Document OJ3k8dM8VOv7G5paM13KYNQyX

tFM recherche $. v i MJ C-W'At OS 5 5>0 000 c A R226-3134 CONFIDENTIAL SPONSOR Elf Atochem S.A. Cours Michelet La Dfense 10 92091 Paris-la-Dfense CEDEX France TEST SUBSTANCE STUDY TITLE SKIN SENSITIZATION TEST IN GUINEA-PIGS (Maximization method of Magnusson, B. and Kligman, A.M.) STUDY DIRECTOR Xavier Manciaux STUD Y COMPLETION DA TE 15 October 1999 PERFORMING LABORA TORY CIT Centre International de Toxicologie BP 563 - 27005 Evreux - France LABORATORY STUDY NUMBER 18744TSG Comoany Sanitized. Does not contain TSCA CBI CENTRE INTERNATIONAL DE TOXICOLOGIE B. R S63 27005 Evreux Cedex France m CONTENTS 2 STATEMENT OF THE STUDY DIRECTOR OTHER SCIENTISTS INVOLVED IN THIS STUDY STATEMENT OF QUALITY ASSURANCE UNIT SUMMARY RESUME 1. INTRODUCTION 2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE AND OTHER SUBSTANCES 2.1.1 Identification of the test substance 2.1.2 Vehicle 2.1.3 Formulation procedure 2.1.4 Other substances 2.2 TEST SYSTEM 2.2.1 Animals 2.2.2 Environmental conditions 2.2.3 Food and water 2.3 TREATMENT 2.3.1 Preliminary test 2.3.2 Main study 2.3.2.1 Preparation of the animals 2.3.2.2 Induction phase by intradermal and cutaneous routes 2.3.2.2.1 Intradermal route 2.3.2.2.2 Cutaneous route 2.3.2.3 Challenge phase 2.4 SUMMARY DIAGRAM Figure I: Treatment sites 2.5 SCORING OF CUTANEOUS REACTIONS 2.6 CLINICAL EXAMINATIONS 2.7 BODY WEIGHT ' 2.8 PATHOLOGY 2.8.1 Necropsy 2.8.2 Skin samples 2.8.3 Microscopic examination 2.9 DETERMINATION OF THE ALLERGENICITY LEVEL Company Sanitized. Does not contain T$CA CBF 4 4 5 6 8 10 10 10 10 11 11 11 11 11 12 12 12 13 13 13 13 13 14 15 15 16 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 3 17 18 18 19 19 19 ' 19 20 20 20 20 20 21 22 23 24 25 26 28 30 32 and 33 Can?,-any S e iz e d . Does no! contain TSCCSI STATEMENT O F 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. . Dcret N 90-206 du 7 mars 1990 concernant les Bonnes Pratiques de Laboratoire (Journal Officiel du 9 mars 1990), Ministre de l'Industrie et de l'Amnagement du Territoire. . 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 (OJ No. L 15 of 17.1.87). 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 Date: 15 October 1999 Study Director Doctor of Pharmacy OTHER SCIENTISTS INVOLVED IN THIS STUDY For Pharmacy: P.O. Guillaumat Doctor of Pharmacy For Toxicology: C. Pelcot Study Supervisor Company Sanitized. Does not contain TSCACBI 5 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. L. Valette-Talbi 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. Com pany Sanited. Does not contain TSC A CBI SUMMARY A tth ^ - e q u e s ^ ^ l^ to c h e m ^ A ., Paris-la-Defense, France, the potential of the test substance t0 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 o f 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: at the concentration of 10% (w/w) in sterile isotonic saline solution (0.9% NaCl), . topical application: f l H H H ^ m u n d i l u t e d . Challenge (all groups)____________ . topical a p p lic a tio n :^ |^ H H IB ^ u n d ilu te d . - 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 com oil. For the challenge application, the reference substance DNCB was applied at the concentration of 1% (w/w) in com oil. Company Sanitized. Does not contain TSC A CB1 7 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 and Kligman, the test substance induces delayed contact hypersensitivity in 14/20 (70%) guinea-pigs. According to the classification criteria laid down in Directive 93/21/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. RESUME A la demande de Elf Atochem S.A., Paris-la-Dfense, France, le potentiel du produit^ __ induire une hypersensibilisation cutane retarde est valu chez le Cobaye 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 (92/69/EEC, B.6, 31 juillet 1992). L'tude est ralise conformment aux rgles de Bonnes Pratiques de Laboratoire. Mthodes Trente cobayes sont rpartis en 2 groupes : un groupe tmoin 1 (5 mles et 5 femelles) et un groupe trait 2 (10 mles et 10 femelles). Au jour 1, des injections intradermiques d'adjuvant de Freund mlang avec le produit (groupe trait) ou le vhicule (groupe tmoin) sont effectues au niveau de la rgion interscapulaire. 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, aprs une priode de repos de 12jours, 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 : 0,9 %, . application cutane : la concentration de 10 % (p/p) dans du NaCl non dilu. Application dclenchante /tous les groupes') . application cutane non dilu. A la fin de l'tude, les animaux sont sacrifis sans examen des organes internes. Des prlvement cutans sont effectus au niveau des sites d'application dclenchante chez tous les animaux. Aucun examen histologique n'est ralis. La sensibilit de la souche de cobayes a t contrle dans un essai rcent ralis dans les mmes conditions exprimentales avec un produit sensibilisant connu, le 2,4-Dinitro Chlorobenzene (DNCB). Pendant la phase d'induction, le produit DNCB est appliqu aux concentrations de 0,1 % (p/p) (jour 1) et 1 % (p/p) (jour 8) dans l'huile de germe de mas. A l'application cutane dclenchante, le produit DNCB est appliqu la concentration de 1 % (p/p) dans l'huile de germe de mas. . Company Sanitized. Does not contain T S C A C B I 9 Rsultats Aucun signe clinique ni aucune mortalit ne sont nots pendant l'tude. Aprs l'application dclenchante, aucune raction cutane n'est observe chez les animaux du groupe tmoin. Dans le groupe trait, la lecture 24 heures, un rythme trs lger, bien dfini ou modr est not chez 5/20, 13/20 et 1/20 animaux, respectivement. Un lger oedme est enregistr chez 4/20 animaux. A la lecture 48 heures, un rythme trs lger ou bien dfini persiste chez 9/20 et 7/20 animaux, respectivement. Une scheresse cutane est observe chez 16/20 animaux. Les rsultats obtenus avec le produit de rfrence montrent des ractions positives d'hyipersensibilit cutane retarde chez 90 % des Cobayes traits avec le DNCB. Conclusion Dans nos conditions exprimentales et selon la mthode de maximisation de Magnusson et Kligman, le produit induit des ractions cutanes attribuables une hypersensibilisation cutane retarde chez 14/20 (70 %) Cobayes. Selon les critres de classification dcrits dans la Directive 93/21/CEE (27 avril 1993) portant dix-huitime adaptation au progrs technique de la Directive 67/548/CEE, le produit est considr sensibilisant par contact avec la peau. Company Sanitized. Does not contain TSC A C B l 1. INTRODUCTION The objective of this study, performed according to the maximization method of Magnusson and Khgman (1), was to evaluate the potential of the test substance 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. 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 s u b s t a n c e u s e d in the study was supplied by Elf Atochem S.A. The test substance was identified as follows: . name: - protocol and labelling:! . batch number - protocol and labelling:! . Elf Atochem filing number 1 . 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 th 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 Frsnius, 92316 Svres 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 TSC A CBI h m 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 (Cooprative Pharmaceutique Franaise, 77000 Melun, France). 2.2 TEST SYSTEM 2.2.1 Animais 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. Breeder: Charles River France, 76410 Saint-Aubin-ls-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: 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 T S C A C B I 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 ml) at different concentrations were performed in the interscapular region, . cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections. 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 formuladon at the chosen concentration(s) was placed on a dry gauze pad (approximately 4 cm2) 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. . Company Sanitized. Does not contain TSC A 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 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 sites Anterior Treated group 1: FCA diluted at 50% (v/v) with 0.9% NaCl Control group 1: FCA diluted at 50% (v/v) with 0.9% NaCl Middle 2: test substance at 10% (w/w) in 0.9% NaCl 2: vehicle Posterior* 3: test substance at 10% (w/w) in a mixture FCA /0.9% NaCl 50/50 (v/v) 3: vehicle at 50% (w/w) in a mixture FCA 10.9% NaCl 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 eroup . 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 o f 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 T S C A 81 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 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) 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% NaCl Company Sanitized. Does not contain T SC A CBI 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............................................................................................................................. .. . Very slight erythema (barely perceptible)................................................................................ . 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............................................................................................................................... .. . Very slight oedema (barely perceptible).................................................................................1 . Slight oedema (visible swelling with well-defined edges).................................................... 2 . Moderate oedema (visible swelling raised more than 1 millimetre).................................... 3 . Severe oedema (visible swelling raised more than 1 millimetre and extending beyond the area of exposure).................................................................................................. .. 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. Com pany Sanitized. Does not contain TSCA CM 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 > 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 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 very weak weak moderate strong very strong According to the Commission Directive 93/21/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 of occlusive 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 Sanitized. Does not contain TSCA CBi * 2.11 PROTOCOL ADHERENCE 18 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. Does notcontainTSp/vcE\S 19 3. RESULTS 3.1 CHOICE OF THE VEHICLE The vehicle chosen was 0.9% NaCl: 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 intradermal 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) Scoring after treatment 24 hours 48 hours 6 days male 01 75 + FCA 75 female 01 75 + FCA 75 male 01 50 + FCA 50 female 01 50 + FCA 50 male 01 25 + FCA 25 female 01 25 + FCA 25 male 02 10 + FCA 10 female 02 10 + FCA 10 male 02 5 + FCA . 5 female 02 5 + FCA 5 male 02 1 + FCA 1 female 02 1 +FCA 1 Oe : oedema . N : necrosis I : irritant LI : slightly irritant A : crusts - : cutaneous examinations not performed FCA: Freund's Complete Adjuvant , N N N N N N N N N N N N I I I I I I I I I I I I N N 'N N N N N N N N N N I/Oe I/Oe I/Oe I/Oe I/Oe I/Oe I/Oe I/Oe I/Oe I/Oe I/Oe I/Oe - - - - - - .- - - - - - A A A A A LI A LI A U A LI Sani,ijei- D o no. coma* tsca c m 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 Concentration of the test substance % male 01 female 01 E : erythema O : oedema RF : right flank LF : left flank 100 50 (w/w) 100 50 (w/w) RF LF RF LF Scoring after removal of the dressing 24 hours 48 hours EOE0 0000 0000 0 0 00 0000 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%. 3 3 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, signs of irritation were observed at the interscapular test site in the control and treated groups. Company Sanitized. Does not contain TSC A CE8 3.3.2.2 Challenge application Scoring of skin reactions was as follows: Control group Sex Animal number 24 hours Erythema Oedema 48 hours Erythema Oedema LF RF LF RF LF RF LF RF Male 61 0 0 0 0 0 0 0 0 62 0 0 0 0 0 0 0 0 63 0 0 0 0 0 0 0 0 64 0 0 0 0 0 0 0 0 65 0 0 0 0 0 0 0 0 Female 76 0 0 0 0 0 0 0 0 77 0 0 0 0 0 0 0 0 78 0 0 0 0 0 0 0 0 79 0 0 0 0 0 0 0 0 80 0 0 0 0 0 0 0 0 Treated group Sex Animal number 24 hours Erythema Oedema 48 hours Erythema 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 10 0 0 1/S 0 0 70 0 2 0 2 0 2/S 0 0 71 0 10 00 0 00 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 0 0 0 0 0 0 0 82 0 2 0 0 0 1/S 0 0 83 0 10 0 0 1/S 0 0 84 0 1 0 * 0 0 0 0 0 85 0 2 0 0 0 1/S 0 0 86 0 2 0 0 0 2/S 0 0 87 0 2 0 0 0 2/S 0 0 88 0 2 0 0 0 1/S 0 0 89 0 2 0 2 0 1/S 0 0 90 0 1 0 0 0 0 0 0 L F : left flank (vehicle) R F : right flank (undiluted test substance) S : dryness of the skin No cutaneous reactions were observed in the animals of the control group. Sompany 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 16/20 animals. 4. CONCLUSION Under our experimental conditions, and accordin g jo 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 93/21/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. figure 2: Male body weight 23 Company Sanitized. Does not contain TSC A CSS Figure 3: Female body weight Company Sanitized. Does not contain T S $ | g | 25 % APPENDICES Com pany Sanitized. D oes not contain T S C A CBS 26 1. Test article description Company Sanitized. Does not contain TSCA CB | TOXICOLOGY DEPARTMENT CONFEDENTLA.L April 99 elf atochem s.a. La dfense 10, cours Michelet 92091 Paris-la-Dfense, France TEST ARTICLE DESCRIPTION IDENTITY' Test article name Chemical name CAS number EINECS number Purity Origin and batch Batch Elf Atochem filing number 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 9 CompaqSM>-Does,,otconlaim SC A e 2. Diet formula not contain T S C A C9* p o m p a* s " 6 4 0 0 ' 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 % MINERALS (calculated in mg/kg) Nat. CMV Cereals....................................... 42 vai. vai. Total Grain byproducts and legumes.. 46 P....... ............... 7400 1400 8800 Vegetable protein (soya bean Ca .................. 5400 5600 11000 meal, yeast).............................. 9 K ..... .............. 12000 0 12000 Vitamin and mineral mixture.... 3 Na .................. 1300 1950 3250 Mg.... .............. 3270 130 3400 AVERAGE ANALYSIS % Mn ................. 60 40 100 Fe..... .............. 170 150 320 Calorific value (Kcal/kg)......... 2600 Cu .................. 10 15 25 Moisture..................................... 10 Zn .................. 40 45 85 Proteins...................................... 17 Co .... .............. 0.1 1.5 1.6 Lipids........................................ 3 I ....... ............. 0 0 0 Carbohydrates (N.F.E.) 49 C l .... ............. 0 0 0 Fibre........................................... 13 Minerals (ash)........................... 8 VITAMINS (calculated per kg) AMINO ACID VALUES Nat. CMV (calculated in mg/kg) vai. vai. Total Vitamin A 3500IU 7500IU 11000 IU Arginine................................ ... 8500 Vitamin D3 3 0 IU 2000IU 2030 IU Cystine.................................. ... 2500 Vitamin B 1 Lysine.................................... ... 7200 Vitamin B2 6 mg 5 mg 6.4 mg 6.4 mg 12.4 mg 11.4 mg Methionine............................ ... 2100 Vitamin B3 Tryptophan............................. ... 2000 Vitamin B6 22 mg 0.7 mg 26 mg 2.7 mg 48 mg 3.4 mg Glycine................................... ... 6000 Vitamin B12 0.003 mg 0.012 mg 0.015 mg Vitamin C 0 mg 400 mg 400 mg FATTY ACID VALUES Vitamin E 1.5 mg 60 mg 75 mg (calculated in mg/kg) Vitamin K3 5 mg 12.6 mg 17.6 mg Vitamin PP 97 mg 14.5 mg 111.5 mg Palmitic acid.......................... ... 3600 Folic acid 2.2 mg 1.3 mg 3.5 mg Palmitoleic acid..................... ... 0 P.A.B. acid 0 mg 2.5 mg 2.5 mg Stearic acid............................. ... 700 Biotin 0.02 mg 0.06 mg 0.08 mg Oleic acid............................... ... 5900 Choline 1010 mg 60 mg 1070 mg Linoleic acid.......................... ... 11200 Meso-Inositol 0 mg 62.5 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 Gallieni, 91360 Villemoisson - T e l: 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. UAR : 1992) Company Sanitized. Does not contain T S C A C B I 3. Individuai body weight values C O m M n,Sm " feeilD '> l m a I,, TSCAM1 31 Groups Sex 1 Male Female 2 Male Female INDIVIDUAL BODY WEIGHT VALUES (g) A nim als------.1 Days 1 (I) 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 1 420 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. SD 347 21 354 24 28 382 18 36 26 408 12 30 49 457 10 33 (1) Body weight gain M Mean SD Standard Deviation Company Sanlized. Does not containTSCA C8J 32 4 Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs Com pany Sanitized. Dons not contain T S C A C H 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 DNCB CIT Study - Date (CIT/Study No. 17335 TSG) - September 1998 Number of animals Induction 1 control group of 5 animals and 1 treated group of 10 animals 0.1% (w/w) 1% (w/w) intradermal route day 1 cutaneous route day 8 Challenge application: 1% (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 Treated Female 161 162 163 164 165 0 0 0 0 2 0 0 0 0 2 166 2 167 2 168 3 169 3 170 2 171 2 172 2 173 1 174 2 : negative 0 2 2 2 0 0 0 0 0 + : hypersensitizing reactions S : dryness of the skin A : crusts 48-hour Erythema Oedema 00 00 00 00 2/S 2/S 2/S 2/S/A 2/S 2/S 2/S 1/S 1/S 1/S 2 0 2 2 0 .0 0 0 0 0 Conclusion m + + + + + + + _ + Company Sanitized.Does notcontainTSCACB