Document 2jBzZJ5LJNoDp0ZaB401geaKr

SPONSOR Elf Atochem SA ' Cours Michelet La Dfense 10 92091 Paris-la-Dfense CEDEX France AR226-3172 TEST SUBSTANCE STUDY TITLE SKIN SENSITIZATION TEST IN GUINEA PIGS (M axim ization method o f M agnusson and Kligman) STUDYDIRECTOR Xavier Manciaux STU D Y COMPLETION DA TE 14Dcember2000 TESTFACILITY err Centre International de Toxicologie BP 563 - 27005 Evreux - France / IFM Recherche S.N.CAUCAPITALDESSS0000F LABORATORYSTUDY NUMBER 20023 TSG 'Company Sa"?*'* n Company Sa'rulka'3. Boss lo i contain TSCA CB CENTRE IN T E R N A T IO N A L D E T O X IC O L O G IE B;R 563 27005 Evreux Cedex France CONTENTS STATEMENT OF THE STUDY DIRECTOR 4 OTHER SCIENTIST INVOLVED IN THIS STUDY 4 STATEMENT OF QUALITY ASSURANCE UNIT 5 SUMMARY 6 RESUME 8 1. INTRODUCTION 10 2. MATERIALS AND METHODS 2.1 2.1.1 2.1.2 2.1.3 2.1.4 TEST SUBSTANCE AND OTHER SUBSTANCES Identification of the test substance Vehicle Dosage form preparation Other substances 2.2 TEST SYSTEM 2.2.1 2.2.2 2.2.3 Animals Environmental conditions 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 2.8 2.8.1 2.8.2 2.8.3 PATHOLOGY Necropsy Skin samples Microscopic examination pM panx Sanitized. Does not contain TSCAC01 10 10 10 10 10 11 11 11 11 12 12 12 13 13 13 13 13 14 15 15 16 16 16 16 16 16 16 2.9 2.10 2.11 2.12 DETERMINATION OF THE ALLERGENICITY LEVEL CHRONOLOGY OF THE STUDY PROTOCOL ADHERENCE ARCHIVING 3. RESULTS 3.1 CHOICE OF THE VEHICLE 3.2 3.2.1 3.2.2 3.3 3.3.1 3.3.2 3.3.3 PRELIMINARY STUDY Administration by intradermal route Application by cutaneous route MAIN STUDY Clinical examinations Body weight 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 Dnkin-Hartley guinea pigs 16 17 18 18 19 19 19 19 20 20 20 20 21 22 23 24 27 29 31 and 32 noicon,a,nTSCACU' STATEMENT O F TH E STUDY DIRECTOR 2 scrited yinWaS Perfbnned ln comPliance with the principles of Good Laboratoiy Practice as (98)T7 PrinCipleS n Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM 'S S rie dU, 31 dcembre 1998 concernant les Bonnes Pratiques de Laboratoire r JSnVier 19991 ******* de l 'Economie, des Finances et de Commission Directive 1999/11/EC of 8 March 1999 adapting to technical progress the Principles of Good Laboratoiy Practice as specified in Council Directive 87/18/EEC on the " zT n i aWj r o ta tio n s and administrative provisions relating to the application of the Principles of Good Laboratoiy Practice and the verification of their applications for tests on chemical substances (OJ No. L 77 of 23.3.1999). st I dedare that this report constitutes a true and faithful record of the procedures undertaken and the results obtained during the performance o f the study. ^ cf dy was Performed at CLT, Centre International de Toxicologie, BP 563, 27005 Evreux, Toxicology X. Manciaux 1 Study Director Date: 14 December 2000 Doctor of Pharmacy OTHER SCIENTIST INVOLVED TN THIS S T im v For Pharmacy: P.O. Guillaumat Doctor of Pharmacy ] l | | M f W W o r n mf i mma n tsc a STATEMENT OF QUALITY ASSURANCE rmnrr Type of inspections Protocol Report Inspections 22 March 2000 22 November 2000 Dates Reported to Study Director (*) 23 March 2000 7 December 2000 Reported to Management (*) 23 March 2000 7 December 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 Q T Management. The inspections were performed in compliance Principles of Good Laboratory Practice. with CIT Quality Assurance Unit procedures and 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: 14 December 2000 Doctor of Biochemistry Head of Quality Assurance Unit (*) The dates indicated correspond to the dates o f signature of audit reports by Study Director and Management. not contain TSC 6 BI SUMMARY A t f c e t ^ u e a r f H f Atochem SA, Paris-la-Dtfense. France, the potential o f the teat substance to induce delayed contact hypersensitivity was evaluate in w s mdtooecd coaducted m compliance with the principles o f Good Laboratory Practice Methods and a treated gProiguSp "o1f t?enalml0atlfe"s and ttwenof^em"aPle5s1. a gTM P o f five males and five femmaaileess S m s -1' 111166 PairS f intradennaI "yections were performed in the interscapular region o f all ` ^ eund's comPIete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups) ' S I ( S o l ou*), C nCentration in 4 6 chosen vehicle (treated group) o r 'vehicle test substance at the chosen concentration in a mixture FCA/0.9% NaCl 50/50 (treated ground S p ) 1CC ^ thC COnCentratl n of 50% ^w/v) " a mixture FCA/0.9% NaCl 50/50 (control a(1n0w%,ayw//w%)thme o"rdTer6 torem^ duncreelfoeciavledirraitattoiPonic.al application o f sodium lauryl sulfate in vvaasseelliinnee On day 8, the test substance (treated group) or the vehicle (control group) was applied toDicallv to the same test site, which was then covered by an occlusive dressing for 48 hours. y On day 22, all animals of the treated and control groups were challenged by a cutaneous t" n of substance to the right flank. The left flank served as control and received 24 houn>Cle n y' T6St Substance 311(1 vehicle were maintained under an occlusive dressing for 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% NaCl), . topical application (day 8):f~ Challenge (all groups') topical application (day 22):i 1at the concentration of 5% (w/w) in sterile fundiluted. ^undiluted. At the end of the study, animals were killed without examination o f internal organs. Skin samples were taken from the challenge application sites of ail the animals of treated group No histological examination was performed. .s Results No clinical signs and no deaths were noted during the study. ^ i ^ p 3" 6" 86 aPP'iCati0n' " " ,aneOUS reaCtinS were 0bSCIVed in "" of the S 8T P' cutaneo" s reactins attributable to delayed contact hypersensitivity fdiscmre or moderate erythema, sometimes associated with oedema) were observed^ all animafs. C on clu sion - " * f t nmxhnization method1o f Magnusson hypersensitivity in 20/20 (100%) guinea pigs. induces delayed contact According, to the classification criteria laid down in Directive 93/21/EEC (27fli April 1993^ adaptmg to techmcal progress for the eighteenth time Council Directive 67/548/EEC the test substance should be considered as a skin sensitizer. ' ne test l ^ n p ^ i y Sanitized- Ones not contain TSCACBr RESUME ^ a d O T d e d e m f A t o c h e m SA, 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 an* 1996) direCtnCeS de 1 CDE (n 406) 17 Juillet 1992> et de la CEE (96/54/EEC, B.6, 30 juillet L tude est ralise conformment aux rgles de Bonnes Pratiques de Laboratoire. M thodes Trente cobayes sont rpartis en 2 groupes : un groupe tmoin de 5 mles et 5 femelle; et nn groupe trait de 10 mles et 10 femelles. uc j maies ei o remeiles et un ^ j_our 1, 3 paires d'injections intradermiques sont effectues au niveau de la rgion mterscapulaire de tous les animaux : rgion ' o ^ r n o S )!61 ^ FfeUnd ^ dU 50 % (V/V) d3nS dU NaCI '9 % ^ ouPe to it ' ( i o ^ e t m S f ^ COnCentrati n Ch0isie dans le vhicule (groupe trait) ou vhicule seul ` Pr d" lt: te!t1er ,la concentration choisie dans une mixture FCA/ NaCI 0,9 % 50/50 (groupe 50/50 (^oupe'tmofn)3 CnCentratKM1 de 50 % <P/v) dans u^e mixture FCA/ NaCI 0,9 % 7' un.e aPPlication cutane de laurylsulfate sodique 10 % (p/p) dans de la vaseline est effectue sur la meme 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. ? q j L jOUl 22; T S lSS, animai!X des t t et tmoin reoivent une application cutane n T H f iU Sm, 16 flanC ^ flanc Sauche * de tmoin et reoit le v h S e ui. Le produit et le vhicul sont maintenus sous pansement occlusif pendant 24 heures. pansement11 ^ reaCnS cutanes est effectue environ 24 et 48 heures aprs l'enlvement du Les concentrations de produit sont les suivantes : Induction (groupe trait! . injections intradermiques (jour i) ;i NaCI 0,9 %, . application cutane (jour 8) : la concentration de 5 % (p/p) dans du non dilu. Application dclenchante /tous les groupes-) . application cutane (jour 22) : non dilu. A la fin de l'tude, les animaux sont sacrifis sans examen des organes internes. Des prlvements cutans sont effectus au niveau des sites d'application dclenchante chez tous les animaux traits. Aucun examen histologique n'est ralis. |?Pitpny Sanitized. o es ifo contain TSCA CBf Rsultats Aucun signe clinique ni aucune mortalit ne sont nots pendant l'tude. Aprs l'application groupe tmoin. dclenchante, aucune raction cutane n'est observe chez les animaux du s e rs C on clu sion une hypersensibilisation cutane retarde chez 2 0 0 ( WO1 ) ' " " h "3" " Selon les critres de classification dcrits dans la Directive 93/21/CFF n i tm'i 100-3% dix-huitime adaptation au progrs technique d e 7 1S | f t ,I11993) portant considr sensibilisant par contact avec la peau.6 67/548/CEE, le produit est Sanffzecf. Doesriofconfafh fSCA CBF 1. INTRODUCTION The objective of this study, performed according to the maximization method o f Khgman (1), was to evaluate the potential of the test substance i----- -------delayed contact hypersensitivity in guinea pigs. lusson and to induce The results of the study are of value in predicting the contact sensitization potential of the test material in humans. e rest 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 o f the test substance The test substance It was identified as follows: . name: used in the study was supplied by the Sponsor. - protocol and labelling: batch number: - protocol and labellingj Elf Atochem filing number description: brown liquid container: one plastic flask date of receipt: 24 March 2000 storage conditions: at room temperature and protected from light composition: see analytical certificate . expiry date: November 2000. Data relating to the characterisation 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 preparaon (for cutaneous application and intradermal injections) and its free passage through a T w fif r mtrade,rma mje^tl0ns)- The hlShest concentrations which satisfied these criteria were called the maximal practicable concentrations. 92316 ^ iv resURanc^|S `9% NaC1' baCh Nos- LR92803 and 2934/1 (Laboratoire Frsnius, 2.1.3 Dosage form preparation AH dosage form preparations were made freshly, on unused material was discarded that same day.1 the morning of administration and any (1) Magnusson B. and Khgman A.M.: The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Derm., 52: 268-276 (1969). 2.1.4 Other substances 0t o , o ta!!CeS Used were Freund's complete adjuvant, batch Nos. 79H8938 and 119H8927 ( igma, 38297 Samt-Quentm-Fallavier, France); sodium lauryl sulfate, batch No. 107H0006 (Sigma, 38297 Samt-Quentm-Fallavier, France) and vaseline, batch No. 1572 (Cooprative Pharmaceutique Franaise, 77000 Melun, France). ^ operative 2.2 TEST SYSTEM 2.2.1 Animals Species and sex: male and female guinea pigs. M o T y F ^ o S ^ V A ^ " ^ BR' W t o ' bM "a i Smtained ' V ins Reason for this choice: species generally accepted by regulatory authorities for this type of study. 1he strain used has been shown to produce a satisfactory sensitization response using known sensl-tizcFS - Breeder: Charles River France, 76410 Saint-Aubin-Ies-Elbeuf, France. Number. . one male and three 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 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 1-3 months old and had a mean body weight standard deviation of 367 22 g for the males and 363 16 g for the females. y Acclimation: at least 2 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 o f 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. unng the acclimation 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 o f 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. | | i p n | Sanitized. Poes not contain TSCCOT 2.2.3 Food and water During the study, the animals had free access to "106 pelleted diet" (UAR, 91360 Villemoissnn sur-Orge, France). Food is analysed regularly by the supplier for composition and contaminant levels lh e 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 l a b o S (Pesticides, heavy metals and nitrosamines), are performed regularly by external The results of these analyses are archived at CIT. No contaminants were known to have been present in the diet, drinking water or beddim* thfstudy31 leVdS WhlCh may be expected t0 have interfered with or prejudiced the outcome of 2.3 TREATMENT 2.3.1 Preliminary test m aiTstuty317 t6St WaS conducted " order to determine the concentrations to be tested in the Ba ^ fa ad c n n d route (tested concentrations: 75%, 50%, 25%, 10%, 5% and 1% (w/w)): 4 hours before treatment, the dorsal region of the animals was clipped, ' S tersc^ far^ S n 1 ^ ^ d Sage ^ preparations ((U ml) were Performed in the inj^tions reaCti nS Were evaluated approximately 24, 48 hours and 6 days after the By cutaneous route (tested concentrations: 100% and 50% (w/w)): ^ hours before treatment, both flank regions of the animals were clipped the filter paper o f a chamber (Finn Chamber) was fully-loaded with the dosage form n erS ^T h ^h W^S * en aPPlied to * e clipped area of the skin (one concentration p r flank). The chamber was held m place by means of an occlusive dressing for 24 hours dressings;5 rCaCtl nS Were evaIuated approximately 24 and 48 hours after removal of the Criteria for selection of concentrations The following criteria were used: . the concentrations should be well-tolerated systemically and locally, - skin/ 6TM 31 injeCtl nS should cause moderate irritant effects (no necrosis or ulceration of the . 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. ll2f1?fZ0C, `0BS a 2.3.2 Main study 2.3.2.1 Preparation o f 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), ' . clipped on day 25, before skin sampling, for the concerned animals (each flank 2 cm x 2 cm). 23.2.2 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 interscanular graduation^ )3 nCedle ^diameter: -50 x 16 TM ) mounted on a 1 ml plastic syringe (0.01 ml Three injections of 0.1 ml were made into each side o f this interscapular region (i.e. three pairs of sites), as follows: F 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 5% (w/w) in 0.9% NaCl 0.9% NaCl 3 Posterior* test substance at 5% (w/w) in a mixture FCA /0.9% NaCl 50/50 FCA: Freuiid 's comolete: adinvanf vehicle at 50% (w/v) in a mixture FCA/0.9% NaCl 50/50 * ' ieSt su^stance y as 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. p e 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 o f sodium lauryl sulfate at the'concentration of 10% (w/w) in vaseline in order to mduce 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 interscapularregion of the animals of the treated group. The animals o f the control group received an application o f 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. SahRlzecf. oes fre eonalrs TSCA CBI 2.3,2.3 Challenge phase On day 22, the animals of treated and control groups received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chamber) was fully-loaded with the undiluted test substance and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. . CeiHpanySanitized. Does not contain TSCA81 2.4 SUMMARY DIAGRAM Figure 1: Treatment sites Induction-site Intradermal injections (day 1)* Cutaneous application (day 7): Sodium lauryl sulfate 10% in vaseline ' Cutaneous application (day 8): vehicle (control group) or test substance at the chosen concentration (treated group) Challenge application sites Cutaneous application (day 22) * Intradermal injections: 50% Freund's complete adjuvant and 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 the mixture Freund's complete adjuvant/0.9% NaCl (50/50) ^ o m m s^ 3 tainfsm L 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......................................................................................................................... . 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 W EIGHT 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 At the end of the study, skin samples were taken from the posterior left and right flanks of all the animals showing skin reactions (all the animals of treated group). The samples were preserved in 10% buffered formalin. 2.8.3 Microscopic examination ' No histological examination was performed. - 2.9 DETERMINATION OF TH E 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. 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. p55iiBnf Sanitized. Poes not contain TSCA CSf % 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, the test substance has sensitization properties and the symbol Xi, the indication of danger "Irritant" and the sentence "R 43: May cause sensitization by skin contact" must be applied. The sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer, MERCAPTOBENZOTHIAZOLE. In a recent study performed under C1T 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 o f the animals and skin samples Date 27 April and 3 May 2000 4 May 2000 5 May 2000 11 May 2000 12 May 2000 14 May 2000 26 May 2000 27 May 2000 Day -9 and -2 -1 1 7 8 10 22 23 28 May 2000 29 May 2000 29 May 2000 24 25 25 not contain "SCA CB' 2.11 PRO TO CO L ADHERENCE The study was performed in accordance with the Study Protocol N o .< ^ H H B ia n d subsequent amendments, with the following deviation from the agreed Study Protocol: the acclimation period was reduced to 2 days for four animals of the control group. This minor deviation was not considered to have compromised the validity or integrity o f 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 o f the in vivo phase of the study. The archived study materials include: . protocol and possible amendments, . raw data, . correspondence, . filial 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 farther period. hi 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. p g B w S a ,i e a .w > ' " WnTSCA0" 3. RESULTS 3.1 CHOICE OF TH E 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 number male 301 female 302 female 303 female 304 Concentration o f the test substance % (w/w) 75 + FCA 75 50 + FCA 50 25 + FCA 25 75 + FCA 75 50 + FCA 50 25 + FCA 25 10 + FCA 10 5 + FCA 5 1+FCA 1 10 + FCA 10 5 + FCA 5 1 +FCA 1 N :necrosis. I : irritation LI : slight irritation A : crusts - : not performed 24 hours N N N N N N N N .N N N N I I I U I II I I I LI I LI Scoring after treatment 48 hours N N N N N N N N N N N N N N I LI I LI . I N I II I LI 6 days _ _ _ _ _ A A I LI I LI A A I LI I LI Sanitized. Does not contain TSCA 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 m ain study was 5% (w/w). 3,2.2 Application by cutaneous route Results were as follows: Animal number male 301 female 302 R F : 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 On removal of the dressing, no residual test substance was observed. In order to respect the criteria for the selection of concentrations (die 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 o f the treated animals was similar to that of the control animals (appendix 3). Sanitized.Does hot contain TSCA CBt 3.3.3 Challenge phase - Scoring of cutaneous reactions On removal of the dressing, no residual test substance was observed. Scoring of skin reactions was as follows: Sex Animal number Control group 24 hours LF RF 48 hours LF RF Male 216 0 0 0 0 217 0 0 0 0 218 0 0 0 0 219 0 0 0 0 220 0 0 0 0 Female 231 0 0 0 0 232 0 0 0 0 233 0 0 0 0 234 0 0 0 0 235 0 0 0 0 Sex Male Animal number 221 222 223 224 225 226 227 228 229 230 Treated group 24 hours LF RF 48 hours LF RF 0 2/Oe 0 2/Oe/S 0 2/Oe 0 2/Oe/S 0 10 1 0 2/Oe 0 2/Oe/S 0 2/Oe 0 2/Oe/S 0 1 0 2/S 0 10 1 0 2/Oe 0 1/S 0 2/Oe 0 2/Oe/S 0 2/Oe 0 2/Oe/S Female 236 237 238 239 240 241 242 243 244 245 0 0 0 0 0 0 0 0 0 0 2/Oe 2/Oe 1 1 1 2/Oe 2/Oe 1 2/Oe 2/Oe LF : left flank (vehicle) RF : right flank (undiluted test substance) Oe : oedema S : dryness of the skin LS : scoring masked by a marked dryness o f the skin 0 2/Oe/S 0 1/S 0 1/S ' 0 1/S 0 1/S 0 2/Oe/S 0 2/Oe/S 0 2/Oe/S 0 1/S 0 LS j5hfHze9.Dees fo! contain TSCACBf No cutaneous reactions were observed in the animals of the control group. In the treated group, a discrete or moderate erythema (grade 1 or 2) was noted in all animals at the 24 and 48-hour readings. An oedema was recorded in 14/20 animals. Dryness of the skin was observed in almost all animals at the 48-hour reading. The observed cutaneous reactions were attributed to delayed contact hypersensitivity. 4. CONCLUSION UnJd?5,.our exPer^menta* conditions and according to the maximization method o f Maenusson and Kligman, the test s u b s t a n c e i n d u c e s delayed contact hypersensitivity m 20/20 ( 100%) guinea pigs. j 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. ' ^ B p fiy S iltz e a i Does flot contain TSCA c m APPENDICES pHHpan Sanitized. Does Hot contain TSCA C8f description and analytical certificate 'g g flP gqriSaBiltlzea. Does liot contain TSCA CB TOXICOLOGY DEPARTMENT elf atochem s.a. CONFIDENTIAL DTI 733 14thMarch 2000 La dfense 10, Cours Michelet 92091 Paris-la-Dfense cedex, France TEST ARTICLE DESCRIPTION ID E N T IT Y Test article name Origin Batch E lf Atochem filing number E lf Atochem Villers-Saint-Paul PHYSICAL AND CHEMICAL PROPERTIES I Appearance | Specific gravity Boiling point j Flash point | Solubility Brown liquid 1070 kg/m3 at 20C (liquid) 95C No flash point in the test conditions: (10 - 100C ) Soluble in : Alcohols, Hydroxylated solvents. Insoluble in : Hydrocarbons, DMSO C3XICOLOGICAL INFORMATION AND USE SAFETY e Material and Safety Data Sheet STORAGE AND DISPOSAL Storage Expiry date Disposal : in dark and at room temperature : November 2000 : incineration l^piHiySaftfflze!I.to e s m contain TSCA eiF atochem W o) Vlllers Saint Paul, le 26 Novembre 1999 CERTIFICAT D'ANALYSE Lot N Extrait Sec Ethanol PH Tension superficielle Densit Foisonnement 0,5% dans eau de ville Drainage 0,5% dans eau de ville Pouvoir ilniognc Sur heptane / eau de ville Unit % % Mn/m Minute Sec. Rsultat Spcification de fabrication 27,3 27 29 Mthode d'analyse LCU622 1,1 0 2,5 LCU 604 8.6 89 LCU642 15,9 0 16,5 LCU 663 1,083 1,06 1,09 LCU662 9 6 50 LCU 670 6 3 50 LCU 670 14 0 40 LCU 668 Le Chef de Service du Laboratoire SIGNATURE: 2. Diet formula *coniata TSCA CW LEtoesno1 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 % C e re a ls ....................................... Grain byproducts and legumes.. Vegetable protein (soya bean meal, yeast)............................... Vitamin and mineral mixture.... AVERAGE ANALYSIS % Calorific value (Kcal/kg).......... Moisture.......................... .......... Proteins.......................... ........... L i p id s ......................................... Carbohydrates (N.F.E.) Fibre........................................... Minerals (ash)............................ MINERALS (calculated in mg/kg) Nat. CMV 42 46 P....... ............. Ca .................. 9 K ..... ............. 3 Na ................. Mg.... ............. Mn ................ 2600 10 17 3 49 13 Fe..... ............. Cu .... ............. Zn .... ............ C o .... ............. I ......... ............. C l ..... ............. vai. 7400 5400 12000 1300 3270 60 170 10 40 0.1 0 0 vai. 1400 5600 0 1950 130 40 150 15 45 1.5 0 0 Total 8800 11000 12000 3250 3400 100 320 25 85 1.6 0 0 8 AMINO ACID VALUES (calculated in mg/kg) A rg in in e ..................................... Cystine..... .............................. Lysine......................................... M eth io n in e................................. Tryptophan................................. Glycine....................................... FATTY ACID VALUES (calculated in mg/kg) Palmitic acid.............................. Palmitoleic acid........................ Stearic acid................................. Oleic acid................................... Linoleic acid.............................. Linolenic acid........................... Vitamin A 8500 Vitamin D3 2500 Vitamin B1 7200 Vitamin B2 2100 Vitamin B3 2000 Vitamin B6 6000 Vitamin B12 Vitamin C Vitamin E Vitamin K3 Vitamin PP 3600 Folic acid 0 P.A.B. acid 700 Biotin 5900 Choline 11200 Vfeso-Inositol 3000 Nat. vai. 3500 IU 30 IU 6 mg 5 mg 22 mg 0.7 mg 0.003 mg 0 mg 15 mg 5 mg 97 mg 2.2 mg 0 mg 0.02 mg 1010 mg 0 mg CMV vai. 7500 IU 2000IU 6.4 mg 6.4 mg 26 mg 2.7 mg 0.012 mg 400 mg 60 mg 12.6 mg 14.5 mg 1.3 mg 2.5 mg 0.06 mg 60 mg 62.5 mg Total il'IU 2030IU 12.4 mg 11.4 mg 48 mg 3.4 mg 0.015 mg 400 mg 75 mg 17.6 mg 111.5 mg 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 o f manufacture. UAR, 7 rue Gallini, 91360 Villemoisson - Tel : 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. UAR : 1992) ' ailza. Dees fit* coMaln tsc ACBf 3. Individual body weight values i8S w tc .^ n T S C A C W C S S p W 5 r f `Md- Groups Sex 1 Male Female 2 Male Female INDIVIDUAL BODY W EIGHT VALUES (g) Days Animals -1 1 (1) 25 216 334 334 192 526 217 340 347 146 493 218 358 368 156 524 219 300 313 147 460 220 375 392 99 491 M 341 351 148 499 SD 28 30 33 27 231 385 391 90 481 232 344 359 120 479 233 353 370 63 433 234 354 379 122 501 235 360 369 111 480 M 359 374 101 475 SD 16 12 25 25 221 367 367 84 451 222 352 364 135 499 223 351 366 177 543 224 349 357 160 517 225 377 386 120 506 226 379 388 138 526 227 364 373 187 560 228 368 374 170 544 229 392 392 122 514 230 377 381 157 538 M 368 375 145 520 SD 14 12 31 31 236 352 358 26 384 237 353 367 144 511 238 371 376 97 473 239 330 337 115 452 240 337 341 ' 49 390 241 350 354 85 439 242 331 338 113 451 243 350 360 76 436 244 360 376 128 504 245 352 366 131 497 M 349 357 96 454 SD 13 15 38 44 ( 1) = Body weight gain M =Mean SD = Standard Deviation Sanitizes. Does m i contain TSCACBt 4. Positive control to check the sensitivity of Dunkin-Hartley guinea pigs ,*contain TSCACB1 Does no' pplripany sanRi1**' Purpose: check the sensitivity o f D unkin-H artley Guinea pigs (Breeder: C harles River France) to a positive control test article Method Test substance CIT Study - Date Magnusson and Kligman MERCAPTOBENZOTHIAZOLE CIT/Study Ni Number of animals Induction Challenge application: one control group of 5 animals and one treated group o f 10 anim als 1% (w/w) 20% (w/w) intradermal route day 1 cutaneous route day 8 20% (w/w) cutaneous route day 22 . C onclusion 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 16 17 - 18 19 20 Treated Female 21 22 23 24 25 26 27 28 29 30 LF : left flank (vehicle) 24-hour LF RF 0 0/C 0 0/C 0 0/C 0 0/C 0 0/C 0 2/C 0 2/C 0 2/C 0 3/C 0 1/C 0 21C 0 3/C 0 1/C 0 2/C 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 2/C/S 0 2/C/S 0 LS/C 0 0/S 0 LS/C 0 LS/C ' 0 LS/C 0 LS/C 0 0/S/C RF : right flank (test substance at the concentration of 20% (w/w)) S : dryness of the skin LS : scoring masked by dryness o f the skin C : yellow coloration o f the skin - : negative + : hypersensitizing reactions Conclusion . _ + + + + + + - + 4-