439 491 477 484 29 466 435 419 450 464 447 20 502 487 529 492 469 539 461 520 519 479 500 26 472 515 432 406 481 378 413 460 505 430 449 45 (1) = Body weight gain M =Mean SD = Standard Deviation 4. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs 32 Purpose: check the sensitivity of Dunkin-Hartley Guinea-pigs (Breeder: Charles River France) to a positive control test article Method Magnusson and Kligman Test substance MERCAPTOBENZOTfflAZOLE CTT Study - Date CIT/Study No. 19306 TSG - December 1999 Number of animals one control group of 5 animals and one treated group of 10 animals Induction l%(w/w) 20% (w/w) intradennal route day 1 cutaneous route day 8 Challenge application cutaneous route day 22 20% (w/w) Conclusion Under our experimental conditions and according to the Magnusson and Kligman method, the test substance MERCAPTOBENZOTHIAZOLE at the concentration of 20% (w/w) induced positive skin sensitization reactions in 80% guinea-pigs. INDIVIDUAL REACTIONS: CHALLENGE PHASE MACROSCOPIC FINDINGS Groups Sex Animals 24-hc5UT LF RF 48-h our LF RF Control Female 16 17 18 0 0/C 0 0/S/C 0 0/C 0 0/S/C 0 0/C 0 0/S/C 19 20 Treated Female 21 0 0/C 0 0/S/C 0 0/C 0 0/S/C 0 2/C 1/S 2/C/S 22 0 2/C 0 2/C/S 23 0 2/C 0 2/C/S 24 0 3/C 0 LS/C 25 0 1/C 0 0/S 26 0 2/C 0 LS/C 27 0 3/C 0 LS/C 28 0 1/C 0 LS/C 29 0 2/C 0 LS/C 30 0 2/C 0 0/S/C LF left flank (vehicle) RF right flank (test substance at the concentration of 20% (w/w)) S dryness of the skin LS . scoring masked by dryness of the skin C yellow coloration of the skin - negative + hypersensitizing reactions ^siv (conclusion + + + + - + + - + +