Document 062E4Xw38ovnXB5m7r3epX5Mb

AR226-3134 CONFIDENTIAL SPONSOR ElfAtochemS.A. Cours Michelet La Defense 10 92091 Paris-la-Defense CEDEX France IFM recherche i ML *L' CWV. () i i>0 OW c STUDY TITLE SKIN SENSITIZATION TEST IN GUINEA-PIGS (Maximization method of Magnusson, B. and Kligman, A.M.) STUDY DIRECTOR Xavier Manciaux STUDY COMPLETION DATE 15 October 1999 PERFORMING LABORATORY err Centre International de Toxicologie BP 563 - 27005 Evreux - France LABORATORY STUDY NUMBER 18744TSG Company Sanitized. Does not contain TSCA CBI CENTRE INTERNATIONAL DETOXICOLOGIE Q. P. 563 27005 Evreux Cedex France CONTENTS STATEMENT OF THE STUDY DIRECTOR OTHER SCIENTISTS INVOLVED IN THIS STUDY STATEMENT OF QUALITY ASSURANCE UNIT SUMMARY RESUME 1. INTRODUCTION 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 Formulation procedure 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 I: Treatment sites 2.5 SCORING OF CUTANEOUS REACTIONS 2.6 CLINICAL EXAMINATIONS 2.7 BODY WEIGHT 2.8 PATHOLOGY 2.8.1 Necropsy 2.8.2 Skin samples 2.8.3 Microscopic examination 2.9 DETERMINATION OF THE ALLERGENICITY LEVEL 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 Scoring of cutaneous reactions 3.3.2.1 End of the induction period 3.3.2.2 Challenge application 4. CONCLUSION Figure 2: Male body weight Figure 3: Female body weight APPENDICES 1. Test article description 2. Diet formula 3. Individual body weight values 4. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs 17 1 g 18 19 19 19 " 19 20 20 20 20 20 21 22 23 24 25 26 28 30 32 and 33 Csw,-?'r^ Sanitized. Does not contain TSCACSI 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 90-206 du 7 mars 1990 concemant les Bonnes Pratiques de Laboratoire (Journal Officiel du 9 mars 1990), Ministere de 1'hdustrie et de 1'Amenagement du Ten-itoire. . Council Directive 87/18/EEC of 18 December 1986 on the harmonization of laws, regulations or administrative provisions relating to the application of the Principles of Good Laboratory Practice and the verification of their applications for tests on chemical substances (OJNo.L15ofl7.1-87). 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: 15 October 1999 OTHER SCIENTISTS INVOLVED W THIS STUDY For Pharmacy: P.O. Guillaumat Doctor of Pharmacy For Toxicology: C. Pelcot Study Supervisor Sanitized. Does not contain TSCA CBI Company STATEMENT OF QUALITY ASSURANCE UNIT Type of inspections Protocol Report Inspections 4 June 1999 29 September 1999 Dates Reported to Study Director (*) 4 June 1999 13 October 1999 Reported to Management (*) 4 June 1999 13 October 1999 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 those used and the results to constitute an accurate and complete reflection of the study raw data. /--; ^-\c^ L. Valette-TaIbi Date: 15 October.1999 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 contain TSCA CBt SUMMARY fHII^IHBRBi^^B At the request of Elf Atochem S.A., 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 (92/69/EEC, B.6, 31st July 1992) 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 1 (five males and five females) and a treated group 2 (ten males and ten females). On day 1. intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were performed in the interscapular region. 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 to the same test site which was then covered by an occlusive dressing for 48 hours. On day 22, after a rest period of 12 days, 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: saline solution (0.9% NaCI), . topical application:; at the concentration of 10% (w/w) in sterile isotonic undiluted. Challenge (all groups) . topical application:^ lundiluted. At the end of the study, animals were killed without examination of internal organs. Skin samples were taken from the challenge application sites of all the animals. No histological examination was performed. The sensitivity of the guinea-pigs in CIT experimental conditions was checked with a positive sensitizer, 2,4-Dinitro Chlorobenzene (DNCB). During the induction period, the reference substance DNCB was applied at the concentrations of 0.1% (w/w) (day 1) and 1% (w/w) (day 8) in corn oil. For the challenge application, the reference substance DNCB was applied at the concentration of 1% (w/w) in corn oil. company Sanitized. Does not contain TSCA St Results No clinical signs and no deaths were noted during the study. After the challenge application, no cutaneous reactions were observed in the animals of the control group. In the treated group, at the 24-hour reading, a very slight, well-defined or moderate erythema was noted in 5/20, 13/20 and 1/20 animals, respectively. A slight oedema was recorded in 4/20 animals. At the 48-hour reading, a very slight or well-defined erythema persisted in 9/20 and 7/20 animals, respectively. Dryness of the skin was observed in 16/20 animals. The species and strain which were used showed a satisfactory sensitization response in 90% animals treated with DNCB. Conclusion Under our experimental conditions and according to the maximization method of Magnusson hypersensitivity in 14/20 (70%) 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 be considered as a skin sensitizer. c.^^--00""011  RESUME mHIBBIHBl A la demande deElfAtochem S.A., Paris-la-Defense, France, Ie potentiel du produiti a induire une hypersensibilisation cutanee retardee est evalue chez Ie Cobaye selon la methode de maximisation de Magnusson et Kligman et conformement aux lignes directrices de 1'OCDE (n 406, 17 juillet 1992) et de la CEE (92/69/EEC, B.6, 31 juillet 1992). L'etude est realisee conformement aux regles de Bonnes Pratiques de Laboratoire. Methodes Trente cobayes sont repartis en 2 groupes : un groupe temoin 1 (5 males et 5 femelles) et un groupe traite 2 (10 males et 10 femelles). Au jour 1, des injections intradermiques d'adjuvant de Freund melange avec Ie produit (groupe traite) ou Ie vehicule (groupe temoin) sont effectuees au niveau de la region interscapulaire. 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, apres une periode de repos de 12 jours, 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 le 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) :^|B^JU^ . injections intradermiques 0,9 %. ____ a I3 concentration de 10 % (p/p) dans du NaCI a . application cutanee r^UI^H^Jpnon dilue. Application declenchante (tous les groupes) . application cutanee rfBBHB non dilue- A la fin de I'etude, les animaux sont sacrifies sans examen des organes internes. Des prelevement cutanes sont effectues au niveau des sites d'application declenchante chez tous les animaux. Aucun examen histoldgique n'est realise. La sensibilite de la souche de cobayes a ete controlee dans un essai recent realise dans les memes conditions experimentales avec un produit sensibilisant connu, le 2,4-Dinitro Chlorobenzene (DNCB). Pendant la phase d'induction, le produit DNCB est applique aux concentrations de 0,1 % (p/p) (jour 1) et 1 % (p/p) (jour 8) dans 1'huile de germe de mai's. A 1'application cutanee declenchante, le produit DNCB est applique a la concentration de 1 % (p/p) dans 1'huile de germe de mals. Company Sanitized. Does not contain TSCA CBS Resultats Aucun signe clinique ni aucune mortalite ne sont notes pendant 1'etude. Apres 1'application declenchante, aucune reaction cutanee n'est observee chez les animaux du groupe temoin. Dans Ie groupe traite, a la lecture 24 heures, un erytheme tres leger, bien defini ou modere est note chez 5/20, 13/20 et 1/20 animaux, respectivement. Un leger oedeme est enregistre chez 4/20 animaux. A la lecture 48 heures, un erytheme ires leger ou bien defini persiste chez 9/20 et 7/20 animaux, respectivement. Une secheresse cutanee est observee chez 16/20 animaux. Les resultats obtenus avec Ie produit de reference montrent des reactions positives d'hypersensibilite cutanee retardee chez 90 % des Cobayes traites avec Ie DNCB. Conclusion Dans nos conditions expenmentales et selon la methode de maximisation de Magnusson et Kligman, !e produit Q^^H^BBiHBHHBBR1 induit des reactions cutanees attribuables a une hypersensibilisation cutanee retardee chez 14/20 (70 %) Cobayes. Selon les criteres de classification decrits dans la Directive 93/21/CEE (27 avril 1993) portant dix-huitieme adaptation au progres technique de la Directive 67/548/CEE, Ie produit est considere sensibilisant par contact avec la peau. Company Sanitized. Does not contain TSCA CBl 10 1. INTRODUCTION The objective of this study, performed according to the maximization method of Magnusson and fUBB----I Kligman (I), was to evaluate the potential of the test substance delayed contact hypersensitivity in guinea-pigs. to induce 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. 92/69/EEC, B.6, 31st July 1992. 2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE AND OTHER SUBSTANCES 2.1.1 Identification of the test substance The test substance fB^----^BI used in the study was supplied by ElfAtochem S.A. The test substance was identified as follows: . name: ____ __ . batch number . Elf Atochem filing number^^UBB . description: brown liquid . quantity and container one smoked glass flask . date of receipt: 5 May 1999 . storage conditions: at room temperature and protected from light . expiry date: May 2000. Data relating to the characterization of the test substance are documented in a test article description (presented in appendix 1) provided by the Sponsor. At the finalisation of the study report, no analytical certificate was available. Characterisation of the test substance, which appropriately defines the tested batch, is under the responsibility of 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. LR82423 (Laboratoire Fresenius, 92316 Sevres, France). (l) Magnusson, B.; Kligman, A.M.: The identification of contact allergens by animal assay. The guinea-pig maximization test. J. Invest. Derm. 52: 268-276 (1969) Company Sanitized. Does not contain TSCA CB1 11 2.1.3 Formulation procedure All preparations were made freshly on the morning of administration and any unused material was discarded that same day. 2.1.4 Other substances The other substances used were Freund's complete adjuvant, batch No. 88H8804 (Sigma, 38297 Saint-Quentin-Fallavier, France); sodium lauryl sulfate, batch No. 107H006 (Sigma, 38297 Saint-Quentin-Fallavier, France) and vaseline, batch No. 9946 (Cooperative Phannaceutique Fran^aise, 77000 Melun, France). 23. 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 . two males and two females 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 1 consisting of ten animals (five males and five females) and a treated group 2 consisting of 20 animals (ten males and ten females). Weight' on day 1, the animals of the main test were approximately 3 months old and had a mean body weight standard deviation of 361 17 g for the males and 358 22 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 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 TSCACBl 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 the 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: . 24 hours before treatment, the dorsal region of the animals was clipped, . intradermal administrations of the test substance formulation (0.1 concentrations were performed in the interscapular region, . cutaneous reactions were evaluated approximately 24, 48 hours and injections. ml) at 6 days different after the By cutaneous route: . 24 hours before treatment, both flank regions of the animals were clipped, . a volume of 0.5 ml of the undiluted test substance or test substance formulation at the chosen concentrations) was placed on a dry gauze pad (approximately 4cm2) which was then applied to the skin and held in place by 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. ;ompany Sanitized. Does notconiain TSCA CBj 13 2.3.2 Main study 2.3.2.1 Preparation of the animals For all animals and before each treatment, 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), . clipped again on day 25 (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 sites Treated group 1:FCA diluted at 50% (v/v) with 0.9% NaCI Control group : FCA diluted at 50% (v/v) with 0.9% NaCI Middle 2: test substance at 10% (w/w) in 0.9% NaCI 2: vehicle Posterior* 3: test substance at 10% (w/w) in a mixture FCA /0.9% NaCI 50/50 (v/v) 3: vehicle at 50% (w/w) in a mixture FCA /0.9% NaCI 50/50 (v/v) 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 cutaneous application to the interscapular region was performed as follows: Control group . application of 0.5 ml of the vehicle. Treated group . application of 0.5 ml of the undiluted test substance. Company Sanitized. Does not contain TSCA Cffi The test substance or the vehicle was placed on a dry gauze pad, which was then applied to the interscapular region. 14 The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster. On removal of the dressing, no residual test substance was observed. The presence of cutaneous irritation was checked 1 hour after removal of the occlusive dressing. 2.3.2.3 Challenge phase On day 22. the animals of both groups received an application of 0.5 ml of the undiluted test substance to the posterior right flank and 0.5 ml of the vehicle to the posterior left flank. This application was performed using a 1 ml plastic syringe (0.01 ml graduations). The test substance or the vehicle was placed on a dry gauze pad, which was then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin. The pads were held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster. On removal of the dressing, no residual test substance was observed. Company Sanitized. Does not contain TSCA 81 15 2.4 SUMMARY DIAGRAM Figure 1: Treatment sites (D (D Vehicle 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 Cutaneous application (day 22) Intradermal injections Q) 50% Freund's complete adjuvant and sterile isotonic solution (0.9% Nad) vehicle (control group) or test substance at the chosen concentration in the vehicle (treated group) vehicle at 50% (control group) or test substance at the chosen concentration (treated group) in a mixture 50/50 (v/v) Freund's complete adjuvant / 0.9% Nad _ Company Sanitized. Does not contain TSCA CBI 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: Erythema and eschar formation . No erythema.......................................................................................................................... 0 . Very slight erythema (barely perceptible)............................................................................ l . Well-defined erythema......................................................................................................... 2 . Moderate to severe erythema................................................................................................ 3 . Severe erythema (beet redness) to slight eschar formation (injuries in depth)..................... 4 Oedema formation . No oedema............................................................................................................................ 0 . Very slight oedema (barely perceptible)............................................................................... 1 . Slight oedema (visible swelling with well-defined edges)................................................... 2 . Moderate oedema (visible swelling raised more than I millimetre).................................... 3 . Severe oedema (visible swelling raised more than 1 millimetre and extending beyond the area of exposure)................................................................................................ 4 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). on days 8 and 15 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 At the end of the study, skin samples were taken from the posterior left and right flanks of all the animals. The samples were preserved in 10% buffered formalin. 2.8.3 Microscopic examination No histological examination was performed. Company Sam-feed. Does not contain TSCACK 17 2.9 DETERMINATION OF THE ALLERGENICITY LEVEL The treated animals show a positive reaction if macroscopic cutaneous reactions are clearly visible (erythema and/or oedema S 2) and if the treated animals have a greater intensity 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. 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 9-28 29-64 65-80 81-100 Allergenicity level I n ffl IV V Classification very weak weak moderate strong very strong 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. 2.10 CHRONOLOGY OF THE STUDY The chronology of the main study is summarized as follows: Procedure Arrival of the animals Weighing and allocation of the animals into groups Weighing, induction by intradermal injection Lauryl sulfate application Weighing, induction by cutaneous route Removal ofocclusive dressings Weighing Challenge cutaneous application Removal of occlusive dressings Scoring of cutaneous reactions after . 24 hours . 48 hours Weighing, sacrifice of the animals and skin samples Date 1 July 1999 8 July 1999 9 July 1999 15 July 1999 16 July 1999 18 July 1999 23 July 1999 30 July 1999 31 July 1999 1 August 1999 2 August 1999 2 August 1999 Day -8 -1 1 7 8 10 15 22 23 24 25 25 Company Sanded. Does not contain TSCA CBS 18 2.11 PROTOCOL ADHERENCE The study was performed in accordance with the Study Protocol No. 18744 TSG and subsequent amendments with the following deviation from the agreed Study Protocol: . the positive control corresponding to the study design was performed more than 6 months ago. This deviation was not considered to compromise the validity or integrity of the study. 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. . histological specimens: - tissues in preservative. 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. Company Sanitized, l 19 ______ ______ 3. RESULTS 3.1 CHOICE OF THE VEHICLE The vehicle chosen was 0.9% Nad: a homogeneous solution was obtained whatever the proportion. The test substance formulation 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 intradennal route In order to determine the concentration to be used in the main study, the following concentrations were tested: Animal number Concentration of the test substance % (w/w) male 01 female 01 male 01 female 01 male 01 female 01 male 02 female 02 male 02 female 02 male 02 female 02 75 + FCA 75 75 + FCA 75 50 + FCA 50 50 + FCA 50 25 + FCA 25 25 + FCA 25 10 + FCA 10 10 + FCA 10 5 + FCA 5 5+FCA 5 1+FCA 1 1+FCA 1 Oe N I LI A FCA oedema necrosis uritant slightly irritant crusts cutaneous examina tions not performed Freund's Complete Adjuvant 24 hours N N N N N N N N N N N N I I I I I I I I I I I I Scoring after treatment 48 hours N N - N N N N N N N N N N I/Oe I/Oe I/Oe VOe I/Oe I/Oe I/Oe VOe VOe VOe VOe I/Oe 6 days - - - . - - - - - - - - - A A A A A U A U A LI A LI CompanySanitized. Do95 not contain T{ 20 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 10%(w/w). 3.2.2 Application by cutaneous route Several concentrations were tested in order to determine the concentration(s) to be used in the main study. Animal number C oncentration of the test substance % male 01 100 RF 50 (w/w) LF female 01 100 RF 50 (w/w) LF E erythema 0 oedema RF right flank LF left flank Scoring after remov al of the dressing 24 hours 48 hours E 0 E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 After removal 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%. 33 MAIN STUDY 3.3.1 Clinical examinations No clinical signs and no mortality were observed during the study. From day 13, crusts associated with bleeding were observed on the site of intradermal injections in all animals of the treated group. The body weight gain of the treated animals was similar to that of the control animals (figures 2 and 3, appendix 3). 3.3.2 Scoring of cutaneous reactions 3.3.2.1 End of the induction period On day 10, after the cutaneous application of the induction period, observed at the interscapular test site in the control and treated groups. signs of irritation were Company Sanitized. Does not contain TSCA CB 21 3.3.2.2 Challenge application Scoring of skin reactions was as follows: Sex Animal number Male 61 62 63 64 65 Female 76 77 78 79 80 Contro)1 grou] } Erythema 24 hours Oedema Erythema 48 hours Oedemai U F Rf LF RF LJ= RF LF RF 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 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 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Sex Animal number Treated group Erythema 24 hours Oedema Erythema 48 hours Oedema LF RF LF RF LF RF LF RF Male 66 0 2 0 0 0 1/S 0 0 67 0 2 0 0 0 2/S 0 0 68 0 2 0 2 0 2/S 0 0 69 0 1 0 0 0 1/S 0 0 70 0 2 0 2 0 2/S 0 0 71 0 1 0 0 0 0 0 0 72 0 2 0 0 0 2/S 0 0 73 0 3 0 2 0 2/S 0 0 74 0 2 0 0 0 1/S 0 0 75 0 2 0 0 0 1/S 0 0 Female 81 0 82 0 83 0 84 0 85 0 86 0 87 0 88 0 89 0 90 0 0 0 0 0 0 0 0 2 0 0 0 1/S 0 0 1 0 0 0 1/S 0 0 1 0 0 0 0 0 0 2 0 0 0 1/S 0 0 2 0 0 0 2/S 0 0 2 0 0 0 2/S 0 0 2 0 0 0 1/S 0 0 2 0 2 0 1/S 0 0 1 0 0 0 0 0 0 LF left flank (vehicle) RF right flank (undiluted test substance) S dryness of the skin No cutaneous reactions were observed in the animals of the control group. Sompanx Sanitized.. Does not contain TSCA CBI 22 In the treated group, at the 24-hour reading, a very slight, well-defined or moderate erythema (grade 1, 2 or 3) was noted in 5/20, 13/20 and 1/20 animals, respectively. A slight oedema (grade 2) was recorded in 4/20 animals. At the 48-hour reading, a very slight or well-defined erythema (grade 1 or 2) persisted in 9/20 and 7/20 animals, respectively. Dryness of the skin was observed in 16/20animals. 4. CONCLUSION QHHHBHHHH^HHHBI Under our experimental conditions, and according to. the maximization method of Magnusson and Kligman, the test substance induces delayed contact hypersensitivity in 14/20 (70%) 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 be considered as a skin sensitizer. Company Sanitized. Does not< 23 Figure 2: Male body weight 700 r(g) 650-600-550-500-- 450 - 400-- 350- 300 0 H----I----\----h 1 234 56 -i----I----------I----h days 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 --Group! -B-Gfoup2 Company Sanitized. Does not contain TSCACB8 Figure 3: Female body weight 700 -r^ 650 600 550 500 450 400 350 300 -I--I--I--1--l--l--1--1--I--I--I--I--l--l--l--I--l--I--l--I--i--l----l--l--l--I 0 1 2 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ---Group I --ia--Group2 CompanySanitized, l 25 APPENDICES CompanySanitized. Does not contain TSCA CBE 26 1. Test article description CompanySanitized. Does not contain TSCA CBJ 27 TOXICOLOGY DEPARTMENT CONFIDENTIAL April 99 elf atochem s.a La defense 10, cours Michelet 92091 Paris-la-Defense, France TEST ARTICLE DESCRIPTION c IDENTITY Test article name Chemical name CAS number EINECS number Purity Origin and batch Batch Elf Atochem filing number :_ ElfAtochem. VSP : CAL 1393/99 PHYSICAL AND CHEMICAL PROPERTIES Appearance Melting point Boiling point Flash point Solubility brownish liquid -22C 95C 50C water TOXICOLOGICAL INFORMATIONS AND USE SAFETY See safety data sheet ________________ __ STORAGE AND DISPOSAL Storage Expiry date Disposal ________ in dark and at room temperature may 2000 incineration .DoesnolconiainTSCACBl CompanX?8"^ 2. Diet formula 29 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 legumesVegetable 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. Stearic acid----.. Oleic acid--...... Linoleic acid...... Linolenic acid.... 42 46 9 3 2600 10 17 3 49 MINIERALS (calcu lated in mg/kg) Nat. CMV val. val. Total p 7400 1400 8800 Ca ....------. K ...-----... Na .....------ M? 5400 12000 1300 3270 5600 0 1950 130 11000 12000 3250 3400 I-------- Mn ----.....- 60 40 100 Fe----------.. 170 150 320 Cu 10 15 25 .-.----..- 40 45 85 Zn .------....... Co ..----... 0.1 0 1.5 0 1.6 0 0 0 0 13 8 8500 2500 7200 2100 2000 6000 3600 0 700 5900 11200 3000 VITAMINS (calculated per kg) Nat. CMV Vitamin A Vitamin D3 Vitamin B 1 Vitamin B2 Vitamin B3 Vitamin B6 Vitamin B 12 Vitamin C Vitamin E Vitamin K3 Vitamin PP Folic acid val. 3500IU 30 IU 6mg 5mg 22 mg 0.7 mg 0.003 mg Omg 15 mg 5mg 97 mg 2.2 mg val. 7500 IU 2000 IU 6.4 mg 6.4 mg 26 mg 2.7 mg 0.012 mg 400 mg 60 mg 12.6mg 14.5 mg 1.3 mg P.A.B. acid Biotin Choline Omg 0.02 mg 1010 mg 2.5 mg 0.06 mg 60 mg Meso-Inositol Omg 62.5 mg Total 11000 IU 2030 IU 12.4 mg 11.4mg 48 mg 3.4 mg 0.015 mg 400 mg 75 mg 17.6 mg HL5mg 3.5 mg 2.5 mg 0.08 mg 1070 mg 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) Company Sanitized. Does not contain TSCACBI 30 A^p^" 3. Individual body weight values .CompanySanitized. Does not contain TSCA CBI 31 Groups Sex 1 Male Female 2 Male Female INDIVIDUAL BODY WEIGHT VALUES (g) Animals Days --------------------------------------------------------------------------------------- '' 1 (1) 8 (1) 15 (1) 25 61 314 328 52 380 46 426 54 480 62 349 362 43 405 31 436 54 490 63 359 373 55 428 0 428 35 463 64 319 334 32 366 52 418 63 481 65 347 366 45 411 23 434 58 492 M 338 353 45 398 30 428 53 481 SD 20 20 9 25 21 7 11 11 76 379 391 47 438 5 443 31 474 77 348 354 46 400 45 445 84 529 78 342 345 51 396 11 407 81 488 79 338 355 49 404 12 416 53 469 80 371 380 57 437 4 441 61 502 M 356 365 50 415 15 430 62 492 SD 18 20 4 21 17 18 22 24 66 360 374 49 423 35 458 51 509 67 363 375 44 419 35 454 65 519 68 371 369 58 427 -5 422 69 491 69 366 379 58 437 40 477 64 541 70 324 340 135 475 -86 389 72 461 71 337 336 21 357 10 367 43 410 72 364 370 52 422 38 460 60 520 73 362 366 41 407 20 427 60 487 74 358 372 47 419 I 420 60 480 75 367 376 47 423 27 450 75 525 M 357 366 55 421 12 432 62 494 SD 15 15 30 29 38 34 10 38 81 344 345 28 373 17 390 60 450 82 332 328 -9 319 39 358 42 400 83 344 355 44 399 18 417 43 460 84 345 359 48 407 24 431 42 473 85 336 344 21 365 24 389 57 446 86 396 406 49 455 6 461 62 523 87 322 329 27 356 26 382 36 418 88 346 351 26 377 25 402 51 453 89 337 342 33 375 38 413 59 472 90 370 379 11 390 44 434 39 473 M 347 354 28 382 26 408 49 457 . SD 21 24 18 36 12 30 10 33 (1) = Body weight gain M - Mean SD - Standard Deviation . enfn-ra<< nnae nrA mntflin Tf! 32 4. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs ompany Sanitized. Does not contain TSCA CBB 33 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 : DNCB CIT Study - Date (CIT/Study No. 17335 TSG) - September 1998 Number of animals : 1 control group of 5 animals and 1 treated group of 10 animals Induction : 0.1% (w/w) intradennal route day 1 1% (w/w) cutaneous route day 8 Challenge application: l%(w/w) Conclusion Under our experimental conditions and according to the Magnusson and Kligman method, the test substance DNCB at the concentration of 1% (w/w) induced positive skin sensitization reactions in 90% guinea-pigs. INDIVIDUAL REACTIONS: CHALLENGE PHASE MACROSCOPIC FINDINGS Groups Sex Animals 24-hour Control Female Erythema Oedema 160 0. 0 161 0 0 162 0 0 163 0 0 164 0 0 Treated Female 165 2 2 166 2 0 167 2 2 168 3 2 169 3 2 170 2 0 171 2 0 172 2 0 173 1 0 174 2 0 : negative + : hypersensitizing reactions S : dryness of the skin A : crusts 48-hour Erythema 0 Oedema 0 0 0 0 0 0 0 0 0 2/S 2 2/S 0 2/S 2 2/S/A 2 2/S 0 2/S 0 2/S 0 1/S 0 1/S 0 1/S 0 Conclusion + + + + + + + + + Company S.^o^'"1""^68'