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AR226-2994 **Vr- .a Baugy, June 17, 1993 STUDY NO 9?0Dfifi F b lU D Y OF CUTANEOUS SENSITIZATION U Mm aAXvI.MI IZvAaMTIAOGNNUTESSST0NINAGNUDINkElAigPmIGa n ! Sponsor : ELF ATOCHEM SA La Dfense 10, cedex 42 92091 PARIS LA DEFENSE FRANCE Company Sanitized. Does not cor>f*n T srA rq? CERB - STUDY NO. 920086 E STUDY COMPOUND Study of cutaneous sensitization using the MAGNUSSON and KLIGMAN test in Guinea Pig. SPONSOR SPONSOR CONTACT ADDRESS ELFATOCHEM SA Mr REGNIER La Dfense 10, Cedex 42 92091 PARIS LA DEFENSE - FRANCE. LOCATION OF STUDY : CENTRE DE RECHERCHES BIOLOGIQUES Chemin de Montifault 1 8 8 0 0 BAUGY-FRANCE RESPONSIBLE STAFF FOR CFRR ; Scientific and Technical Director : S. RICHARD Pharmacien, Docteur de 3me Cycle Matre en Pharmacologie Date .. / = Signature Head of Toxicoloav Department : C. AUDEVAL-GERARD Docteur Vtrinaire CES d'Ophtalmologie Studv Director : C. BESSON DUT de Biologie Applique Date Signature Quality Assurance : C. VIGIER Signature The authentic results of the experiment form the basis of the present report. r ittiii TuA CBi CERB - STUDY NO. 920086 E j This report consists of 42 pages numbered from 1 to 42 including 5 appendices. CONTENTS' I. SUMMARY II. EXPERIMENTAL PROTOCOL ADOPTED 11.1 AIM II.2 METHOD II.3 COMPOUND II.4 GENERAL CHARACTERISTICS OF STUDIES 11.4.1 ANIMALS II.4.2 PRINCIPLE II.5 EXPERIMENTAL PROTOCOL 11.5.1 PRE-EXPERIMENTAL PROCEDURE II.5.2 PRELIMINARY STUDIES 11.5.2.1 Intradermal administration II.5.2.2 Epicutaneous application II.5.3 FINAL STUDY 11.5.3.1 Study design II.5.3.2 Primary induction II.5.3.3 Sensitization II.5.3.4 Challenge II.6 CLINICAL MONITORING OF ANIMALS II.7 READING OF SKIN REACTIONS II.8 DATA ENTRY OF RESULTS II.9 REPORT 11.10 QUALITY ASSURANCE 11.11 RECORDS III. III. 1 III.2 III.2.1 III.2.2 III.3 III.3.1 III.3.2 III.3.3 III.3.4 RESULTS DEVIATION FROM PROTOCOL N 92.04.10.05 PRELIMINARY STUDIES INTRADERMAL ADMINISTRATION EPICUTANEOUS ADMINISTRATION FINAL STUDY DATES MONITORING OF ANIMALS READING OF SKIN REACTIONS CLASSIFICATION OF COMPOUND IV. CONCLUSION APPENDIX 1 APPENDIX 2 APPENDIX 3 APPENDIX 4 APPENDIX 5 Protocol n 92.04.10.05 References and batch numbers of reagents and equipment used Technical data and analytical certificate concerning compound Analytical certificate concerning foodstuff Analytical certificate concerning water Page 4 5 6 6 7 8 8 ' 10 10 10 10 10 12 13 13 14 15 15 16 16 17 17 17 17 18 19 19 19 19 20 20 20 20 21 22 23-33 34-35 36-38 39-40 41-42 pompany Sanitized. Does ho! contain TSCA CERB - STUDY NO. 920086 E I. SUMMARY Study of the sensitizing capacity of compound in the Guinea Pig using the technique of as undertaken IAGNUSSON and KLIGMAN in comparison w ith a negative control group receiving water for injection and a positive control group receiving dinitrochlorobenzene (D.N.C.B., 1 %). The maximum slightly irritant concentration determined by intradermal a r t m in ie tr a tir m orrJ j-1_____:_____ ^ Under the experimental conditions adopted, 1 % D.N.C.B. showed a degree of allergenicity class V at 24 hours. Its sensitizing capacity was very stronq at 24 hours in the Guinea Pig. Water for injection showed a degree of allergenicity o f class I at 24 hours. It may be considered as being free of any sensitizing capacity in the Guinea Pig. 24 hours. CERB - STUDY NO. 920086 E !i- e x p e r im e n t a l p r o t q c o i a d o p t e d I 'm CERB - STUDY NO. 920086 E 0 11.1 AIM : The aim of th ^ s tu d y was to evaluate the possible delayed sensitization capacity experimental m e t h o d w / a s m s p i r e d o ^ ^ A ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^T^AN^ ^ ^(1^9^6^9^) and GUILLOT (1983). II.2 METHOD : The method described can be applied to all compounds in liquid, or semi-liquid. The protocol complied with the requirements of O.E.C.D. Guideline No. 406 (Mav 12, 1981). y The protocol n 92.04.10.05 signed by the legal responsible for the sponsor of the study and by the experimenters may be found in appendix 1. The study was carried out according to Good Laboratory Practices as published by : - the French M inistry o f Social Affairs and National Solidarity : State Secretariat for Health. Guideline concerning Good Laboratory Practices (G.L.P.) in the area of experimental toxicology. Guideline dated May 31, 1983, official text No. 1065 ; reference SN-S83/25. E.E.C. Directive 87/18. This directive refers to recommendation CC81/30 Appendix 2 o f the O.E.C.D. Food and Drug Administration : G.L.P. 21 C.F.R. Part 58 dated December 22, 1978 and amendments of April 11, 1980 and September 4, 1987. i CERB - STUDY NO. 920086 E report in A ppendix^.10316 concernm9 the compound is supplied w ith the, study - Name - Batch No. - Record number (CAL) - Origin --Date received - Amount - Appearance pH ELF ATOCHEM SA Saturday April 18 1992 1 bottle of 107 g Pip=ny Sanitized. Dees not contain TSCA CBr Co CERB - STUDY NO. 920086 E 1L4, GENERAL CHARACTERISTICS OF S T iim cc ; 1L-4.1 A N IM A IS ; - SPECIES : Hartley strain albino Guinea Pigs. - o n s ~ - 37600 SAINT-JEAN - SEX : Males and females. Females were nulliparous and non-gravid. - NUMBER : Preliminary studies : 8 Intradermal administration : 4 (2 males + 2 females) Epicutaneous administration : 4 (2 males + 2 females). Final study : 40 animals divided into three groups : 10 S I8 {|S i +5" } - 20 treated w ith the study compound (10 males + 10 females) - DATE RECEIVED : On 22.04.1992. ' eA5 e r i 3 w a r t o N,ai<eplaoe3 d* y* bef0re the S,Udy in the * * the ' sofuton ofAp TM c acWteaygg7nge idemified Per C33e' individuallV bV ^ e c u s I IIi CERB - STUDY NO. 920086 E These cages were placed in an air-conditioned animal house (17C-21 p i L.on* at a constant relative humidity of between 45 % and f i l % P v rJ n tw P ton8S Peri0dS in W5i.Ch " O ^ y o l e d filtered air was changed a n i m a t e " ? hours d a r k ie s Ur' art,f,Clal dayli9ht cycla was 12 h urs flht and 12 An analytical certificate ; Tap water distributed "ad libitum " in polycarbonate feed months and l e M ^ h . 8? 61 ? at' ,A w| ter sample was obtained every three Malet 18014 Rn?Rr^QDr 5 tl0 n r:doef . ^ ervlces Vtrinaires, 216, rue Louis cert fic a tl 7,, Cedex' FRANCE for analysis. The water analytical close ta e s tPaPrt S?tXhe studrr P * * " SamP'e btained n the date Does noi contain TSf'fi CERB - STUDY NO. 920086 E H.4.2 PRINCIPI F ; Animals were treated three times (Figure 1). The test consisted of three phases : - ^ g j J y i O N first contact between the body and compounds Li - SENSITIZATION : second contact with compound / f l B H H H H B V l Recognition of allergen and multiplication of c e llu /a r a n d /o ^ u lm o r ^ l J transformations. - CHALLENGE : third contact with com poundlj to the finding of a clinical manifestation. leading or not II.5 EXPERIMENTAL PROTOCOI ; II-5.1 PRE-EXPERIMENTAL PROCFDIIRF ; - PREPARATION OF ANIMALS : the day prior to each application Guinea Pigs used were shaved using AESCULAP FAVORITA II GT 104 clipper. An area of approjiimately 24 cm2 was exposed on the back, behind the head, at the level of the shoulders and flanks. - SELECTION OF ANIMALS : only healthy animals, w ith a skin free of any trace of cutaneous lesions, were selected. il- 5.2 PRELIMINARY STHniFS : H.5.2.1 Intradermal administration : Determination of the maximum concentration causing slight irritation (with neither scab nor necrosis) in all animals by intradermal injection. - Animals : 2 males and 2 females. - Treatment : Intraderm alm jechonsw ere administered in the dorsal region using 0.1 ml of c o m p o u n d j^ ^ H B H ^ ^ j^ o r of its dilutions. Injections were administered at 4 sites using one concentration per site, i.e. 4 concentrations per animal. Water tor injection was used as a non-irritant and non-sensitizing vehicle. . vC ii/.vvT. hUi >'OisiJH f oOA 001 CERB - STUDY NO. 920086 E - Concentration : concentrations selected were compound undiluted and diluted 3/4, 1/2 and 1/4. . - Grading of results : Lesions were evaluated for each concentration 2 * hours after injections using the scale published by MAGNUSSON and KLIGMAN : No reaction Slight erythema (scarcely visible) Moderate erythema (clearly visible) Intense erythema w ith edema 11.5.2.2 Epicutaneous application ; 0 1 2 3 a. Determination of M .N .I.C . (Maximum Non-Irritant Concentration) by epicutaneous application under an ELASTOPLASTE non occlusive dressing - Animals : 2 males and 2 females. - Treatm ent: Epicutaneous application of 0.5 ml of c o m p o u n d il^ ^ ^ ^ ^ H ^ ^ ^ H o r of it; dilution over an area of 8 cm 2 using one c o n c e n tra tio rH J r^ a c f^ B ? i e tw o concentrations per animal. Le- TWO - Concentrations : X TM orT which h * * 48 " S S ? " 9 0f r?SU't f : ,sions were evaluated for each concentration one hour after removal of dressings using the scale already described in 11.5.2.1. 15. .max,'lnum slightly irritant concentration determined by intradermal administration was used during the primary induction phase. 'ntradermal The maximum non-irritant concentration (M .N.I.C.) determined bv emcutaneo..; administration was used during the sensitization and challenge phases. Does no! contain TSCA CB CERB - STUDY NO. 920086 E FIGURE N 1 SITES OF ADMINISTRATION Site of application j for sensitization Sites of intradermal injection or epicutaneou Application for primary. induction . Site of. application for 24 hours for challenge i Cj_j CERB - STUDY NO. 920086 E 1L5-3 FINAL STUDY ; Jl.5.3.1 Study design ; . Into 3tUgiodueps9SnCt e f a s if o M h e ' according to their general condition^ 1 S ' each sex| o m ta d f b dy We'9ht and se,ected The 3 groups o f animals involved in the study consisted of : was indeed evidence of an allergic reaction ^ negat,ve cor>tro1 9rouP ' ^ e n S T o M h ^ 'm iS io d u s fd "T h is 'c o n firm e d "thai D N c V 0 / ' ^ 16 * 5 a a - g y y a - asssa usld to * d ltlrdmmeh w 3 i ^ t l ! 5 S H i * ^ M & Shadf T h Se* '' propert.es under the experimental c o n d W o f f i S S f t S P 9 a"er0emC fe m p a n y Sanitized. Does no? confab T f* a CERB - STUDY NO. 920086 E 11-5.3.2 Primary induction (time D1) ; received 6 hiecftfns^n 'theTeffoscapula^reoio ^ ^ triaL Each Guinea Pi9 column, using a previously shaved area of a p p r o x i m a t e * 2 W M cm x ^ c m h preparations."013* inieCti nS f 0 J ml TREATMENT OF NEGATIVE CONTROLS : administered with three different ' Pyro^jen-free isotonic sodium c N o d i^s o lu ^l^^p e r^s ite 6^ 5 % ,n Sterile a" d - Sites 2 : 0.1 ml of water for injection, per site. complete FR EU N D 's^djuTM ^^ sodium chloride solution per site TREATMENT OF POSITIVE CONTROLS : 7 ?f wa*er for injection and of and P^ogen-free isotonic - Sites 2 : 0.1 ml of 1 % D.N.C.B., per site. > % *- - riia * complete FREUND*^adjuvant d ilu te d ifo %* in s te ^ f f J % D-N' C' B and of sodium chloride solution, per site. n sten e and Pyrogen-free isotonic TREATMENT OF TREATED ANIMALS : 5 0 % in * a n d - Sites 2 : 0.1 ml o f compound irritant concentration determined f f lS ^ S V r 'y ^ T ^ s T e . S'i9h,ly adjuvant'dilutedn 5 0 f r r i n ejte r ile nandf solution and of compound f Complete F U N D 'S ,sot?nic sodium chloride concentration determined d S S iih ^ S S iy s iu d ^ p T s T e .3" 9*1*1'' irritant ; *>!.!'.^main fSCAOSi CERB - STUDY NO. 920086 E ^L^-3i 3_Sensiti7atinr>(timp ng | ; Preparatory phase (time D8) : creation of local irritation. - Actual sensitization (time D9): Sensitization involved cutaneous application at injection sites of D 1 . p r o te c iE ^ ^ 1^ 0^ ^ ^ ^ ^ Sauze' l a s u r i n ^ s T m " 0'The OU,a,neous application place ^ P ^ ^ It P u r s ^ y ^ ^ E L A S T o V L A ^ T E ^ n o n - o t ^ lu ^ ^ ^ ^ J ^ ^ P P ^ ^ ^ P TM" h" d ' 5 of I % O.N.C.B. solution under the same 6s S ^ - Rest phase : D 11 to D21. Animals were left at res. from D , I ,o D21, i.e. for 1 , days. 1UL.3 .4 Challenge (tim Q p o o ) . On D22, 0.5 ml of compound concentration determined d u r in a H h ^ ^ T iv !^ ^ ^ TM ^ 1 the maximun non-irritant the nght |?tera| abdominal Teg S r in an ama ,, " y ^ were W le d topical y to compound. 9 n' ,n an araa never previously in contact w ith the Application was as described at time D9 to previously shaved shin. non-occlusive dressing.38 he,d m place for 24 hours using an ELASTOPLASTE & n % S e C: o ^ Company Sanffizeii. Does Wo! contain TSCA CBI CERB - STUDY NO. 920086 E 1L_6. CLINICAL MONITDRIMG OF a n im a i c . e TM n l,i07 w % ST s S e ed In case of m ortality, the cause was defined whenever possible. 117-READING OF SUM P n r . n r n (times D M anrf nog, , Determination of degree of allergenicity : ^ Z v i l o t i h / d r e f ^ g . * " StUdiSd 6 hours' 24 I TM and 48 hours after Results were graded for each animal and by group using the following scale: No reaction Slight erythema (scarcely visible) Moderate erythema (clearly visible) Intense erythema w ith edema. 0 1 2 3 or any g e n e ra ? b e h a v io ra l dryness or other lesions) clinical case report form. 9 were recorded daily for each animal using a the p T r c e n ta ^ severity of the latter. 24 hurs was based upon 9 Up showm9 a reaction rather than the Classification involving 5 degrees, used the following scale : Percentage of animals sensitized U- 8 9 - 28 29 - 64 65 - 80 _ 81 - 100 Degree of allergenicity (classification) 1 II III IV V . _ Sensitizing capacity Very slight Slight Moderate Strong Very strong CERB - STUDY NO. 920086 E JI.8 DATA ENTRY OF RESULTS ; study d u m b e r'6 reC rded 35 and When btained using ft>rms identified by the 11.9 REPORT : l ^ f XperJ mental. re,port included all findings noted during the course of the study, and m particular information concerning : - observation o f animals - recording of m ortality and of its causes - skin reactions - classification of the compound 11.10 QUALITY ASSURAMCF : tin Assurance ,U nit ensured that working procedures relative to this th rc o irs e Uof th e r?eSarnC V C mP'ied Wi` h by Peri di inspections at random over .n the experimental report were audited by the Quality Assurance Unit in accordance w ith standard operating procedures at the Centre. ' II. 11 RECORDS ; The protocol, raw data, the correspondence, the report and a samDle of th<=> compound have been stored for five years at CERB - 18800 BAUGY PFRANCE starting from the date of the final report. ' de\eTM ine?oertheiS: Peri d' CERB Wi" C ntaCt the Sp0ns0r in order - continue storage of records, - return the data to the sponsor, - destroy the data. Jointly Company Sanitized. Does not contain TSCA CB! CERB - STUDY NO. 920086 E III. RESULTS I CERB - STUDY NO. 920086 E 11I..1 DEVIATION FROM PRQTOnm N 92.04 10 OR : The3 weigh! of males was more than 550 g on the day of randomization (586 g 1(1.2 PRELIMINARY STIiniF.Q ; Mj.2.1 INTRADERMAL ADMINISTRATION : - Dates of study : Start: 28.04.1992. End : 29.04.1992. The la3^rdiryrnj-jr.j!^mgf Xv 1Um tio r^ a 5 d 'shty used ,rritant concentration determined during the primary induction phase by intradermal was compound 3 /4 in water for injection. 1U;,2.2 EPICUTANEOUS ADMINISTRATION ; - Dates of study : . Start: 28.04.1992. End : 30.04.1992. administration1 a n r f l S w ' ` h" '? ' (M.N.I.C.) determined by epicutaneous o o m** p o u( ^n^dSi ii ^*ii i Ay 'llur utewdj h1?/,2,S?innsw'talzt,eatrl?fonr i?n"jedcti?ohna.llen9e phases was i f f i f r any Sanitized. Does not contain TSCA CERB - STUDY NO. 920086 E 11I.3 FINAL S T Iin v UL 3.1 DATFS ; Induction Preparatory phase Sensitization Challenge Readings 05.05.1992 (D1) 12.05.1992 (D8) 13.05.1992 (D9) 26.05.1992 (D22) 27.05.1992 (D23) 28.05.1992 (D24) 29.05.1992 (D25) HL3.2 MONITORING OF ANIMAi g . - Animals were subjected to daily clinical monitoring throughout the study period. group!7' a We'9ht l0SS was noted in the TM le No 920341 of the negative control loss was noted norgans ^ ere* ir^ adJ a n c e d ^ r T A ' neCropsy' a 9rea< weight intestines were empty. cec^ aut0*ysis stage. The stomach and edema of hindlegs^ :eSSee3oo7ff3motric,ty due to a paWrai,lhysis and an On D23, no symptomatology was recorded. 11L3 .3 RFADIIMfi OF SKIN R F A r jin ^ ig . Results of findings are summarized in the table overleaf (Table 1). I III j 'fjpmpaniy SaniilzedL Does rtof confaln TSCA r** CERB - STUDY NO. 920086 E TABLE 1 DETERMINATION OF DEGREE OF ALLERGENICITY ALLERGEN TIME NEGATIVE 6H CONTROLS 24 H (1) 48 H POSITIVE 6H CONTROLS (2) 24 H 48 H FORAPERLE 6H 321 24 H _ _ _ _ _ _ L 48 H No. of animals/grade 0 9 9 9 - 1 6 20 20 20 12 -- -_ -- '1 9 63 22 . `- -- -- 3 .. _ - _ - (1) water for injection. (2) 1 % dinitrochlorobenzene. % o f animals sensitized 0 0 0 100 90 40 0 0 0 Lll.3.4 CLASSIFICATION OF COMPOUND : CLASS I V | C o m p o u n c ^ ^ H l^ ^ m ^ Q h o w e d a degree of allergen of class I at 24 hours. Under the experimental conditions adopted, c o m p o u n d f f l i l | ^ ^ B h L p V nsidered as being free o f any sensitizing capacity in t h ^ L ^ ^ U ^ p * V IS&fflpany Sanitized. Does not contain TSCA CBf CERB - STUDY NO. 920086 E IV . CONCLUSION ; S S liS positive control group receiving 1 % dV b Vl" 9 Watr f r lnject,on and a frfpliilluutteedd 3^/M4 -in'0S9wa+the'er fPotrririnnh?jercytieoinnd.uctionp phSase ' was '"compMouSnd ,M; N-1-C-' de!?rmined ^ epicutaneous oomp6Un d J * j M | l 9J ^ 1// 2? inntw:"aftetrloffonr i?nnjedcti?ohna.llen9e Phases was a T r^ n W tv T /c lT s T v a?24 h o u r e ^ K ? ' ! % D' N-C4 showed a da9rse o f 24 hours in the Guinea Pig24 h sensitizing capacity was very strong at 24^oursn d ^ ^ ^ ^ ^ ^ Sh Wed a degree of allergenicity of class I at CERB - STUDY NO. 920086 E APPENDIX 1 Protocol n 92.04.10.05 Company Sanitized. Does nn) aov CERB - PROTOCOL N 92.04.10.05 TRIAL Page 1 of 10 f j^ d y of cutaneous sensitization usina the MAGNUSSON and KLIGMAN test in Guinea Pig. SPONSOR ELF ATOCHEM SA La Dfense 10, Cedex 42 92091 PARIS LA DEFENSE _SJTE OF TRIAI CENTRE DE RECHERCHES BIOLOGIQUES Chemin de Montifault 18800 BAUGY, FRANCE APPROBATION ; SPONSOR CONTAOT : Mr REGNIER 22/04/92 .RESPONSIBLE STAFF FOR OFRR Date Signed Scientific and Technical Director ; S. RICHARD 16/04/92 Head of Toxicology Department ; Date Signed C. AUDEVAL-GERARD 16/04/92 Study Director ; Date Signed C. BESSON 14/04/92' Quality Assurance : Date Signed C. VIGIER 16/04/92 Date Signed 24 j j Comoaov San*?,** Doas nn?ewfj) iri tsca CRi CERB - PROTOCOL N 92.04.10.05 Page 2 of 10 STUDY OF CUTANEOUS SENSITIZATION USING THE MAGNUSSON AND KLIGMAN MAXIMIZATION TEST IN GUINEA PIG I - AIM : The aim of t l ^study i^ ^ e v a lu ^ e the possible delayed sensitization capacity of compound -------- --^ ^ M B B rtT h e experimental method is inspired of MAGNUSSOl - KLIGMAN (1969) and GUILLOT (1983). II - METHOD The method described can be applied to all compounds in liquid, or semi-liquid. The protocol complies w ith the requirements of OECD Guideline No. 4 0 6 (Mav 12, 1981). 1 The study is carried out according to Good Laboratory Practices as published by : - the French Ministry of Social Affairs and National Solidarity : State Secretariat for Health. Guideline concerning Good Laboratory Practices (G.L.P.) in the area of experimental toxicology. Guideline dated M ay 3 1 , 1983, official text No. 1065 ; reference SN -S 83/25. - E.E.C. Directive 8 7 /1 8 . This directive refers to recommendation C C 81/30 Appendix 2 of the O.E.C.D. - Food and Drug Administration : G.L.P. 21 C.F.R. Part 58 dated December 22, 1 9 78 and amendments of April 11, 1980 and September 4, 1987. Ill - COMPOUND DATA SUPLIED BY THE SPONSOR : The substance information sheet and the analytical certificate were received, on April 10 1992. DATA TO BE SUPPLIED BY THE SPONSOR : Please fill in the attached substance information form. AMOUNT OF SUBSTANCE TO BE SUPPLIED : 50 ml of substance study. jfjw ill be necessary for carrying out this u CERB - PROTOCOL N 92.04.10.05 Page 3 of 10 IV GENERAL.CHARACTERISTICS OF STUDIFS IV. 1 ANIMALS : - SPECIES : Hartley strain albino Guinea Pigs. - ORIGIN : From a specialized breeding station. - WEIGHT : Between 350 g and 550 g on the day of randomization (D-1). - SEX : Males and females. Females were nulliparous and non-gravid. - NUMBER : Preliminary studies : 4 males, 4 females. Final study : 20 males, 20 females. - ACCLIMATIZATION : for at least 5 days before the study in the area where the experiment is to take place. - IDENTIFICATION : the animals are identified by an aqueous solution of picric acid tagging. HOUSING : Five-male or female animals from each treatment group are kept per cage of standard size, on dust-free white wood shavings as bedding. These cages are placed in an air-conditioned animal house (17C -21C ) kept at a constant relative humidity of between 45 % and 65 %, except during cleaning periods in which no recycled filtered air is changed approximately ten times per hour. The artificial daylight cycle is 12 hours light and 12 hours darkness. FOOD : UAR 106 foodstuff. An analytical certificate concerning foodstuff is included in the study report. I i C iii CERB - PROTOCOL N 92.04.10.05 Page 4 of 10 - DRINKING WATER : Tap water distributed "ad libitum" in polycarbonate feed bottles with a stainless steel teat. A water sample is obtained every three months and sent to the Direction des Services Vtrinaire, 216, rue Louis Mallet, 18014 BOURGES Cedex, FRANCE for analysis. A water analytical certificate is included the study report. IV-2 PRINCIPLE : The test consists of 3 phases. mDUCTION : first contact between the body and'com pound - SENSITIZATION : second contact with compound jjH I H B H H } Recognition of allergen and multiplication of cellular and/or humoral tra n s fo rm a tio n s . - CHALLENGE : third contact with compound to the finding of a clinical manifestation. pleading or not V - EXPERIMENTAL PROTOCOL : V.1 PRE-EXPERIMENTAL PROCEDURE : - PREPARATION OF ANIMALS : . The day prior to each application Guinea Pigs used are shaved using AESCULAP FAVORITA II GT 104 clipper. An area of approximately 24 cm2 is exposed on the back, behind the head, at the level of the shoulders and flanks. - SELECTION OF ANIMALS : . Only healthy animals, w ith a skin free of any trace of cutaneous lesions, are selected. Company Sanitized. Does not contain TSCA CB# CERB - PROTOCOL N 92.04.10,05 Page 5 of 10 28 V-2, PRELIMINARY STUDIES : . - Intradermal administration : Determination of the maximum concentration causing slight irritation (with neither scab nor necrosis) in all animals by intradermal injection. - Animals : 2 males and 2 females. - Treatment : Intradermal injections are administered in the dorsal region using 0.1 ml of compound or of its dilutions. Injections are administered at 4 sites using one concentration'per site, i.e. 4 concentrations per animal in a* non irritant and non-sensitizing vehicle. - Concentration : concentrations used will be undiluted substance as well as 1/4, 1/2, 3/4 dilutions of the finished product. Dilutions w ill be subsequently increased in a second phase if the substance is found to be irritant at these concentrations. - Grading of results : Lesions are evaluated for each concentration 24 hours after injections using the scale published by MAGNUSSOIM and KLIGMAN : No reaction Slight erythema (scarcely visible) Moderate erythema (clearly visible) Intense erythema w ith edema 01 2 3 - Epicutaneous application : Determination of M .N .I.C . (Maximum Non-Irritant Concentration) by epicutaneous application under an ELASTOPLASTE non occlusive dressing. - Animals : 2 males and 2 females. -Treatm ent : 0.5 ml of compound or of its dilution are deposited on a piece of gauze of approximately 8 cm2 (4cm x 2 cm). The piece of gauze is applied to the skin at the level of the flanks and held in place for 48 hours using an ELASTOPLASTE non-occlusive dressing. -Concentrations : Compound is applied undiluted or diluted 1/2 in the chosen vehicle. Then, the' dilutions are increased if the compound is irritant at these concentrations. CERB - PROTOCOL N 92.04.10.05 Page 6 of 10 28 - Grading of results : lesions are evaluated for each concentration one hour after removal of dressings using the scale already described in V-2. The maximum slightly irritant concentration determined by intradermal administration is used during the primary induction phase. The maximum non-irritant concentration (M .N.I.C.) determined by epicutaneous administration is used during the sensitization and challenge phases. V-3 FINAL STUDY : V-3.1 Study design : ' The study design calls for the use of 40 animals (20 of each sex) randomized into 3 groups on the basis of the criterion of body w eight and selected according to their general condition. The 3 groups of animals involved in the study consist of : 1. A negative control group of 5 animals of each sex, used to confirm tha t under the experimental conditions adopted, the appearance of skin lesions in the treated group in the absence of any lesion in the negative control group is indeed evidence o f an allergic reaction. 2. A positive control group of 5 animals of each sex, serving to validate the sensitivity of the method used. This confirms tha t D.N.C.B.*, a compound recognized as being allergenic in the Guinea Pig, fully exerts its properties under the experimental conditions adopted here. 3. A group treated w ith c o m p o u n d lB B B im B O o f 10 animals of each sex, used to determine whether compound has allergenic properties under the experimental conditions adopted here. * V .3.2 Primary induction (time D ll : The induction phase takes place on D1, the first day of the final study. Each Guinea Pig receives 6 injections in the retroscapular region on either side of the vertebral column, using a previously shaved area of approximately 24 cm2 (4 cm x 6 cm). * D.N.C.B. : Dinitrochlorobenzene (chloro 1-dinitro 2.4-benzene). Use of D.N.C.B. : D.N.C.B. is used in 1 % alcoholic solution. Company Sanitized. Does not contain TSCA CE 30 CERB - PROTOCOL N 92.04.10.05 Page 7 of 10 - Injection technique : tw o intradermal injections of 0.1 ml are administered for each of the three following preparations, i.e. one injection per site : . Complete Freund adjuvant diluted 50% in sterile and pyrogen-free isotonic sodium chloride solution. . Vehicle for the n e g a tiv ^ ^ o n tm ^ r o u ^ r D.N.C.B. for the positive control group, or substance the group treated w ith the maximum slightly irritant concentration, if this concentration has been determined in preliminary experiments, . 50/50 (v/v) mixture of complete FREUND's adjuvant diluted 50% in sterile and pyrogen-free isotonic sodium chloride solution, w ith the addition of the vehicle for the negative c o n tro lg ro u p o ro f D.N.C.B. for the positive control group or of substance y flH M H H H B jfo r the group treated w ith the maximum slightly irritant concentration previously adopted. ` V .3.3 Sensitization (time D9) : Preparatory phase (time D8) : - Creation of local irritation. On D8, topical application of 0.5 ml of a 10 % suspension of sodium lauryl sulfate (dodecylhydrogenosulfate sodium salt) in paraffin oil was performed to previously shaved skin, at 6 injection sites of D1. Actual sensitization (time D9) : Sensitization involves cutaneous application at 6 injection sites of D 1. Negative controls receive 0.5 ml of vehicle by cutaneous application. Positive controls receive 0.5 ml of 1 % D.N.C.B. solution under the same conditions, Treated animals receive under the same conditions 0.5 ml of compound| at the maximun non-irritant concentration (CMNI). Application method : 0.5 ml of s u b s ta n c e ||H [H IH H iB ^ re deposited on a piece of gauze of approximately 8 cm 2 (4 cm x 2 cm). The piece of gauze is applied to the skin and held in place for 48 hours using an ELASTOPLASTE non-occlusive dressing. - Rest phase : D 11 to D 21. Animals are left at rest from D 1 1 to D21, i.e. for 11 days. mrnrnAChallenge (time D22) : On D22, 0.5 ml of compound ta the maximun non-irritant concentration determined during the preliminary study are applied topically to the right lateral abdominal region, in an area never previously in contact w ith the compound. Application is performed as described at time D9 to previously shaved skin. The piece of gauze is held in place for 24 hours using an ELASTOPLASTE non occlusive dressing. CERB - PROTOCOL N 92.04.10.05 Page 8 of 10 Application method : Application is as described at time D9, to previously shaven skin. The piece of gauze is kept in place by a non-occlusive ELASTOPLAST tape for 24 hours. VI CLINICAL MONITORING OF ANIMALS : ' Animals are monitored daily throughout the study period. This clinical examination is designed to seek any possible abnormalities due to treatment. In case of m ortality, the cause is defined whenever possible. VII READING OF SKIN REACTIONS (times D23, D24 and D25) : Possible skin reactions are studied 6 hours, 24 hours and 48 hours after removal of the dressing. Results are graded for each animal and by group using the following scale: No reaction Slight erythema (scarcely visible) Moderate erythema (clearly visible) Intense erythema w ith edema 0 1 .2 3 Any other cutaneous abnormality (thickening, vesicles, dryness or other lesions) or any general behavioral changes are recorded daily for each animal using a clinical case report form. Determination of the degree of allergenicity is based upon the percentage of animals in the group showing a reaction rather than the severity of the latter. Classification involving 5 degrees, uses the following scale : Percentage of animals sensitized 0- 8 . 9 - 28 29 - 64 65 - 80 81 - 100 Classification I II III IV V Sensitizing capacity Very slight Slight Moderate Strong Very strong Company Sanitized. Does not contain TSCA CBt CERB - PROTOCOL N 92.04.10.05 Page 9 of 10 VIII - REFERENCES : GUILLOT (J.P), GONNET (J.F), CLEMENT (C.) et FACCINI (J.M.) Etude comparative de diffrentes mthodes choisies par l'AFNOR pour l'valutation du pouvoir sensibilisant chez le Cobaye. Informations Chimie, 1982, 228. 1177-192. Comparative study of the different methods chosen by the AFNOR for the evaluation of the sensitizing potential in the guinea-pig. Food an Chemical Toxicology, 1983, 21 (n6), 795-805. MAGNUSSON (B) & KLIGMAN (A.M.) The identification o f contact allergens by animal assay. The guinea-pig maximization test. J. Invest. Derm., 1969, 52, 268-276. . IX - RESULTS : All results are recorded as and when obtained using forms identified by the study number. X - REPORT : The experimental report includes all findings noted during the course of the study, and in particular information concerning : - observation of animals - recording of m ortality and of its ca - skin reactions - classification of studied compound XI - QUALITY ASSURANCE : The Quality Assurance Unit ensures that working procedures relative to this type of study are strictly complied with by periodic inspections at random over the course of the year. Data in the experimental report are audited by the Quality Assurance Unit, in accordance w ith standard operating procedures at the Centre. XII RECORDS : . The protocol, any possible amendments, raw data, the correspondence, the report and a sample of the compound are stored for five years at CERB - 18800 BAUGY, FRANCE, starting from the date of the final report. A t the end of this period, CERB will contact the sponsor in order to jointly determine to either : - continue storage of records, - return the data to the sponsor, - destroy the data. CO CERB - PROTOCOL N 92.04.10.05 Page 10 of 10 XIII - PLANIFICATION : Date of start of study : week of April 27 1992. Results by telex : at the end of the study. Date of submission of draft report : at the end of June 1992. Date of submission of the English summary : at the final report submission. ICempany Sanfzed. Does no conain TSC CBf CERB - STUDY NO. 920086 E APPENDIX 2 References and batch numbers of reagents and equipment used V Company Sanitized. Does no! conlam TSCA CR CERB - STUDY NO. 920086 E REFERENCES AND BATCH NUMBERS OF REAGENTS ANS EQUIPMENT USED REAGENTS: Complete FREUND's adjuvant : SIGMA - Reference F-4258 o f batch 106 F- 8860. . Sterile and pyrogen-free isotonic sodium chloride solution : MERAM of batch 56243. D.N.C.B. : Dinitrochlorobenzene (1-chloro-2;4-dichlorobenzene). D.N.C.B. was used in 1 % alcoholic solution. SIGMA C-3762 of batch 10 H 05341. 90% (V/V) ethanol, Cooper of batch A 245589090152. PEG 400 : polyoxyethylene glycol 400 : COOPER of batch A 232 20 91130. Sodium lauryl sulfate (sulfuric acid monododecyl ester sodium salt) : Merck Reference 13760 of batch 029 L 765960. Paraffin oil : COOPER reference 3108. COMPOUND SOLVENT : MERAM water for injection of batchs 56671 and 56663. EQUIPMENT : Absorbant gauze : SHV type 17 fils reference 010118RA. ELASTOPLASTE REF. 6 HB : of batchs 20775S and 1423S. Syringes Terumo 1 ml of batch 91H19B5. Terumo 5 ml of batch 91C29C5. Terumo 10 ml of batch 91 C13S7. Sherwood 2 ml of batch 91E022. Henke - sass 2 ml of batch 9129451. Needles Terumo code NN-2125R of batch 8891G10G. <.T':'H U I O ` Lf. CERB - STUDY NO. 920086 E APPENDIX 3 Technical data and analytical certificate concerning compound Company SanfYFzedf. Does not con W n TgcA SERVICE DE TOXICOLOGIE CONFIDENTIEL Avril 1992 elf atochefri sa La Dfense 10, cdex 42 92091 Paris-La Dfense France FICaE D 'INFORMATIONS IDENTIFICATION Nom commun Nom chimique Origine; n de lot N d'archivage (CAL) PROPRIETES PHYSICO-CHIMIQUES Apparence Viscosit Temprature bullition pH Densit Point d'clair S o lu b ilit INFORMATIONS TOXICOLOGIQUES ET PRECAUTION D'EMPLOI Aucune information disponible. (CONDITIONS DE CONSERVATION ET DE DESTRUCTION Conservation Stabilit Destruction : A l'obscurit et l'abri de la chaleur. : Stable jusqu'en Avril 1993 dans ces conditions de stockage : Incinration. | :com as ' i o C h CB e!f atochem sa 4, cours Michelet, cedex 42 92091 Paris La Dfense 10 France BULLETIN D'ANALYSE <J 0 ATO Jean-Franois Rgnier Service de Toxicologie Company Sanfzea. Does no! eoMarsT'R t m CERB - STUDY NO. 920086 E 3a APPENDIX 4 Analytical certificate concerning foodstuff Jiofi i'vtrt m&WPWi q i ALIMENT COMPLET C O B A Y E S - E N T R E T IE N Granuls : 4.5 m m Ration Journalire du Cobaye : en fonction de son ge et poids, de 35 50 g par jour. Eau a v FORMULE % Crales, sucre .................................... Issues et Lgumineuses................ - - Protines Vgtales (Tourteaux, Levure)----Protines Animales (V ian d e).............. Compos Minral Vitaminis . ........... 44.50 40 9.50 2 4 ANALYSE MOYENNE % Valeur calorifique (en C a l/k g ).........2.600 Eau ................................ - - - - - 10 Protides .................... -.......................... ^ Lipides .................................................. Glucides (EJiA.) ............................ 3 49 Cellulose (Weende) ..................'-- . . . 13 Minraux .............................................. 8 ACIDES AMINES (calculs en rog/kg) Arginine ................i................. 1. Cystine ........................................ Lysine ................................ '.___ Mthionine .................................. Tryptophane ................ ............... Glycine ...........: ............... .......... 8.500 ' 2500 . 7.200 2.100 .2.000 6.000 MINERAUX (calculs en mg/kg) App. Not. (moy.) App. por C M TOTAUX P ................... ____ 7.400 C * .................. ............. 5.400 K ............................. 12.000 ........... 1 .3 0 0 . ...........: ' 3 .2 7 0 ........... 60 F e ................ ........... 170 Cu* ............... .. 10 7 n ____I . ____ -40 rv . : . . . ....................0 .1 0 1 ............. 1.400 5.600 0 ISSO 130 40 ISO 15 45 1.50 8.800 11.000 12.000 3250 3.400 100 320 2S 85 1.60 loon, assimilable par slguei merir*ce. VITAMINES (calcules eu kg) App. Nat. (moyj App. Syntht. totaux V itam . A . 3.400 . m 03- 30 B1 B2 6 5 m B3 B6 .. .1. m B 12 : 22 o;7o 0.003 C ------ o E .... K3 PP -.1 15 s 87 A c . Follgue . i 2.20 A c. P-AJB. B iotloe .........J 0.02 C ho line ..-- 1 1.010 M eso4oositol \ Ul 6.000 - 2.000 . mg 6.40 6.40 26 2.70 0.012 . ; 160 60 * ' 1260 14.50 1.30 2J50 * i . 0.06 60' > j 62.50 Ul 9400 - 2.030 mg 12.40 11,40 * 48 - - 3.40 - 0.01S 160 *; 7S 17.60 ` -111.50 *: 3.50 2.S0 0.08 1.070 * j 62.50 Ul * mg * - * * L supplmentation de cet aliment avec de la vitamine C stabilise enrobe supprime la ncessite d e tout autre apport (verdure, acide ascorbique) pendapt les deux premiers mois suivant fabrication. Company Sanitized. Does nof contain TSCA CBi CERB - STUDY NO. 920086 E APPENDIX 5 Analytical certificate concerning water IT Sa I Gnrai du laboratoire dpartemental VTRINAIRE RESULTAT D'ANALYSE D'EAU agr par le Ministre de la sant Nd'AnaIyse: 920794 *Type d'analyse: C7B3 N d'Ordre: 1236 Prlvemnt d'eau parvenu le: Lundi 11Mai 1992 OEfrfiegcitnue:pan C.E.R.B. Agent Commune:L i t ZONE PROTEGEE LAPIN COt JLOIR PROPR F. BAUGY Physicochimie Turbidit en U Jackson Conductivit pS/cm PH Oxydabilit KMn04 mg/1 Duret en 'Franais T.A.C en 'Franais Ammoniaque en mg/l N itrites en mg/1 Nitrates en mg/1 Sulfates en mg/1 1 Chlorures en mg/1 F er en mg/1 Manganse en mg/1 Fluor en mg/1 Rsultats Normes B actriologie 0 .3 652 17 . 5 37 <2 U 6 .5 -9 <5 Coliformes therm otolrants/100m l Streptocoques fcaux/ 100ml C o lifo rm es /1 00 m l Dnombrement des bactries arobies revivifiables 3 7 'C /m l 2 5 " ' 22*C/ml 0 < 0.5 Spores de bactries anarobies 0 < 0.1 su!fto-rductrices/20m I 57 < 50 ' ' /100m l 6 5 < 250 Salmonelles 2 0 < 200 Staphylocoques pathognes 0 < 0.2 < 0.05 - < 1.5 Rsultats O O 2 <1 2 Normes* 0 0 0-95% il 0 0 0 0 *:Selon decret N89-3 et suivants. <5 Bourges.le: Lundi 18 Mai 1992 Adj. Dir. du Laboratoire Le Directeur du Laboratoire i-