Document 3QdnjD14kEQO9kVmxXbapyvgD

SPONSOR Elf Atochem SA Cours Michelet La Dfense 10 92091 Paris-la-Dfense CEDEX France AR226-3158 CONFIDENTIAL TEST SURSTANC.F STUDY TITTP SKIN SENSITIZATION TEST f*/r . , EV GUINEA-PIGS (Maximization method of Magnusson and Kligman) STUDY DTRFrrnp Xavier Manciaux STUDY COMPLETION DA T P 28 August 2000 TESTFACTTJTV CIT C^ temationaJ de Toxicologie BP 563 - 27005 Evreux - France LABORA TORY STUD Y NTIMn I IFMRecherche S-N.CAl/ CAPITAI D S SSO 0 00 F S * S5>QAt -'^WEREr 2700S EVREUX Company Sanitized. Does not contain TSCA CBf CENTRE INTERNATIONAL DE TO XICO LOGIE B.P. 563 27005 Fvmnv ( ~ ~ c __ _ CONTENTS STATEMENT OF THE STUDY DIRECTOR OTHER SCIENTIST INVOLVED IN THIS STUDY STATEMENT OF QUALITY ASSURANCE UNIT SUMMARY RESUME l.JNTRODUCTION 2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE AND OTHER SUBSTANCES 2.1.1 Identification of the test substance 2.1.2 Vehicle 2.1.3 Dosage form preparation 2.1.4 Other substances 2.2 TEST SYSTEM 2.2.1 Animals 2.2.2 Environmental conditions 2.2.3 Food and water 2.3 TREATMENT 2.3.1 Preliminary test 2.3.2 Main study 2.3.2.1 Preparation of the animals 2.3.2.2 Induction phase by intradermal and cutaneous routes 2.3.2.2.1 Intradermal route 2.3.2.2.2 Cutaneous route 2.3.2.3 Challenge phase * 2.4 SUMMARY DIAGRAM Figure 1: Treatment sites 2.5 SCORING OF CUTANEOUS REACTIONS 2.6 CLINICAL EXAMINATIONS 2.7 BODY WEIGHT 10 10 10 10 11 11 11 11 12 12 12 13 13 13 13 13 14 15 15 16 16 2.8 PATHOLOGY 2.8.1 Necropsy 2.8.2 Skin samples 2.9 DETERMINATION OF THE ALLERGENICITY LEVEL 16 16 16 16 iSpmpanjr Sanitized. Does not contain TSC dal 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-Hartley guinea-pigs 17 18 18 19 19 19 19 20 20 20 20 21 22 23 24 27 29 31 and 32 Company Sanitized. Does not contain TSCA C B I 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 dcembre 1998 concernant les Bonnes Pratiques de Laboratoire (Journal Officiel du 1er janvier 1999), Ministre de l'Economie, des Finances et de Industrie. Commission Directive 1999/11/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 harmonization 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 -o n 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 OTHER SCIENTIST INVOLVED IN THIS STUDY For Pharmacy: P.O. Guillaumat Doctor of Pharmacy '* * Company Sanitized. Does no! contain TSCA CB? STATEMENT OF QUALITY ASSURANCE TTNTT 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 CIT 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 thse used and the results to constitute an accurate and complete reflection of the study raw data. L. Valette-Talbi 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 not confam t ^ a r R f SUMMARY Paris-la-Defense, France, the potential of the test substance to induce delayed contact hypersensitivity 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% NaCl (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% NaCl 50/50 (treated group) or vehicle at the concentration of 50% in a mixture FCA/0.9% NaCl 50/50 (control group). On day 7, the same region received a topical application of sodium lauiyl 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 1): isotonic saline solution (0.9% . topical application (day 8) Challenge fall groups'! . topical application (day 22): at the concentration of 25% (w/w) in sterile ndiluted. ndiluted. 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 Sanifizad, Does fib?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 co n d itio n ^n ^acco rd in ^^d i^n ax im izatio n method of Magnusson and Kligman, the test s u b s t a n c e ^ m m m m i H H p does not induce delayed contact hypersensitivity in guinea-pigs. According to the classification criteria laid down in Directive 93/21/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. IC P RESUME A la demande de Elf Atochem SA, Paris-la-Dfense, France, le potentiel du produit . induire une hypersensibilisation cutane retarde est valu chez le Jobaye selon la mthode'de maximisation de Magnusson et Kligman et conformment aux lignes directrices de l'OCDE (n 406, 17 juillet 1992) et de la CEE (96/54/EEC, B.6, 30 juillet 1996). L'tude est ralise conformment aux rgles de Bonnes Pratiques de Laboratoire. Mthodes Trente cobayes sont rpartis en 2 groupes : un groupe tmoin de 5 mles et 5 femelles et un groupe trait de 10 mles et 10 femelles. Au jour 1, trois paires d'injections intradermiques sont effectues au niveau de la rgion interscapulaire de tous les animaux : . Adjuvant complet de Freund (FCA) dilu 50 % (v/v) dans du NaCl 0,9 % (groupe trait et groupe tmoin), . produit tester la concentration choisie dans le vhicule (groupe trait) ou vhicule seul (groupe tmoin), . produit tester la concentration choisie dans une mixture FCA/ NaCl 0,9 % 50/50 (groupe trait) ou vhicule la concentration de 50 % dans une mixture FCA/ NaCl 0,9 % 50/50 (groupe tmoin). Au jour 7, une application cutane de laurylsulfate sodique 10 % (p/p) dans de la vaseline est effectue sur la mme zone dans le but d'induire une irritation locale. Au jour 8, le produit (groupe trait) ou le vhicule (groupe tmoin) sont appliqus sur le mme site, qui est ensuite recouvert d'un pansement occlusif pendant 48 heures. Au jour 22, tous les animaux des groupes trait et tmoin reoivent une application cutane dclenchante de produit sur le flanc droit Le flanc gauche sert de tmoin et reoit le vhicule seul. Le produit et le vhicule sont maintenus sous pansement occlusif pendant 24 heures. L'valuation des ractions cutanes est effectue environ 24 et 48 heures aprs l'enlvement du ' pansement. Les concentrations de produit sont les suivantes : Induction (groupe trait) . injections intradermiques (jour 1) NaCl 0,9 %, . application cutane (jour 8) : /p) dans du Application dclenchante (tous les . application cutane (jour 22) aon dilu A la fin de l'tude, les animaux sont sacrifis sans examen des organes internes. Aucun prlvement cutan n'est effectu au niveau des sites d'application dclenchante. Company Sanitized. Does not contain TSCA CBf Rsultats Aucun signe clinique ni aucune mortalit ne sont nots pendant l'tude. Aucune raction cutane bien dfinie n'est observe aprs l'application dclenchante. Conclusion Dans nos condition^xprimentales et selon la mthode de maximisation de Magnusson et Kligman, le produit n'induit pas de raction cutane attribuable une hypersensibilisation chSeeretam eecneHeCobaye. Selon les critres de classification dcrits dans la Directive 93/21/CEE (27 avril 1993) portant du-huitime adaptation au progrs technique de la Directive 67/548/CEE, le produit est considr non sensibilisant par contact avec la peau. SJompany Sanitize'. Does hot contain TSCA CBi 10 1. INTRODUCTION The objective of this study, performed according to the maximizatioiMnethocUj^Magmisson and Kligman (1), was to evaluate the potential of the test s u b s t a n c e ^ H H ^ ^ m ^ 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. 4 0 6 ,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 used in the study was supplied by the Sponsor. It was identified as follows: .name: - protocol and labelling:! . batch number - protocol and labelling:! . Elf Atochem filing number . 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% NaCl, batch No. LR92011 (Laboratoire Fresenius, 92316 Svres, France). 2.1.3 Dosage form preparation ,A11 dosage form preparations were made freshly on the tmorning 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). Company SaRtzecf, "Does not contain 1 BC CB 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-Quentin-Fallavier, France) and vaseline, batch No. 1144 (Cooprative Pharmaceutique Franaise, 77000 Melun, France). 2.2 TEST SYSTEM 2.2.1 Animals Species and sex; male and female guinea-pigs. Strain and sanitary status: Hartley Crl: (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. Breeden 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: 12h/12h . 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 botde. 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 TSCA 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 th"study. 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% (w/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. Com pany Sanitized. Does not contain TSCA 061 13 9 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 16 mm) 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 1 Site Anterior Treated group FCA at 50% (v/v) in 0.9% NaCl Control group FCA at 50% (v/v) in 0.9% NaCl 2 Middle test substance at 25% (w/w) in 0.9% NaCl 0.9% NaCl 3 Posterior* test substance at 25% (w/w) in a vehicle at 50% (w/v) in a mixture mixture FCA IQ.9% NaCl 50/50 FCA /0.9% NaCl 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 substalice 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. , - 'pgffipaby Sanitized. Does hot contain TSCA CB5 14 23.2.3 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 Sanitize?. Does fret contain TSCA CBr 2.4 SUMMARY DIAGRAM Figure 1: Treatment sites 15 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% NaCl) vehicle (control group) or test substance at the chosen concentration in the vehicle (treated group) (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% NaCl 50/50 ^pMpahyBhiiiiia uoes m i contain TSCA 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 CBP 17 Determination of the allergenicity 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 - 28 29 - 64 65 - 80 81 - 100 Allergenicity level I n m IV V Classification weak mild moderate strong extreme According to the Commission Directive 93/21/EEC, when the reactions are positive in at least 30% of the treated animals, th 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 intradermal injection Sodium lauryl sulfate application Induction by cutaneous route ' Removal of occlusive 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 SJswparty SaniriTed Dors rio?c o n ia i TSr:A 18 2.11 PROTOCOL ADHERENCE The study was performed in accordance with the Study Protocol N o . ^ m ^ f ^ R i d 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 CIT 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. Campany Sanitized. Does not cantata TSOa o b i 3. RESULTS 3.1 CHOICE OF THE VEHICLE The vehicle chosen was 0.9% NaCl: 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 Concentration of the number test substance % (w/w) Scoring after treatment 24 hours 48 hours male 301 75 + FCA I 75 I 50 + FCA I 50 I 25 + FCA I 25 I female 302 75 + FCA I 75 I 50 + FCA I 50 I 25 + FCA I 25 I FCA: mixture Freund%Complete Adjuvant/0.9% NaCl 50/50 (v/v) N : necrosis I : irritation * LI : slight irritation A : crusts N N N I I LI N N N I I U 6 days A A A A A LI A A A A A U 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 CB! 3.2.2 Application by cutaneous route Results were as follows: Animal number male 301 female 302 RF: right flank L F : left flank Concentration of the test substance % 100 50 (w/w) 100 50 (w/w) RF LF RF LF Scoring after removal of the dressing 24 hours 48 hours 00 00 00 00 Offremoval of the dressing, no residual test substance was observed. In 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 t r o a c * r 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: Animal number Control group 24hours LF RF LF Male . ' Female 121 122 123 124 125 136 137 138 139 140 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 0 0 oex Animal number 24 hours LF Male 126 127 128 129 130 131 132 133 134 135 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 04 LF : left flank (vehicle) RF : right flank (undiluted test substance) S : dryness of the skin Treated group RF 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 0 1 0 0 0 LF 0 0 0 0 0 0 0 0 0 0 0 0 0 o/s 0 0 0 0 0 0 ^Thorns o o 0 0 0 o o o o 0 48 hours RF o 0 o o o/s o o o 0 0 o o 0 0 0 o o/s 0 0 0 Company Sanitized. Dees hoi contain TSO& 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 co n d itio n ^ n ^acco rd in ^ ^tii^ n ^d m izatio n method of Magnusson and Kligman, the test substance does not induce delayed contact hypersensitivity in guinea-pigs. According to the classification criteria laid down in Directive 93/21/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 not contain T s r a m V APPENDICES i Pp/npany Sanitized. Does not contain TSC CB. 1. Test article description and analytical certificate Company Sanitized. Does not contain TSCA CRt 25 TOXICOLOGY DEPARTMENT elf atochem s.a. CONFIDENTIAL DTI 421 La dfense 10, Cours Michelet 92091 Paris-la-Dfense cedex, France 2ndDecember 1999 TEST ARTICLE DESCRIPTION IDENTITY Test article name Purity Origin Batch Elf Atochem filing number 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 P&fttpny Sanitized. Poes not contain TSC C^i eiF atochem 26 ViOers Saint Paul, le 18 Novembre 1999 CERTIFICAT D'ANALYSE Lot N ... m Extrait Sec Unit % Rsultat Spdfcation de fabrication 39,5 39,5 40,5 Mthode d'analyse LCU622 pH 7,3 78 LCU642 Pouvoir moussant ml 80 60 100 LCU664 Tension superficielle Taux de 1145 H202 Mn/ra % .% 15,2 M <0,01 0 16 07 0 0,2 LCU663 LCU639 LCU637 Pouvoir filmogne Sur cyclo hexane sur eau de ville Pouvoir filmogne Sur cydo hexane sur eau de mer Sec. Sec. 11 14 O 20 LCU668 0 60 LCU 668 Le Chef de Service du Laboratoire SIGNATURE: NOM : Robert Gruppo Company Sanitized. oes not contain TSCACB 0 2. Diet formula SanUitioesWolco^pSWpsny 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 % MINERALS (calculated in mg/kg) Nat. CMV Cereals...................................... Grain byproducts and legumes.. Vegetable protein (soya bean meal, yeast).............................. Vitamin and mineral mixture.... 42 46 P..................... .. C a ................. . 9 K ................... . 3 N a ................. . vai. 7400 5400 12000 1300 vai. 1400 5600 0 1950 Total 8800 11000 12000 3250 AVERAGE ANALYSIS % M g................. . Mn ................ Fe................... 3270 60 170 130 3400 40 100 150 320 Calorific value (Kcal/kg)......... 2600 C u ................. 10 15 Moisture.................................... 10 Zn ................. 40 45 Proteins..................................... 17 C o ................. 0.1 1.5 Lipids........................................ 3 I ..................... 0 0 Carbohydrates (N.F.E.) 49 C l .................. 0 0 25 85 1.6 0 0 Fibre.......................................... 13 Minerals (ash)........................... 8 VITAMINS (calculated per kg) AMINO ACID VALUES (calculated in mg/kg) Vitamin A Nat. vai. 3500IU CMV vai. 7500IU Total 11000IU Arginine.................................... Cystine...................................... Lysine........................................ Methionine................................ Tryptophan................................ Glycine....................................... 8500 Vitamin D3 2500 Vitamin B1 7200 Vitamin B2 2100 Vitamin B3 2000 Vitamin B6 6000 Vitamin B12 Vitamin C 3 0 IU 6 mg 5 mg 22 mg 0.7 mg 0.003 mg Omg 2000 IU 6.4 mg 6.4 mg 26 mg 2.7 mg 0.012 mg 400 mg 2030IU 12.4 mg 11.4 mg 48 mg 3.4 mg 0.015 mg 400 mg FATTY ACID VALUES (calculated in mg/kg) Vitamin E Vitamin K3 Vitamin PP 15 mg 5 mg 97 mg 60 mg 12.6 mg 14.5 mg 75 mg 17.6 mg 111.5 mg Palmitic acid............................. Palmitoleic acid........................ Stearic acid................................. Oleic acid................................... Linoleic acid.............................. 3600 Folic acid 0 P.A.B. acid 700 Biotin 5900 Choline 11200 Meso-Inositol 2.2 mg Omg 0.02 mg 1010 mg Omg 1.3 mg 2.5 mg 0.06 mg 60 mg 62.5 mg 3.5 mg 2.5 mg 0.08 mg 1070 mg 62.5 mg Linolenic acid........................... 3000 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 Gallini, 91360 Villemoisson Tel: 01.69.04.03.57-Fax : 01.69.04.81.97 (Ref. Doc. UAR : 1992) {Company Sanitized. Does not contain TSCA Cfi! # 3. Individual body weight values `fppily'sahltzed. tides fol tonWm * c* INDIVIDUAL BODY WEIGHT VALUES (g) Groups Sex 1 Male Female 2 Male Female Animals 121 122 123 124 125 M SD 136 137 138 139 140 M SD 126 127 128 129 130 131 132 133 134 135 M SD 141 142 . 143 144 145 146 147 148 149 150 M SD -1 344 376 362 377 360 364 14 354 333 353 331 330 340 12 337 350 366 393 360 341 360 350 375 353 359 17 333 340 340 373 347 343 325 332 368 340 344 15 Days 1 (1) 349 143 383 136 355 84 384 107 364 113 367 117 16 24 355 111 330 105 347 72 339 111 352 112 345 102 10 17 346 156 349 138 371 158 387 105 372 97 346 193 358 103 357 163 376 143 361 118 362 137 14 31 330 142 346 169 337 95 366 40 350 131 319 59 314 99 328 ' 132 371 134 331 99 339 110 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 30 i 31 4. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs Purpose: check the sensitivity of Dunkin-Hartley Guinea-pigs (Breeder: Charles River France) to a positive control test article Method Test substance Magnusson and Kligman MERCAPTOBENZOTHIAZOLE CIT 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 1% (w/w) 20% (w/w) intradermal route day 1 cutaneous route day 8 Challenge application 20% (w/w) cutaneous route day 22 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 Control Female Treated Female 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 24-hour LF RF 0 01C 0 0/C 0 0/C 0 0/C 0 0/C 0 21C 0 21C 0 21C 0 31C 0 1/C 0 2JC 0 3/C 0 1/C 0 21C 0 2/C 48-hour LF RF 0 0/S/C 0 0/S/C 0 0/S/C 0 0/S/C 0 0/S/C 1/S 2/C/S 0 vas 0 vas 0 LS/C 0 o/s 0 LS/C 0 LS/C 0 LS/C 0 LS/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 Conclusion - + + + + - + + + +