Document B534BzXmLgkk4RRmqVJjK3z3X

AR226-2994 Baugy, June 17, 1993 STUDY NO 920086 E STUDY OF CUTANEOUS SENSITIZATION USING THE MAGNUSSON AND KLIGMAN MAXIMIZATION TEST IN GUINEA PIG Sponsor : ELF ATOCHEM SA La Defense 10, cedex 42 92091 PARIS LA DEFENSE FRANCE Company Sanitized. Does noi conta'n T<^A 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 ELF ATOCHEM SA Mr REGNIER La Defense 10, Cedex 42 92091 PARIS LA DEFENSE - FRANCE. LOCATION OF STUDY CENTRE DE RECHERCHES BIOLOGIQUES Chemin de Montifault 18800BAUGY-FRANCE RESPONSIBLE STAFF FOR CERB : Scientific and Technical Director : S. RICHARD Pharmacien, Docteur de Seme Cycle MaTtre en Pharmacologie C^-l--?.^!--?.? Date Ssiiggnnaature Head of Toxicology Department : C. AUDEVAL-GERARD Docteur Veterinaire CES d'Ophtalmologie Study Director : C.BESSON DUT de Biologie Appliquee _^l06jq5 Date Quality Assurance : C. VIGIER ^/aQB5 D^te ' The authentic results of the experiment form the basis of the present report. t , --.-;,,...a. ,y ^i;tf,,iy^, l..t.cS hv; (..Olliai'i To^A CBl' CERB - STUDY NO. 920086 E j This report consists of 42 pages numbered from 1 to 42 including 5 appendices. CONTENTS Page I. SUMMARY 4 II. EXPERIMENTAL PROTOCOL ADOPTED 5 11.1 AIM 6 11.2 METHOD 6 11.3 COMPOUND 7 11.4 GENERAL CHARACTERISTICS OF STUDIES 8 11.4.1 ANIMALS 8 11.4.2 PRINCIPLE 10 II.5 EXPERIMENTAL PROTOCOL 10 11.5.1 PRE-EXPERIMENTAL PROCEDURE 10 11.5.2 PRELIMINARY STUDIES 10 11.5.2.1 Intradermal administration 10 11.5.2.2 Epicutaneous application 12 II.5.3 FINAL STUDY 13 11.5.3.1 Study design 13 11.5.3.2 Primary induction 14 11.5.3.3 Sensitization 15 11.5.3.4 Challenge 15 11.6 CLINICAL MONITORING OF ANIMALS 16 11.7 READING OF SKIN REACTIONS 16 11.8 DATA ENTRY OF RESULTS 17 11.9 REPORT 17 II. 10 QUALITY ASSURANCE 17 II.11 RECORDS 17 III. RESULTS 18 111.1 DEVIATION FROM PROTOCOL N 92.04.10.05 19 111.2 PRELIMINARY STUDIES 19 111.2.1 INTRADERMAL ADMINISTRATION 19 111.2.2 EPICUTANEOUS ADMINISTRATION 19 III.3 FINAL STUDY 20 111.3.1 DATES 20 111.3.2 MONITORING OF ANIMALS 20 111.3.3 READING OF SKIN REACTIONS 20 111.3.4 CLASSIFICATION OF COMPOUND 21 IV. CONCLUSION APPENDIX 1 : Protocol n 92.04.10.05 APPENDIX 2 : References and batch numbers of reagents and equipment used APPENDIX 3 : Technical data and analytical certificate concerning compound APPENDIX 4 : Analytical certificate concerning foodstuff APPENDIX 5 : Analytical certificate concerning water 22 23-33 34-35 36-38 39-40 41-42 W companySanHfeetf. Does nofcoma?n JSCA ^ CERB - STUDY NO. 920086 E I. SUMMARY Study of the sensitizing capacity of compoundpUI^^HH^Ivas undertaken in the Guinea Pig using the technique of 'TVIAGNUSSON and KLIGMAN in comparison with 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 .administraliflftaqd used during the primary induction phase jiluted 3/4 in water for injection. by intradermal was compound The maximmuumni ni louin1--1i1r1rintaainILt ct.i-o11n11centration (M.N.I.C.) determined by epicutaneous ^ii----qdiii administraatitojnonandc^jussee^cLLdduurriinr g the'sensitization and challenge phases was compoundJiH--^^^Hrjdiluted 1/2 in water for injection. Under the experimental conditions adopted, 1 % D.N.C.B. showed a degree of altergenicity class V at 24 hours. Its sensitizing capacity was very strong at 24 hours in the Guinea Pig. Water for injection showed a degree of allergenicity of class I at 24 hours. It may be considered as being free of any sensitizing capacity in the Guinea Pig. CompoundJUH^^BB9\showed a degree of allergenicity of class I at ' 24 hours. " ~ . Under the experimental conditions adopted, compound[flUBBIIIBBHi9 sensitizing capacity in the Guinea Pig." ;;;;;;',);'i:^^^ i&i CERB - STUDY NO. 920086 E II. EXPERIMENTAL PROTOCOL ADOPTED ^%^anife^9r%$$:^^^ CERB - STUDY NO. 920086 E 11.1 AIM : The aim of this. study was to evaluate thejjossible delayed sensrtization capacity GUILLOT(1983). 11.2 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 (May 12, 1981). 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 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 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 of 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. CERB -STUDY NO. 920086 E 11.3 COMPOUND : rTehpeortaniinnalAytpicpaelndciexrt3if.icate concerning the compound is supplied with the, study - Name - Batch No. - Record number (CAL) - Origin -Date received - Amount - Appearance : : ELF ATOCHEM SA : Saturday April 18 1992 : 1 bottle of 107 g -pH CompanySanfti'zed. Does nof contain TSCA CBt CERB - STUDY NO. 920086 E 11.4 GENERAL CHARACTERISTICS OF STUDIES : 11.4.1 ANIMALS : - SPECIES : Hartley strain albino Guinea Pigs. - SOARIINGTIN- G: EINRTMEARINFAUNA FRANCE S.A.R.L. -les Sillons" - 37600 SAINT-JEAN- specialized breeding station. - mWeEaInGHwTei:ghMtaolef 5a1n3imgals h2a1d ga omnetahne wdaeyigohft roafnd5o8m6 izgation32(D-g1,).and females a - 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 negative controls 10 positive (5 males + 5 females) - 20 treated controls with the study (5 males + 5 females) compound (10 males +10 females) DATERECElVED.-On22.04.1992. eAxCpCeLriImMeAnTt IZwAasTItOoNtak: efoprlac1e3. days before the study in the area where the sIDolEuNtiToInFIoCfApTicIOriNc a:ciTdhteayggwinegre. identified per cage, individually by an aqueous '^fir^f. .- CERB - STUDY NO. 920086 E - pHeOr UcSaIgNeGof: sFtiavnedmaradlesiozre,feomn adleusat-nfirmeealswhfritoemweoaocdh sthreaavtimngesntasgrboeudpdiwnge.re kept These cages were placed in an air-conditioned at a constant relative humidity animal house (17C-21 C) kept cleaning periods in which of between 45 % and 65 %, except during hteonurstimdeasrknpeesrs.hour. The anrotinfi-crieacl ycdlaeydligfihltterceydclaeir wwaass 1c2hanhoguedrs alpigphrot xaimndate1l2y FOOD : UAR 106 special Guinea Pig foodstuff. concerning foodstuff may be found in Appendix 4. An analytical certificate DRINKING WATER bottles with : Tap water distributed "ad libitum" in polycarbonate feed months and a stainless steel teat. sent to the Direction A'water sample des Services was obtained every three Mallet, 18014 BOURGES Cedex, FRANCE for Veterinaires, 216, rue Louis analysis. The water analytical ccleorstiefsictaotef thine Astpaprtenodf itxhe5 stcuodryre. sponds to the sample obtained on the date I i Does no! confaFn TSC.^ '"" CERB - STUDY NO, 920086 E : [j 11.4.2 PRINCIPLE : Animals were treated three times (Figure 1). The test consisted of three phases ; INDUCTION : first contact between the body and compound SENSITIZATION Recognition of transformations. ^^UUHBlr : second contact with compound allergen and multiplication of cellular and/or humoral - CHALLENGE : third contact with compoundUH^UBIIIfJIeading or not to the finding of a clinical manifestation. II. 5 EXPERIMENTAL PROTOCOL : II. 5.1 PRE-EXPERIMENTAL PROCEDURE : - PREPARATION OF ANIMALS : the day prior to each application Guinea Pigs used were shaved using AESCULAP FAVORITA II GT 104 clipper. An area of approximately 24 cm2 was exposed on the back, behind the head, at the level of the shoulders and flanks. - SELECTION OF ANIMALS : only healthy animals, with a skin free of any trace of cutaneous lesions, were selected. II.5.2 PRELIMINARY STUDIES : 11.5.2.1 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 : Intradermaj. injections were administered in the dorsal region using 0.1 ml of compound^BB----MB^ of its dilutions. Injections were administered at 4 sites using one concentration per site, i.e. 4 concentrations per animal. Water for injection was used as a non-frritant and non-sensitizing vehicle. T^;,-;;'--; "J:^;..^i-^.. ,j,;.,^n^s,t.mi[amTSCAC8i 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 24 hours after injections using the scale published by MAGNUSSON and KLIGMAN : No reaction Slight erythema (scarcely visible) Moderate erythema (clearly visible) Intense erythema with edema II. 5.2.2 Epicutaneous application : a. Determination of M.IM.I.C. (Maximum Non-Irritant Concentration) by epicutaneous application under an ELASTOPLASTE non occlusive dressing. - Animals : 2 males and 2 females. - Treatment: Epicutaneous application of 0.5 ml of compound||HHHUUBor of its dilution over an area of 8 cm2 using one concentration on each flank, i.e. two concentrations per animal. - Concentrations : CompoundjIIHHHBBBIj/vas applied undiluted or diluted 1/2 in water for injection to the skin prepared for this purpose. The piece of gauze on which the compound or its dilution was deposited was held in place for 48 hours using an ELASTOPLASTE non-occlusive dressing. - Grading of results : lesions were evaluated for each concentration one hour after removal of dressings using the scale already described in 11.5.2.1. The maximum slightly irritant concentration determined by intradermal administration was used during the primary induction phase. The maximum non-irritant concentration (IV1.N.I.C.) determined by epicutaneous administration was used during the sensitization and challenge phases. KompanySanitized. Does no! contain T8CA CB CERB - STUDY NO. 920086 E FIGURE ?1 SITES OF ADMINISTRATION ||i%| Site of application for sensitization .___ Sites of intradermal injection or epicutaneous application for primary induction Site of application for 24 hours for challenge ij CERB - STUDY NO. 920086 E U.5.3 FINAL STUDY : 11.5.3.1 Study design : The into study 3 design called for the use of 40 animals (20 of each sex) randomized groups on the basis of the according to their general condition. criterion of body weight and selected The 3 groups of animals involved in the study consisted of : LA negative under the control group of 5 animals of each sex, used to confirm that experimental conditions adopted, the appearance of skin lesions in wthaes tirnedaeteedd egvroiduepncine tohfeanabaslelenrcgeic of any lesion in the negative con-trol group reaction. 2. A positive control sensitivity of the group of 5 animals of each sex, serving to validate the recognized as method used. This being allergenic in the confirmed that D.N.C.B., Guinea Pig, fully a compound under the experimental conditions adopted here. exerted its properties 3. A group used to treated with determine compoundqmBjUBKpf 10 animals of whether compound {f--------------) each sex, properties under the experimental conditions adopted here. had allergenic companySanitized. Does not conWr> T"" a '"""" CERB - STUDY NO. 920086 E 11.5.3.2 Primary induction (time D1) : The induction phase took place on D1, the first day of the trial. Each Guinea Pig received 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). Two intradermal injections of 0.1 ml were administered with three different preparations. TREATMENT OF NEGATIVE CONTROLS : - Sites 1 : 0.1 ml of complete FREUND's adjuvant diluted 50 % in sterile and pyrogen-free isotonic sodium chloride solution, per site. - Sites 2 : 0.1 ml of water for injection, per site. - Sites 3:0.1 ml of an emulsion of equal volume of water for injection and of complete FREUND's adjuvant diluted 50 % in sterile and pyrogen-free isotonic sodium chloride solution, per site. TREATMENT OF POSITIVE CONTROLS : - Sites 1 : 0.1 ml of complete FREUND's adjuvant diluted 50 % in sterile and pyrogen-free isotonic sodium chloride solution, per site. - Sites 2 : 0.1 ml of 1 % D.N.C.B., per site. t^i - Sites 3 : 0.1 ml of an emulsion of equal volume of 1 % D.N.C.B. and of 'y complete FREUND's adjuvant diluted 50 % in sterile and pyrogen-free isotonic sodium chloride solution, per site. TREATMENT OF TREATED ANIMALS : - Sites 1 : 0.1 ml of complete FREUND's adjuvant diluted 50 % in sterile and pyrogen-free isotonic sodium chloride solution, per site. JBHU^BvJ^ - Sites 2 : 0.1 ml of compound tne maximum slightly irritant concentration determined during the preliminary study, per site. - Sites 3 : 0.1 ml of an emulsion of equal volume of complete FREUND's adjuvant diluted 50 % in ^sterile and pyrogen-free isotonic sodium chloride solution and of compound p^--------------1b.tthe maximum slightly irritant concentration determined during the preliminary study, per site. ) .;<, .,-'; ~^- ^ ^^.-iil TSCA CBf 15 CERB - STUDY NO. 920086 E 11.5.3.3 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 in paraffin oil was performed to previously shaved skin, at 6 injection sites of D1. - Actual sensitization (time D9): Sensitization involved cutaneous application at injection sites of D1. Negative controls received 0,5 ml of water for injection by cutaneous application on a piece of absorbant gauze measuring 8 cm2. The application site was protected by a piece of absorbant gauze measuring approximately 24 cm2 held in place for 48 hours by an ELASTOPLASTE non-occlusive dressing. Positive controls received 0.5 ml of 1 % D.N.C.B. solution under the same conditions. ^Treated animals received under the same conditions 0.5 ml of compound |,--------------------thel3amtaximun non-irritant concentration determined during the preliminary study. - Rest phase : D11 to D21. Animals were left at rest from D 11 to D21, i.e. for 11 days. 11.5.3.4 Challenge (time D22) : iflHHIBBHIpt On D22, 0.5 ml of compound the maximun non-irritant concentration determined during^he preliminary study were applied topically to the right lateral abdominal region, in an area never previously in contact with the compound. Application was as described at time D9 to previously shaved skin. The piece of gauze was held in place for 24 hours using an ELASTOPLASTE non-occlusive dressing. Under the same conditions as above, negative control animals received water torinjection. Positive control animals'received 1 % D.N.C.B. solution. company San,fee-d. Ooes-rfot con^n TSCA W CERB - STUDY NO. 920086 E 11.6 CLINICAL MONITORING OF ANIMALS : Animals were monitored daily throughout the study period. This clinical examination was designed to seek any possible abnormalities due to treatment. In case of mortality, the cause was defined whenever possible. 11.7 READING OF SKIN REACTIONS (times D23. D24 and D25) : Determination of degree of allergenicity : Possible skin reactions were studied 6 hours, 24 hours and 48 hours after removal of the dressing. 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 with edema. Any other cutaneous abnormality (thickening, vesicles, dryness or other lesions) or any general behavioral changes were recorded daily for each animal using a clinical case report form. Determination of the degree of allergenicity at time 24 hours was based upon the percentage of animals in the group showing a reaction rather than the severity of the latter. Classification involving 5 degrees, used the following scale : Percentage of animals sensitized 0- 8 9- 28 29- 64 65- 80 81 - 100 Degree of allergenicity (classification) 1 11 III IV V Sensitizing capacity Very slight Slight Moderate Strong Very strong CERB - STUDY NO, 920086 E 11.8 DATA ENTRY OF RESULTS : All results were recorded as and when obtained using forms identified by the study number. 11.9 REPORT : The experimental report included all findings noted during the course of the study, and in particular information concerning : - observation of animals - recording of mortality and of its causes - skin reactions classification of the compou<n^d^J^IB^B^|^|B^I^H^B1j - 11.10 QUALITY ASSURANCE : The Quality Assurance Unit ensured that working procedures relative to this type of study were strictly complied with by periodic inspections at random over the course of the year. Data in the experimental report were audited by the Quality Assurance Unit, in accordance with standard operating procedures at the Centre. 11.11 RECORDS : The protocol, raw data, the correspondence, the report and a sample of the compound have been stored for five years at CERB - 18800 BAUGY, FRANCE, starting from the date of the final report. At 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. company Samt-ized. Does not contain TSCA CW CERB - STUDY NO. 920086 E I.RESULTS CERB - STUDY NO. 920086 E 111.1 DEVIATION FROM PROTOCOL N 92.04.10.05 : The weight of males was more than 550 g on the day of randomization (586 g -t 32 g) 111.2 PRELIMINARY STUDIES : 111.2.1 INTRADERMAL ADMINISTRATION : - Dates of study : Start.-28.04.1992. End : 29.04.1992. The maximum slightly irritant concentration determined administration and used during the primary induction phase ^'""""""''"^""'^iluted 3/4 in water for injection. by intradermal was compound III.2.2 EPICUTANEOUS ADMINISTRATION : - Dates of study : Start: 28.04.1992. End : 30.04.1992. The maximum non-irritant concentration (M.N.l.C.) determined by epicutaneous administratior^andusedduring the sensitization and challenge phases was compoundUimUHM^iIuted 1/2 in water for injection. 'Sanitized. Doss not contain TSCA CBf CERB - STUDY NO. 920086 E 2u 111.3 FINAL STUDY : III.3.1 DATES: 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) 111.3.2 MONITORING OF ANIMALS : Animals were subjected to daily clinical monitoring throughout the study period. gOrnouDp.17, a weight loss was noted in the male No 920341 of the negative control On D18, loss was this animal noted, was found dead in its cage. At necropsy, a great weight intestines organs were empty. were in an advanced autolysis stage. The stomach and , From D18 to D22^ th J--------------B^h oe wfeemda l e No 920373 of the group treated with 'edema of hindlegs. a decrease of motricity due to a compound paralysis and an On D23, no symptomatology was recorded. 111.3.3 READING OF SKIN REACTIONS : Results of findings are summarized in the table overleaf (Table 1). 'R^pany'Sanitizeci. Does not contain TSCA rw CERB - STUDY NO. 920086 E TABLE 1 DETERMINATION OF DEGREE OF ALLERGENICITY ALLER6EM NEGATIVE CONTROLS (1) POSITIVE CONTROLS (2) FORAPERLE 321 TIME 6 H 24 H 48 H 6H 24 H 48 H 6H 24 H 48 H No). of ani mals/gr ade 0 1 2 3 9 - - - 9 - - - 9 - - - 9 '1 - - 1 6 3 - 6 2 2 - 20 - - - 20 - - - 20 - - - % of animals sensitized 0 0 0 100 90 40 0 0 0 CLASS - 1 - V - I - (1) water for injection. (2) 1 % dinitrochlorobenzene. III.3.4 CLASSIFICATION OF COMPOUND : Compounopii^BIIHBHu^^^d a degree of allergen of class I at 24 hours. Under the experimental conditions adopted, compoundVlBHBIUHgmaybe considered as being free of any sensitizing capacity in the Guinea Pig. %lpanySahWz8d. Does nof confam TSCA CRf CERB - STUDY NO. 920086 E IV. CONCLUSION : The sensitizing capacity of compoundlUBUBfwas studied in the male and female Guinea Pig using the technique of MAGNUSSON and KLIGMAN, in comparison with a negative control group receiving water for injection and a positive control group receiving 1 % D.N.C.B. The maximum slightly irritant concentration determined ^UHlUH^J^uted .administration and used during the primary induction phase 3/4 in water for injection. by intradermal was compound The maximum non-irritant concentration (M.N.I.C.) determined by epicutaneous administration and used during the sensitization and challenge phases was compoundgHIIIB----lbiluted 1/2 in water for injection. Under the experimental conditions adopted, 1 % D.N.C.B. showed a degree of allergenicity of class V at 24 hours. Its sensitizing capacity was very strong at 24 hours in the Guinea Pig. Water for injection showed a degree of allergenicity of class 1 at 24 hours. It may be considered as being free of any sensitizing capacity in the Guinea Pig. jmmUFjshowed Compound 24 hours. a degree of allergenicity of class I at l--------fBHIMBI^may Under the any sensitizing capacity in the Guinea Pig. be considered as being fTrreee ol r--~-7^~~ ---------------,.,.-----':-" ----_----ik,." .. W^-"'^ "' "-: ""' "-' ' ' '.;./,' S/ba =,::,' '' ' .i;*',-^"^ " '.>-.;y;;r?'; "" ' ~{'-~ y " CERB - STUDY NO- 920086 E u APPENDIX 1 Protocol n 92.04.10.05 v Company Sanitized. Does n" w^i^ ryr-p, r-vii- 24 CERB - PROTOCOL N 92.04.10.05 TRIAL Page 1 of 10 Study of cutaneous sensitization using the MAGNUSSON and KLIGMAN test in Guinea Pig. SPONSOR : ELF ATOCHEM SA La Defense 10, Cedex 42 92091 PARIS LA DEFENSE SITE OF TRIAL : CENTRE DE RECHERCHES BIOLOGIQUES Chemin de Montifauft 18800 BAUGY, FRANCE APPROBATION : SPONSOR CONTACT : Mr REGNIER 22/04/92 RESPONSIBLE STAFF FOR CERB : Scientific and Technical Director : S. RICHARD Date 16/04/92 Signed Head of Toxicology Department : C. AUDEVAL-GERARD Date 16/04/92 Signed Study Director : C. BESSON Date 14/04/92' Signed Quality Assurance : C. VIGIER Date 16/04/92 Signed Date Signed Comnanv Sa"W?sd. Does r>nf wnf^n rSff\ CW 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 thjsjjtudy is to evaluate the possible delayed sensitization capacity of compound ijplBHl^^------nThe experimental method is inspired of MAGNUSSON - KLIGMAN (1969) and GUILLOT (1983). II - METHOD : The method described can be applied to all compounds in liquid, or semi-liquid. The protocol complies with the requirements of OECD Guideline No. 406 (May 12, 1981). 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 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 of 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. 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 ]mUJl|will be necessary for carrying out this CERB - PROTOCOL N 92.04.10.05 Page 3 of 10 IV GENERAL.CHARACTERISTICS OF STUDIES :, 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 a'nd 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 g^ 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. .sn, , yiAi.diii ii u ^ri 'io'tii 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 Veterinaire, 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. ) fiiHIIHIHBBj DUCTION : first contact between the body and'compoun SENSITIZATION : second contact with compound Recognition of allergen and multiplication of cellular and/or humoral transformations. CHALLENGE : third contact with compoundMHBBHj|\leading or not to the finding of a clinical manifestation. 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, with a skin free of any trace of cutaneous lesions, are selected. Somparsy San?HEed. Does not contain TSCA CBil CERB - PROTOCOL N 92.04.10.05 Page 5 of 1 0 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'nonirritant 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 will 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 MAGNUSS01M and KLIGMAN : No reaction Slight erythema (scarcely visible) Moderate erythema (clearly visible) Intense erythema with edema - 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. -Treatment : 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. P CERB - PROTOCOL N 92.04.10.05 23 Page 6 of 10 - 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 weight 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 that 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 of 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 that 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 with compoundBBBBB^^Jo_(_animals of each sex, used to determine whether compound n------------I^BI&Jhaasllergenic properties under the experimental conditions adopted here. V.3.2 Primary induction (time PI) : 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-dinit'ro 2.4-benzene). Use of D.N.C.B. : D.N.C.B. is used in 1 % alcoholic solution. 'Company Sanitized. Does noi contain TSCA CE CERB - PROTOCOL N 92.04.10.05 Page 7 of 1 0 - Injection technique : two 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. QU||HfUR]for . Vehicle for the negative_control group or D.N.C.B. for the positive control group, or substance the group treated with 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, with the addition of the vehicle for the negative control group or oj D.N.C.B. for the positive control group or of substance^--------fjfor the group treated with 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 D1. 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^J"reated animals receive under the same conditions 0.5 ml of compoundf at the maximun non-irritant concentration (CMNI). Application method : 0.5 ml of substance^HBiH^B^B^re deposited on a piece of gauze of approximately 8 cm2 (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 : D11 to D21. Animals are left at rest from D11 to D21, i.e. for 11 days. Challenge (time D22) : On D22, 0.5 ml of compound(j^----B----Bjatthe 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 with the compound. Application is performed as described at time D9 to previously shaved skin. ^ The piece of gauze is held in piace 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 mortality, 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 with edema 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 Pompsmy SanHSze'd. Does not contain TSCA CBIr 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 differentes methodes choisies par I'evalutation du pouvoir sensibilisant chez Ie Cobaye. Informations Chimie, 1982, 228. 1177-192. Comparative study of the different methods chosen by the evaluation of the sensitizing potential in the guinea-pig. Food an Chemical Toxicology, 1983, .21 (n6), 795-805. I'AFNOR pour AFNOR for the MAGNUSSON (B) & KLIGMAN (A.M.) The identification of 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 mortality and of its causes - 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 with 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. At 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. CERB - PROTOCOL N 92.04.10.05 33 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. companySaniiized. Does not contain TSCA CRT CERB - STUDY NO. 920086 E ^ i, APPENDIX 2 References and batch numbers of reagents and equipment used Pompany Sanitized. Does not contain TSCA CR; CERB - STUDY NO. 920086 E -.'J REFERENCES AND BATCH NUMBERS OF REAGENTS ANS EQUIPMENT USED REAGENTS: Complete FREUND's adjuvant : SIGMA - Reference F-4258 of batch 106 F8860. 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 A2322091130. Sodium lauryl sulfate (sulfuric acid monododecyi 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 01011 BRA. 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. ' K;-"""-----------;-.:--.^ ,',. ,' .^"T;-'.''1 i.^'5'^- * -'"A CL'- CERB - STUDY NO. 920086 E .:: 0 APPENDIX 3 Technical data and analytical certificate concerning compound 'Company SanRFzed. Does no? confab T.5C.A ^'a.f SERVICE DE TOXICOLOGIE CONFIDENTIEL Avril 1992 u elf atochehi sa La Defense 10, cedex 42 92091 Paris-La Defense France FICEE D' INFORMATIONS INFORMATIONS TOXICOLOGIQUES ET PRECAUTION D'EMPIiOI Aucune information disponible. ____ C01JDITIONS DE CONSERVATION ET DE DESTRUCTION Conservation Stabilite Destruction : A 1'obscuri'te et a 1'abri de la chaleur. : Stable jusqu'en Avril 1993 dans ces conditions de stockage : Incineration. "^^StrsckW elf atochem sa 4, cours Michelet, cedex 42 92091 Paris La Defense 10 France BULLETIN D'ANALYSE J ATO ^8-=.-?Cwril 1992 Jean-Francois Regnier Service de Toxicologie 'Company Sanitized Does hot eoNaIn TSC'K CBF CERB - STUDY NO. 920086 E APPENDIX 4 Analytical certificate concerning foodstuff COBAYES ^i.^^aluaos^^aa 4 ^ ALIMENT COMPLET - ENTRETIEN Granules : 4.5 mm Ration .joumaliere dii Cobaye : en fonction de son age et poids, de 3S a 50 g par jour. -- Eau a \/ FORMULE % Cereales. sucre ................... Issues et Legumineuses ............ Proteines Vegetales (TourteauxL. ewre).... Proteines Animales -(Viande) ........ Compose Mineral Vitammise ..'-..-.. 44.50 40 9.50 2 4 ANALYSE MOYENNE % Valeur calorifique (en Cal/kg) Eau ....................... Protides ..-..-..--.-_....-.- Lipides -..-.--.-........... Glucides "(EJ^A.) ........... Cellulose fWcende) .......... Mineraux ..-..-..-...-.-..- 2.600 10 17 3 49 13 8 Arginine Cystine Lysine .... Methionine Tryptophane Glycine ACIDES AMINES (calcut^s en mg/kg) 8.500 "2^00 7.200 2-100 .2-000 6.000 MINERAUX (calcules en <"g/kg) ^r-' App.p^CM TOTAUX P ............. Car ........... K ............. Na ........... Mg .......... Mn .......... Fe ........... Cu ........... Zn ........... Co ...;...... 7.400 5.400 12.000 1.300. 3.270 60 170 10 40 . 0.10 1.400 5.600 0 1350 130 40 ISO 15 45 1.50 8.800 11.000 12.000 3.2SO 3.400 100 320 2S 85 1.60 (onna ascltnilable F>ar tgueR VITAM1NES (calculees t.u kg) App- Nrt. (noyj App. SyntMt. TOTAUX Vitam. A. .-.-. 3.400 . Ill 6.000 Ul 9400 U D 3 30 2.000 . . - 2-030 -- 6 mg 6.40 mg 12,40 m 81. . B 2 5 6.40 " 11,40 . . . B 3 . . 22 26 48 8 6 . 0:70 2.70 - 3.40 B 12 0,003 0.012 0.015 . C 0 e ... 15 K 3... 5 . PP .. Ac. Folique . Ac. PAB; .: Biotloe .....,' Choline .,..1J Mesch4oosttol i 87 2.20 0.02 1.010 160 60 12.60 : 14.50 ; 1.30 : -2^0 1 . 0.06 j '60' 1 62,50 ; 160 ; 7S 17.60 -. --111.SO = 3.50 . i 2.50 . . .; 0.08 i 1.070 ! 62.50 .---------------- . La supplementation de cet aliment avec de la vitamine C stabllisee enrobee supprime la necessite de tout autre apport (verdure, aclde ascorfaiquel pendant les deux premiers mois suivant fabrication. Company Sanitized. Does not contain TSCA CBf CERB - STUDY NO. 920086 E APPENDIX 5 Analytical certificate concerning water LABORAT01RE DEPAF1TEMENTAL VETER1NA1RE agree par Ie Minislere de la sante N''d'An.aIyse: 920794 *Type d'analyse: C7B3 RESULTAT D'ANALYSE D'EAU N" d'Ordre: 1236 Prelevement d'eau parvenu Ie: Lundi 11 Mai 1992 Effectuepar C.E.R.B. Origine: Agent lieu: ZONE PROTEGEE LAPIN COULOm PROPRE Commune: BAUGY Physicochimie Turbidite en U Jackson Conductivite pS/cm PH Oxydabilite KMn04 mg/1 Durete en "Fran(;ais T.A.C en 'Francais Ammoniaque en mg/1 Nitrites en mg/1 Nitrates en mg/1 Sulfates en mg/1 Chlorures en mg/1 Fer en mg/1 Manganese en mg/1 Fluor en mg/1 Resultats Nonncs* Bacteriologie 0.3 652 7.5 1 37 25 0 0 57 65 20 0 <2U 6,5-9 <5 < 0.5 < 0.1 < 50 < 250 < 200 < 0,2 < 0.05 < 1.5 Coliformes thermotolerants/lOOmI Streptocoques fecaux/ 100ml Coliformes/100ml Denombrement des bacteries aerobics revivifiables a 37'C/ml a 22'C/ml Spores de bacteries anaerobies suirito-reductrices/20ml /100ml Salmonelles Staphylocoques palhogenes Resultats <1 2 <5 *:SeIon decrct ?89-3 et suivants. Bourgcs.le: Lundi 18 Mai 1992 Adj. Dir. du Laboratoire Le Du-ecteur du Laboratoire Erici PRENGERE Jean'Marie GUERAUD