Document N2G6Eo6OEKdQQ1ma1wo0VM0aQ

SPONSOR ElfAtochemS.A. La Defense 10 - Cedex 42 92091 Paris-la-Defense France STUDY TITLE SKIN SENSITIZATION TEST IN GUINEA-PIGS (Maximization method of Magnusson, B. and Kligman, A.M.) AR226-3013 '--'-'I'^X XJ--'J_,11 Jl JLrtJ-, STUDY DIRECTOR Stephane de Jouffrey STUDY COMPLETION DATE 24th March 1995 PERFORMING LABORATORY Centre International de Toxicologie (C.I.T.) Miserey - 27005 Evreux - France. LABORATORY STUDY NUMBER 12407TSG Company Sanitized. Does not contain TSCA CBI. CIT/Study No. 12407TSGMMMBBBBjElfAtochem 2 CONTENTS STATEMENT OF THE STUDY DIRECTOR OTHER SCIENTISTS INVOLVED IN THIS STUDY STATEMENT OF QUALITY ASSURANCE UNIT SUMMARY 1. INTRODUCTION 7 2. MATERIALS AND METHODS 2.1. TEST AND CONTROL SUBSTANCES 2.1.1 Test substance 2.1.2 2.1.3 Vehicle Preparation 2.1.4 Other substances 2.2. TEST SYSTEM 2.2.1 Animals 2.2.2 Environmental conditions 2.2.3 Food and water 2.3. TREATMENT 2.3.1 Preliminary test 2.3.2 Main study, 2.3.2.1 Preparation of the animals 2.3.3 Induction phase by intradermal and cutaneous routes 2.3.3.1 Intradermal route 2.3.3.2 2.3.3.3 Cutaneous route Challenge phase 2.4. SCORING OF CUTANEOUS REACTIONS 2.5.. CLINICAL EXAMINATIONS 2.6. BODY WEIGHT 2.7. PATHOLOGY 2.7.1 Necropsy 2.7.2 Cutaneous samples 2.7.3 Microscopic examination 2.8. DETERMINATION OF THE ALLERGENICITY LEVEL 2.9. SUMMARY DIAGRAMS Figure 1: control group 7 7 7 8 8 8 8 8 8 9 9 9 9 9. 9 10 10 11 11 11 11 11 11 11 12 13 13 Figure 2: treated group 2.10. CHRONOLOGY OF THE STUDY 14 conlam_TS^C&AGcfBiKP Company eSa,.nitu^.-d-".-uDoes not 2.11. ARCHIVES 3. RESULTS 3.1. PRELIMINARY STUDY 3.1.1 3.1.2 Administration by intradermal route Application by cutaneous route 3.2. MAIN STUDY 3.2.1 Clinical examinations 3.2.2 Scoring of cutaneous reactions 3.2.2.1 End of the induction period 3.2.2.2 Challenge application 4. CONCLUSION Figure 3: Male body weight gain (g) Figure 4: Female body weight gain (g) APPENDICES 1. Analytical certificate 2. Diet formula 3. Individual body weight values 4. Individual observation of cutaneous reactions 5. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs 15 16 16 16 16 17 17 17 17 17 17 lg, 19 20 ' 2 123 25 27 29 and 30 -x^-'"^09' coiw53^"1n ^ - 0 0 ' 3 CIT/Study No. 12407 TSCf^l^Mp^ Atochem 4 STATEMENT OF THE STUDY DIRECTOR The study was performed in compliance with the principles of Good Laboratory Practice Regulations: . O.E.C.D. principles of Good Laboratory Practice, C(81)30(final) Annex 2. May 12, 1981. I declare that this report constitutes a true and faithful record of the procedures undertaken and the results obtained during the performance of the study. This study was performed at the Centre International de Toxicologie (C.I.T.), Miserey, 27005 Evreux, France. Toxicology Study Director Doctor of Veterinary Medicine Head of Short-term and Environmental Toxicology OTHER SCIENTISTS INVOLVED IN THIS STUDY For Pharmacy: J. Richard Doctor of Pharmacy For Toxicology: C. Pelcot Study Supervisor ^^w"kcBl ^^^ CIT/Study No. 12407 TSGlW----------U^ Atochem 5 STATEMENT OF QUALITY ASSURANCE UNIT 1. Specific study inspections Type of inspections Protocol Report Inspections 24.10.94 14.3.95 Dates (day/month/year) Report to Study Director / Management (*) 26.10.94 14.3.95 2. Routine inspections performed on other studies of the same type according to a frequency defined in Q.A.U. procedures Inspected phase Inspections Dates (day/month/year) Report to Study Director / Management (*) Animals/housing Treatment Test substance/preparation 13.10.94 15.11.94 23.11.94 17.10.94 16.11.94 25.11.94 The inspections were performed in compliance with C.I.T. Quality Assurance Unit procedures and the Good Laboratory Practice Regulations. (*) The dates mentioned correspond to the dates of signature of audit reports by Study Director / Management. M. Labiche Pharmacist Date: 24.3.95 Head of Quality Assurance Unit and Scientific Archives .oicon^nTSC^CBl ' Co^^006 Or/Study No. 12407 TSGJf--------QElf Atochem 6 SUMMARY At the request ofjglf Atochem S.A., Paris-la-Defense, France, the potential of the test substance, ' W------------||to induce delayed contact hypersensitivity following intradennal injection and ' cutaneous application was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligman. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Methods Thirty guinea-pigs (15 males and 15 females) were allocated to 2 groups: a control group 1 (5 males and 5 females) and a treated group 2 (10 males and 10 females). The sensitization potential of the test substance was evaluated after a 10-day induction period during which time the animals were treated with sterile isotonic saline solution (0.9% Nad) (control group) or the test substance (treated group). On day 1, in presence ofFreund's complete adjuvant, 0.1 ml of the test substance at a concentration of 1% (w/w) in the vehicle was admi nistered by intradennal route. On day 8, 0.5 ml of the test substance in its original form was applied by cutaneous route during 48 hours by means of an occlusive dressing. After a period of 12 days without treatment, a challenge cutaneous application of 0.5 ml of the vehicle (left flank) and 0.5 ml of the test substance in its original form (right flank) were administered to all animals. The. test substance and the vehicle were prepared on a dry gauze pad then were applied to the skin and held in place for 24 hours by means of an occlusive dressing. Cutaneous reactions on the challenge application sites were then evaluated 24 and 48 hours after removal of the dressing. After the final scoring period, the animals were killed. Due to the absence of cutaneous reactions, no skin samples were taken from the challenge application sites from all the animals. The sensitivity of the guinea-pigs in C.I.T. experimental conditions were checked in a recent study with a positive sensitizer: Dinitro 2.4 Chlorobenzene. During induction period the test substance was applied at 0.1% (day 1) and 5% (day 8) concentrations. At cutaneous challenge application, 1% (w/w) was tested on the right flank. Results No clinical signs and no deaths were noted during the study. After 24 and 48 hours following removal of the dressing of the cutaneous challenge application of the test substance, no cutaneous reactions were recorded. The guinea-pigs which were used in a recent study showed a satisfactory sensitization response in 95% animals using a positive sensitizer (appendix 5). Conclusion Under our experimental conditions and according to the maximization method established by y 3 ^ ^ Magnusson anjijCligman, no cutaneous reactions attributable to the sensitization potential oft test substancepBBHBHBHBf1'1ite original form were observed in guinea-pigSAgin ?SCA* CoWP5^..san^6 CIT/Study No. 12407TSCj(||gBB^JKplfAtochem 7 1. INTRODUCTION l U t 0 The objective of this study, performed according to maximization method established by ^Aasmisson and Kligman (i), was to evaluate the potential of the test substance]]----|B| induce delayed contact hypersensitivity in guinea-pigs, (i^^^^^r The results of the study are of value in predicting the contact sensitization potential of the test material in Man. During the induction period, the test substance was administered by intradennal route (together with an adjuvant to maximise potential reactions) and cutaneous route. After a rest period of 12 days, a challenge application with the test substance was performed in order to provoke a cutaneous sensitization reaction. The study was conducted in compliance with: . O.E.C.D. guideline No. 406,17th July 1992. . B.C. Directive No. 92/69/E.E.C., Bg, 31st July 1992. 2. MATERIALS AND METHODS 2.1. TEST AND CONTROL SUBSTANCES 2.1.1 Test substance_____g^ The test substance^lUmiJused in the study was supplied by Elf Atochem S.A. Documentation supplied by the Sponsor identified the test substance as follows: . depnormoitnoatcioon:l:yllH__^^^^--^^m - IabeIling'H|UU|j| . description: browmshTiqmd . container: 1 plastic flask .dateofreceipt.-28.11.94 . storage conditions: at room temperature. Data relating to the characterization of the test substance are documented in an analytical certificate (presented in appendix 1) provided by. the Sponsor. 2.1.2 Vehicle The vehicle used was sterile isotonic saline solution (0.9% Nad), batch No. 3126 (Biosedra, 92240 Malakoff, France). (1) Magnusson, B.; Kligman, A.M.: The identification of contact allergens by animal assay. ^.con^CACBt The guinea-pig maximization test. J. Invest. Derm. 52: 268-276 (1969). ^Sb a^-006 Cowpa^ CIT/Study No. 12407 TSCJ--------B----^EAItfochem g 2.1.3 Preparation The test substance was prepared in an appropriate vehicle. 2.1.4 Other substances The other substances used were Freund's complete adjuvant, batch No. 063H8800 (Sigma, 38297 Saint-Quentin-Fallavier, France); sodium laurylsulphate, batch No. 83H0238 (Sigma, 38297 Saint-Quentin-Fallavier, France) and vaseline, batch No. 0015 (Cooperative Pharmaceutique Frangaise, 77000 Melun, Prance). 2.2. TEST SYSTEM 2.2.1 Animals Species and strain: Dunkin-Hartley guinea-pigs. Reason for this choice: species recommended by the international regulations for sensitization studies. The strain used has been shown to produce a satisfactory sensitization response using known positive sensitizers. Breeder: Centre d'EIevage Lebeau, 78950 Gambais, France. Number: 30 animals (15 males and 15 females). Allocation of the animals to the groups: on day -1, the animals were weighed and randomly allocated to 2 groups: a control group 1 consisting of 10 animals (5 males and 5 females) and a treated group 2 consisting of 20 animals (10 males and 10 females). Weight: on day -1, the animals had a mean body weight of 359 17 g for the males and 345 26 g for the females. Acclimatization: at least 5 days before the beginning of the study. Identification of the animals: the animals were identified individually by an ear-tattoo. 2.2.2 Environmental conditions During the acclimatization period and throughout the study, the conditions in the animal room were as follows: . temperature: 21 2C . relative humidity: 30 to 70% . light/dark cycle: 12h/12h . ventilation: about 12 cycles/hour of filtered, non-recycled air. During the acclimatization period and throughout the study, the animals were housed indivi dually in polycarbonate cages (48 x 27 x 20 cm) equipped with a polypropylene bottle. Cali brated and dust-free sawdust was provided as litter (SICSA, 92142 Alfortville, Prance). An analysis of potential residues and major contaminants is performed periodically (Laboratoire Wolff, 92110 Clichy, France). 2.2.3 Food and water During the study, the animals had free access to 106 diet" (U.A.R., 91360 Villemoisson-surOrge, France). Food was periodically analysed (composition and contaminants) by the supplier. The diet formula is presented in appendix 2. Animals had free access to drinking water, filtered by a F.G. Millipore membrane (0.22 mi cron). Bacteriological and chemical analysis of the water and detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed periodically. Results are archived at C.I.T. . ^<;f*ft ffSf A POS5 rl0 W^San^-03 CIT/Study No. 12407 TSG----------------lf Atochem 9 There were no contaminants in the diet, water or sawdust at levels likely to have influenced the outcome of the study. 2.3. TREATMENT 2.3.1 Preliminary test A preliminary test was performed to define the concentration to be tested in the main study. By intradermal route Determination of the Minimum Irritant Concentration (M.I.C.): . 24 hours before treatment, the dorsal region of the animals was clipped, . the test substance was prepared in an appropriate vehicle, . intradermal administration of the test substance (volume 0.1 ml) at increasing concentrations was performed in order to determine the minimum concentration which causes an irritation, . evaluation of the potential cutaneous reactions, 24 and 48 hours after injection. By cutaneous route Determination of the Minimum Irritant Concentration (M.I.C.) and Maximum Non-Irritant Concentration (M.N.I.C.): . 24 hours before treatment, the dorsal region of the animals was clipped, . 0.5 ml of the test substance in its original form was applied to a dry gauze pad of. approxi mately 4 cm2 and then held in place by an occlusive dressing for 24 hours, . potential cutaneous reactions were evaluated 24 and 48 hours after removal of the gauze pads. 2.3.2 Main study 2.3.2.1 Preparation of the animals For all animals and'before each treatment, the application sites were: . clipped on days -1 and 7 (scapular area 4 cm x 2 cm), . clipped and shaved on day 21 (each flank 2 cm x 2 cm). 2.3.3 Induction phase by intradermal and cutaneous routes 2.3.3.1 Intradermal route On day 1, 6 intradermal injections were made into a clipped area (4 cm x 2 cm) in the scapular region, using a needle (diameter: 0.50 x 16 mm, Terumo: C.M.L., 77140-Nemours, France) mounted on at ml glass syringe (0.01 ml graduations, Record: Carrieri, 75005 Paris, France). Three injections of 0.1 ml were injected into each side of the animal, as follows: Control group (figure 1) . Freund's complete adjuvant diluted to 50% (0.9% NaCI), . vehicle, . a mixture of 50/50 (w/v) Freund's complete isotonic aqueous NaCI solution and the vehicle. (v/v) with a sterile isotonic saline adjuvant diluted to 50% (v/v) with solution a sterile A eoffita'sn TS Comp^r 5an^.D='"s r.s CIT/Study No. 12407 TSGJBBBI--------^A^tlofchem 10 Treated group (figure 2) . Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCI), test substance at a concentration of 1% (w/w) in the vehicle, . . a mixture 50/50 (w/v) of Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCI), and, the test substance at a concentration of 1% (w/w) in the vehicle. 2.3.3.2 Cutaneous route On day 7, the scapular area was clipped. As the test substance is shown to be non-irritant after occlusive cutaneous treatment during preliminary test, the animals were treated with 0.5 ml of sodium laurylsulphate (10%) in vaseline to provoke local irritation. On day 8, a cutaneous application on the 6 injection areas (4 cm x 2 cm) of the scapular region was performed. Control group . application of 0.5 ml of the vehicle. Treated group . application of 0.5 ml of a non-irritant concentration of the test substance i.e. in its original form. The test substance and the vehicle were prepared on a dry gauze pad (Semes Prance, 54183 Heillecourt, France), which was then applied to the scapular region and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicates, 92240 Malakoff, France). No residual test substance was observed at removal of the dressing. One hour after removal of the occlusive dressing, cutaneous reactions were recorded. 2.3.3.3 Challenge phase At the end of the rest period on day 22, the test substance was applied at the Maximum NonIrritant Concentration (M.N.I.C.) i.e. in its original form. On day 22, the animals from both groups received an application of 0.5 ml of the M.N.I.C. of the test substance on the posterior right flank, and 0.5 ml of the vehicle on the posterior left flank. This application was performed using a 1 ml plastic syringe (0.01 ml graduations, Terumo: C.M.L., 77140 Nemours, France). The test substance and vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Che nove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France). No residual test substance was observed at removal of the dressing. ^-.-"otc"ta!"TSCACB' Company sanl1"- CIT/Study No. 12407 TSGOB------I------PA^tochem 11 2.4. SCORING OF CUTANEOUS REACTIONS Twenty-four and 48 hours after removal of the dressing from the challenge application site, the both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions, according to the following scale: Erythema and eschar formation .No erythema ........................................................................................................................... 0 . Very slight erythema (barely perceptible) .............................................................................. 1 . Well-defined erythema ........................................................................................................... 2 . Moderate to severe erythema 3 ................................................................................................ . Severe erythema (beet redness) to slight eschar formation (injuries in depth)........................ 4 Oedema formation No oedema ............................................................................................................................. 0 . Very slight oedema (barely perceptible) ................................................................................ 1 . Slight oedema (visible swelling with well-defined edges)...................................................... 2 . Moderate oedema (visible swelling raised more than 1 millimetre)........................................ 3 . Severe oedema (visible swelling raised more than 1 millimetre and extending beyond the area of exposure) ....................................................................................................4 Any other lesions were noted. 2.5. CLINICAL EXAMINATIONS The animals were observed twice a day during the study in order to record clinical signs and to check for mortality: 2.6. BODY WEIGHT The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1), then on days 8, 15 and 25. 2.7. PATHOLOGY 2.7.1 Necropsy On day 25, after the 48-hour observation period, the animals were killed by C0 inhalation in excess. 2.7.2 Cutaneous samples On day 25, no skin samples were taken. 2.7.3 Microscopic examination No histological examinations were performed, ^^85nTS . QOSStt" -./.a f,Ql ^ ^ ^ s&s-"3U- r CIT/Study No. 12407 TSGpH------------MAEtIofchem 12 2.8. DETERMINATION OF THE ALLERGENICITY LEVEL The treated animals show a positive reaction if macroscopic cutaneou? reactions are clearly visible (erythema and/or oedema >. 2) and more marked than the most severe reactions of the control animals, or, if "doubtful" macroscopic reactions are confirmed at microscopic examination as being due to the sensitization process. Sensitization reactions are characterized at microscopic examination by basal spongiosis, reactional acanthosis of the epidermis and infil tration of mononucleated cells into the dermis (1). Determination of the allergenicity level The allergenicity level of the test substance is calculated by comparing the number of animals showing positive reactions with the number of surviving treated animals at the end of the study. % of animals showing a reaction Allergenicity level Classification 0 - 8 I 9 - 28 n 29 - 64 in 65 - 80 rv 81 - 100 V very weak weak moderate strong very strong According to the E.E.C. directive 93/21/E.E.C. published in the Journal Officiel des Communautes Europeennes, when the reactions are positive in at least 30% of the treated animals, the test substance has sensitization properties and the sentence "R 43: May cause sensitization by skin contact" must be applied. (1) Duprat, P. ; Delsaut. L. ; Gradiski, D. ; Lepage, M. : Investigations histo-pathologiques et cytologiques lors de la mise en evidence, chez Ie cobaye, d'une allergic cutanee de type retarde. Revue Med. Vet. 127: 7, 1083-1101 (1976). ^ contain TSCA CM Company Sam., tW, ---- CIT/Study No. 12407 TS 2.9. SUMMARY DIAGRAMS Figure 1: control group Chronology IfAtochem Day-1 Clipping of the scapular region Day 1 Day? Intradermal injection Clipping + Sodium laurylsulphate Day 8 Application covered by an occlusive dressing Day 10 Removal of dressing and scoring after one hour Day 21 Clipping and shaving of the flanks Day 22 Challenge application covered by an occlusive dressing Day 23 Day 24 Day 25 Removal of dressing First scoring Second scoring, sacrifice of the animals 13 Induction site 1-- 2 intradennal injections 3_ cutaneous application (4 cm x 2 cm) Challenge application cutaneous application (2 cm x 2 cm) Intradermal injections 0 1 - 50% Freund's complete adjuvant and Nad at 0.9% solution ^ 2 vehicle 0 3 - 1 + 2,50/50 (w/v) ant^"11 COWP5^ Sa . ^ n*.^-- . ^ ^ CIT/Study No. 12407 TS IfAtochem 14 Figure 2: treated group Chronology Day -1 Clipping of the scapular region Day 1 Day 7 Intradermal injection Clipping + Sodium laurylsulphate Day 8 Day 10 Application covered by an occlusive dressing Removal of dressing and scoring after one hour Day 21 Clipping and shaving of the flanks Day 22 Challenge application covered by an occlusive dressing Day 23 Removal of dressing Day 24 First scoring Day 25 Second scoring, sacrifice of the animals 0 @ (g) Vehicle Induction site 0 1 - @ 2 intradermal injections 3 cutaneous application (4 cm x 2 cm) Test substance at a concentration o/700% /Challenge application cutaneous application (2 cm x 2 cm) 0 Intradermal injections ^ 0 i 50% Freund's complete adjuvant and Nad at 0.9% solution 2 test substance at a concentration of 1% (w/w) in the vehicle 3 1 + 2,50/50 (w/v) Company Samdzee;. Dsss not contain TSCA CE! CIT/Study No. 12407 TS If Atochem 2.10. CHRONOLOGY OF THE STUDY The chronology of the study is summarized as follows: Procedure Arrival of the animals Allocation of the animals into groups Weighing, induction by intradermal injection Laurylsulfate application Weighing, induction by cutaneous route Removal of occlusive dressings and scoring of local reactions after 1 hour Weighing Challenge cutaneous application Removal of occlusive dressings Scoring of cutaneous reactions after . 24 hours . 48 hours Weighing, sacrifice of the animals Date 1.12.94 8.12.94 9.12.94 15.12.94 16.12.94 18.12.94 23.12.94 30.12.94 31.12.94 1.1.95 2.1.95 2.1.95 15 Day -8 -1 1 7 8 10 15 22 23 24' 25 25 2.11. ARCHIVES The study archives: . protocol and possible amendments, . raw data, . correspondence, . final study report and possible amendments, are stored on the premises of C.I.T., Miserey, 27005 Evreux, France, for 5 years after the end of the in vivo study. At the end of this period, the study archives will be returned to the Sponsor. SamW,D^not^NnTSCACB5 Company CU/Study No. 12407 TS ,lf Atochem 16 3. RESULTS 3.1. PRELIMINARY STUDY 3.1.1 Administration by intradermal route Several tests were performed to determine the minimal irritant concentration which did not provoke necrosis or ulceration. Animal number Concentration of the test substance % (w/w) Scoring after treatment 24 hours 48 hours Male No. 01 5 1 0.1 irritation irritation irritation irritation irritation irritation Female No. 01 5 1 0.1 irritation irritation irritation Concentration used in the main study was 1% (w/w). 3.1.2 Application by cutaneous route irritation irritation irritation Animal number Concentration of the test substance Scoring 24 hours Scoring 48 hours after removal of the dressing (1) E 0 E 0 Male No. 01 100 RF LF Female No. 01 100 RF 0 LF 0^ M.N.I.C. is 100% of the test substance. E erythema 0 oedema RF right flank LF left flank (1) no residual test substance was observed. CoiBpan"ys Sanitized. Does noi coniain TSCA CM CIT/Study No. 12407 TSC^|BB--------||EIfAtochem 17 3.2. MAIN STUDY 3.2.1 Clinical examinations No clinical signs or mortalities were observed during the study. The body weight gain of the treated animals was normal when compared to that of the control animals (figures 3 and 4, appendix 3). 3.2.2 Scoring of cutaneous reactions Observations of cutaneous reactions are presented in appendix 4. 3.2.2.1 End of the induction period On day 10, after removal of me dressing, signs of irritation in control and treated groups were observed at the intradermal injection sites. 3.2.2.2 Challenge application No cutaneous reactions were observed 24 and 48 hours after removal of the dressing of the challenge cutaneous application of the test substance, 4. CONCLUSION Under our experimental conditions and according to the maximization method established by Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substancdHBll----liHHHin its original form were observed in guinea-pigs. ^\ .^contai"^04 ' ^^Dos3 CIT/Study No. 12407 TSG--------------RElf Atochem 19 Figure 4: Female body weight gain (g) B^notoo"-.*^081 Company SaM. i",*01'- CIT/Study No. 12407TS If Atochem 20 APPENDICES ^~\ ^oo^5^ san^d.^'--0 Co^^.58-- - CIT/Study No. 12407TSGp----------iBfclfAtochem 21 1. Analytical certificate ^\ ^^s^c6l .nol""""" ^s^^-00"' CoTOT11"1 - CIT/Study No. 12407TS IfAtochem 22 ^onfcrole laboratoire N" 57/176. Bif-Atochero. ATELIER FORAFAC DE OISSEL LE 29/7/94. A. 1'afcfcention do MMB VAESKEN. (D.C.F.S) de MR DELLB CASE. (V.S.P) Les controles ana^ytiquea et \QB teats applioatifs ef fee-hues sur 1'operation Buivantel 176 de|flBBH----HP|ont ^ ^ ctonn<5 lea r6sultats ft MATIBRB SECHE a TftDX D'BTHAMOlj * TAUX D'BAU 6 TEMSION 80PERPICIBLLE i & 1000 pprn on oau bidiefca-.lldo apr^s 30 minutes d'^quilibre & 1^ 25C 27.0 % 34 -0 % 390 % (Norme i de 26.5% & 27%) (Norroe ; de 32.0% & 35.0%) (Norrne i de 38.0% & 41.S^) 15.35 niTO/to (Norrne ; < 16 mN/m }" * PODVOIR MOUB8ANT & 25C) en eau de roer. aprfee 30 e'eoondee; api-<Ss 3 minufcea i apr^a 5 roinufces t 290 ral 280 ml 2ft9. rol (Norroe : > 150 ml ) (Norme : > 150 ml ) (Norme i > 150 ml ) ^ a MASSE VOLOMIQUE & 25C i 1.039 kg/m3 (Norme : 1.038+/-0.010 kg/ro3 Lee valeurs donn^es par lea fcestB applicafcifs (y compris les tests d'^fcaleraent) sorA conformea aux norroes. L'op6rafcion 176 de-| i- ^ est commercialisable. & MR GARCIA G. (CAD 6 MR GUILLAOD JL/Mn FREMy ft MR DURUAL P. Salutations. //?^/(- MORfiAU Jean-Francois. >ICOO^' ;a^ osP'.,^8ac' 00^ fSC^ CIT/Study No. 12407 TS Atochem 23 2. Diet formula ^^NnTSCAGffl co^-^" CIT/Study No. 12407 TS Atochem 24 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 libitwn. FORM^ULAA%% CerealIss .................................... 42 Grain biproducts and lleegguummeess...... 46 Vegetaable protein (soya bean meal, yeast) ............................ 9 Vitamuin and mineral mixttuurree.. 3 AVER^AAGGE ANALYSIS % fie value (KCal/kg) ..... ..... ure ............................... ..... ns ................................. ..... hydrates (N.F.E.) .......... ..... als (ash) ....................... 2600 10 17 3 49 13 8 0 ACID VALUES ated in mg/kg) MINERALS (calculated in mg/kg) Nat. CMV val. val. Total P.............. Ca K............. Na Mg.......... Mn Fe Cu .......... Zn Co I Cl.............. ........... 7400 5400 12000 1300 3270 60 170 10 40 0.1 0 0 1400 5600 0 1950 130 40 150 15 45 1.5 0 0 8800 11000 12000 3250 3400 100 320 25 85 1.6 0 0 VITAMINS (calculated perkg) Nat. CMV val. val. Total Argininnee .................................. .... Cystinee .................................... .... Lysine .. .... Methio)nniinne ............................. .... Tryptopphhan ............................ .... Glycinee ................................... .... 8500 2500 7200 2100 2000 6000 f ACID VALUES ated in mg/kg) c acid ........................... .... 3600 )leic acid ...................... 0 acid .............................. ... 700 cid ................................ ... 5900 c acid ........................... ... 11200 ic acid ......................... ... 3000 Vitamin A Vitamin D3 Vitamin B 1 Vitamin B2 Vitamin B3 Vitamin B6 Vitamin B 12 Vitamin C Vitamin E Vitamin K3 Vitamin PP Folic acid P.A.B. acid Biotin Choline vteso-Inositol 3500 IU 30 IU 6mg 5mg 22 mg 0.7 mg 0.003 mg Omg 15 mg 5mg 97 mg 2.2 mg Omg 0.02 mg 1010 mg Omg 7500 ru 2000 IU 6.4 mg 6.4 mg 26 mg 2.7 mg 0.012 mg 400 mg 60 mg 12.6mg 14.5 mg1.3mg 2.5 mg 0.06 mg 60 mg 62.5 mg 11000IU 2030 IU 12.4 mg 11.4mg 48 mg 3.4 mg 0.015mg 400 mg 75 mg 17.6mg 111.5mg 3.5 mg 2.5 mg 0.08 mg 1070 mg 62.5 mg This food is supplemented with stabilized coated vitamin C, avoiding the need of other food substances (greenery, ascorbic acid) if used within 4 months of date of manufacture. U.A.R., 7 rue Gallieni, 91360 Villemoisson (Ref. Doc. UAR: 1992) Tel: 69.04.03.57 - Fax : 69.04.81.97 w^y- ISCftCBl eonte1" I I-IQSS,S,,,0-A' err/Study No. 12407 TSd|--------HH||EIf Atochem 25 3. Individual body weight values , ^ t B ^ . ..,-sd.^0'1""" Sot^5^ CIT/Study No. 12407TS If Atochem 26 Groups Sex 1 Male Female 2 Male Female (1) = Body weight gain M = Mean on -- c**^^-/i na<n'^f;^M INDIVIDUAL BODY WEIGHT VALUES (g) AnimalsI Days '-------- -1 ^------------------------------------------------------------------------------------------''--------------------------------------------""-- " 1 (1) 8 (1) 15 (1) 25 111 342 351 61 412 64 476 62 538 112 375 382 83 465 63 528 82 610 113 370 376 38 414 70 484 76 560 114 369 385 67 452 60 512 72 584 115 335 346 59 405 37 442 70 512 M 358 368 62 430 59 488 72 561 SD 18 18 16 27 13 33 7 38 126 334 331 50 381 41 422 54 476 127 301 317 72 389 34 423 73 496 128 307 308 57 365 50 415 79 494 129 328 325 44 369 29 398 54 452 130 316 320 57 377 61 438 53 491 M 317 320 56 376 43 419 63 482 SD 14 9 10 10 13 15 12 18 116 358 369 43 412 33 445 41 486 117 344 364 43 407 56 463 59 522 118 360 371 64 435 37 472 49 521 119 367 372 49 421 63 484 64 548 120 317 337 63 400 62 462 111 573 121 348 343 50 393 47 440 51 491 122 336 338 67 405 44 449 68 517 123 336 337 73 410 45 455 56 511 124 341 350 76 426 52 478 85 563 125 347 359 62 421 36 457 81 538 M 345 354 59 413 48 461 67 527 SD 14 15 12 13 11 14 21 29 131 317 321 40 361 54 415 70 485 132 364 363 70 433 50 483 26 509 133 393 399 48 447 69 516 68 584 134 367 368 . 36 404 38 442 47 489 135 349 342 70 412 30 442 48 490 136 352 354 56 410 33 443 61 504 137 345 344 85 429 15 444 49 493 138 368 370 68 438 18 456 32 488 139 339 329 31 360 12 372 35 407 140 385 378 62 440 27 467 52 519 M 358 357 57 413 35 448 49 497 SD 22 24 17 31 18 38 15 43 company Sanifesd. Doss nst contain TSCA CB! CIT/Study No. 12407 TSC{|--------------A|t^oclfhem 27 4. Individual observation of cutaneous reactions 'i-\ , ^^ainTSCAA Compaq S^^0'- CIT/Study No. 12407TS ,lf Atochem 28 MACROSCOPIC EXAMINATION OF CUTANEOUS REACTIONS Challenge application Group Sex Animals; Control 1 Male Female Treated 2 Male ,^ Female LF: left flank (control) RF: right flank (treated) 111 112 113 114 115 126 127 128 129 130 116 117 118 119 120 121 122 123 124 125 131 132 133 134 135 136 137 138 139 140 Da:Y 24 sec)dng pe:nod (after 2 4 hours;) Erytltiema Oedlema LF RF LF RF 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -0 0 0 0 0 0 0 0 0 0 0 0 0 Da:y 25 sec)ring pe;riod (after A1-8hours0 Eryt hema LF RF Oedlema LF RF 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Company SanlM. Does not contain TSCA CB1 5. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs v\ Company Sanife^ D^s "^ co"^" TSCA CBf CrT/Study No. 12407 TS If Atochem 30 Purpose; check the sensitivity of Dunkin-Hartley Guinea-pigs (Centre d'elevage Lebeau) to a positive control test article Method Magnusson and KUgman' Test substance : DINTTRO 2.4 CHLOROBENZENE C.I.T. Study - Date : Number of animals : December 1994 (CIT/Study No. 12437 TSG) 20 females Induction : 0.1 % intradennal route day 1 5% cutaneous route day 8 Challenge application : 1% right flank paraffin oil left flank Conclusion Under our experimental conditions and according to the Magnusson and Kligman method, DINTTRO 2.4 CHLOROBENZENE at a concentration of 1% (w/w) induced positive skin sensitization reactions in 95% of the guinea-pigs. INDIVIDUAL REACTIONS: CHALLENGE PHASE MACROSCOPIC FINDINGS Group Treated Sex Animals Female ^11 ' 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 24-hour scoring period Erythema LF RF Oedema LF RF 0 2 0 2 0 1 0 3 0 2 0 4 0 3 0 2 0 2 0 3 0 3 0 3 0 2 0 3 0 2 0 3 0 2 0 3 0 3 0 3 0 0 0 0 0 0 0 2 0 2 0 2 0 2 0 0 0 0 0 2 0 2 0 2 0 0 0 2 0 0 0 2 0 0 0 2 0 2 0 2 48-hour scoring period Erythema LF RF Oedema LP RF 0 2/S 0 0 0 2/S/A 0 0 0 1/S 0 0 0 3/S 0 0 0 2/S 0 0 0 4 0 0 0 1/S 0 0 0 2/S 0 0 0 2/S 0 0 0 3/S 0 2 0 3/S 0 0 0 3/S - 0 0 0 1/S 0 0 0 2/S 0 0 0 2/S 0 0 0 2 0 0 0 1/S 0 0 0 2 0 0 0 3/S 0 2 0 3/S 0 2 Conclusion LP RF - + - + - +/- - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + negative + hypersensitizing reaction +/- borderline reactions S drynessoftheskin A crust LF left flank RF right flank Does n si contain TSCA Compaq 5"^"'