Document qdYBbyxKa9me0EwajQ5M0rmQk

SPONSOR Elf Atochem S.A. La Dfense 10 - Cdex 42 92091 Paris-la-Dfense France STUDY TITLE SKIN SENSITIZATION TEST IN GUINEA-PIGS (Maximization method of Magnusson, B. and Kligman, A.M.) TEST SUBSTANCE STUDY DIRECTOR Stphane de Jouffrey STUDY COMPLETION DA TE 24th March 1995 PERFORMING LABORA TOR Y Centre International de Toxicologie (C.I.T.) Miserey - 27005 Evreux - France LABORATORY STUDY NUMKER 12407TSG AR226-3013 ULVO-,1'i JL1H II Company Sanitized. Does not contain TSCA CBI err/StudyN o. 12407 TS1 ilf Atochem 2 CONTENTS STATEMENT OF THE STUDY DIRECTOR OTHER SCIENTISTS INVOLVED IN THIS STUDY STATEMENT O F QUALITY ASSURANCE UNIT SUMMARY 4 4 5 6 1. INTRODUCTION 7 2. MATERIALS AND METHODS 2.1.TEST AND CONTROL SUBSTANCES 2.1.1 Test substance 2.1.2 Vehicle 2.1.3 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 Cutaneous route 2.3.3.3 Challenge phase 2.4. SCORING OF CUTANEOUS REACTIONS 2.5. CLINICAL EXAMINATIONS 2.6. BODY WEIGHT w- 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 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 Saniti: Does not contain TSCACIlf CIT/StudyNo. 12407 TS' ilf Atochem 2.11. ARCHIVES 3. RESULTS 3.1. PRELIMINARY STUDY 3.1.1 Administration by intradermal route 3.1.2 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 observationof cutaneous reactions 5. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs 3 15 16 !6 16 16 17 17 17 17 17 i7 lg. 19 20 '2 1 23 25 27 29 and 30 oniain QQttSl^ Sawt1f*ed.B esn0tC ' err/Study No. 12407 TS If 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 CIT/Study No. 12407 TS Ilf Atochem 5 STATEM ENT O F QUALITY ASSURANCE UNTT 1. Specific study 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 Dates (day/month/year) Inspections 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. Head of Quality Assurance Unit and Scientific Archives Compaq noi conWi TSCA CB* CIT/Study No. 12407 TS< ilf Atochem 6 SUMMARY , At the request ofjglf Atochem S.A., Paris-la-Defense, France, the potential of the test substance, induce delayed contact hypersensitivity following intradermal 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% NaCl) (control group) or the test substance (treated group). On day 1, in presence of Freund's complete adjuvant, 0.1 ml of the test substance at a concentration of 1% (w/w) in the vehicle was admi nistered by intradermal 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 Magnusson antM O gm anjiocutaneous reactions attributable to the sensitization potential oftha. test Jiinn tift\sg norriitgriinnnall ffnorrmm were observed in guinea--- - t s c a CWi A tJO5 r'^ ***** CIT/StudyNo. 12407 TSi ilf Atochem 7 1. INTRODUCTION The objective of this study, performed according to maximization method established b\ (i*M amusson and Kligman (i), was to evaluate the potential of the test substance,^ T H R o induce delayed contact hypersensitivity in guinea-pigs. The results of the study are of value in predicting the contact sensitization potential of the test material in Man. During the induction period, the test substance was administered by intradermal 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. 4 0 6 ,17th July 1992. . E.C. Directive No. 92/69/E.E.C., B6, 31st July 1992. 2. MATERIALS AND METHODS 2.1. TEST AND CONTROL SUBSTANCES 2.1.1 Test substance The test substance. ised in the study was supplied by Elf Atochem S.A. Documentation supplied by the Sponsor identified the test substance as follows: . denominatii - protocoll - labelling!j . batch numb - protocol:) - labelling!!_____________ . description: brownish liquid . container: 1plastic flask . date of receipt: 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% NaCl), batch No. 3126 (Biosedra, 92240 Malakoff, France). (1) Magnusson, B.; Kligman, A.M.: The identification of contact allergens by animal assay. The guinea-pig maximization test. J. Invest. Derm. 52: 268-276 (1969). contain 1 SCACE ooes CIT/StudyNo. 12407 TS' If Atochem 8 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 (Cooprative Pharmaceu tique Franaise, 77000 Melun, France). 2.2. TEST SYSTEM 2.2.1 Animais 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 'Elevage 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 o f 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, France). 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-sur- Orge, 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.LT. CIT/Study No. 12407 TS If 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 o f 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 a l ml glass syringe (0.01 ml graduations, Record: Carried, 75005 Paris, France). Three injections of 0.1 ml were injected into each side o f the animal, as follows: Control group (figure 1) . . Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCI), . vehicle, . a mixture of 50/50 (w/v) Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCI solution and the vehicle. Compaq CIT/Study No. 12407 TS< ilf Atochem 10 Treated group (figure 2) . Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCl), . 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 o f 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 France, 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'Hygine, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Labora toire des Professions Mdicales, 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 Non Irritant 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, Trumo: 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 cm (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'Hygine, 21300 Che nove, France) and an adhesive anallergenic waterproof plaster (Laboratoire dtes Professions Mdicales, 92240 Malakoff, France). . No residual test substance was observed at removal of the dressing. InTSCACB CIT/Study No. 12407 TSA a m m m m l E i f Atochem U Jk 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-definededges)......................................................... 2 . Moderate oedema (visible swelling raised more than 1millimetre).............................................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 C 0 2 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. CIT/Study No. 12407 TSG :if Atochem 12 2.8. DETERMINATION OF THE ALLERGENICITY LEVEL The treated animals show a positive reaction if macroscopic cutaneous reactions are clearly visible (erythema and/or oedema > 2) and 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. % o f animals showing a reaction Allergenicity level Classification 0- 8 I very weak 9 - 28 n weak 29 - 64 m moderate 65 - 80 IV strong 81 - 100 V very strong According to the E.E.C. directive 93/21/E.E.C. published in the Journal Officiel des Commu nauts Europennes, 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. (!) Duprat, P. ; Delsaut. L. ; Gradiski, D. ; Lepage, M. : Investigations histo-pathologiques et cytologiques lors de la mise en vidence, chez le cobaye, d'une allergie cutane de type retard. Revue Md. Vt. 127: 7, 1083-1101 (1976). Company Saniti^ ol CO'nisln TSCA CBf CIT/Study No. 12407 TSG 2.9. SUMMARY DIAGRAMS Figure 1: control group Chronology ;if Atochem Day -1 Clipping of the scapular region Day 1 Intradermal injection Day 7 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 Removal of dressing Day 24 First scoring Day 25 Second scoring, sacrifice of the animals 13 Induction site intradermal injections cutaneous application (4 cm x 2 cm) Challenge application cutaneous application (2 cm x 2 cm) intradermal injections O 1 50% Freund's complete adjuvant and NaCl at 0.9% solution vehicle o 3 - 1+ 2,50/50 (w/v) C o P,a*y ,,.,,,,y. DOS3.W,, o^ TSCACE CIT/Study No. 12407 TS If Atochem 14 Figure 2: treated group Chronology Day -1 Clipping of the scapular region Day 1 Intradermal injection Day 7 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 Removal of dressing Day 24 First scoring Day 25 Second scoring, sacrifice of the animals intradermal injections Intradermal injections o o 50% Freund's complete adjuvant and NaCl at 0.9% solution test substance at a concentration of 1% (w/w) in the vehicle 1 + 2,50/50 (w/v) Companwy Sanitized. Dess not contain TSCA CBJ CIT/Study No. 12407 TS' ilf 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 intraderm al 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. Company Sanitized, Ppes not ontain TSCA CBl CIT/Study No. 12407 TS< 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 irritation irritation irritation Concentration used in the main study was 1% (w/w). 3.1.2 Application by cutaneous route Animal number Male No. 01 Female No. 01 Concentration of the test substance % 100 RF LF 100 RF LF Scoring 24 hours Scoring 48 hours after removal of the dressing ( 1) EO EO 00 00 00 00 00 0^ 0 00 00 N.I.C. is 100% of the test substance. : erythema : oedema : right flank - LF: left flank ' ( 1) no residual test substance was observed. S WO S Sanitized. Dos noi coiniain TSCGift Company CIT/Study No. 12407 TS< ilf Atochem 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 o f 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 the 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 su b sta n c d E H H H IH H H H in its original form were observed in guinea-pigs. CoTRP3^ ^ c o n l^ rS C A CIT/Study No. 12407 TSG ilf Atochem Figure 4: Female body weight gain (g) 19 ySCACBl Company S an$^ D o e . * * * * CIT/StudyNo. 12407 TS' I ilf Atochem 20 APPENDICES CQJP;m l Does c,CAC CIT/Study No. 12407 TSG ilf Atochem 21 1. Analytical certificate Saw GoiW?: pos fS 1 CnVStudyNo. 12407 TS' ilf Atochem 22 Controle laboratoire N 57/176. . Blf-Atochem, ATELIER FORAFAC DE OISSEL LE 29/7/94. A l'attention de MME VAESKEN. (D.C.F.S) de MR DELLE CASE. (V.S.P) Les controles anal y tiques et sur l'opration 176 de esuivantsi ;s tests applicatifs ffectus ont donn les rsultats * MATIERE SECHE a TAUX D 'ETHANOL * TAUX D'EAU * TENSION SUPERFICIELLE : A 1000 ppm on oau bidiefcillde aprs 30 minutes d 'quilibre 25C . 27.0 % 34.0 % 39.0 % (Norme i de 26,5% 27%) (Norme : de 32.0% 35.0%) (Norme i de 38,0% 41.5%) 15.35 mN/rn (Norme < 16 mN/m }' * POUVOIR MOUSSANT 25Ci en eau de mer. _ aprs 30 secondes: aprs 3 minutes i aprs 5 minutes : 290 ml 280 ml 2&R ml (Norme : > 150 ml ) (Norme : > 150 ml ) (Norme i > 150 ml ) : MASSE VOLUMIQUE 25 C i 1.039 kg/m3 (Norme : 1.03B+/-0.010 kg/m3 Les valeurs donnes par les tests applicatifs (y compris les tests d'talement) sont conformes aux normes. L ' opration 176 est commercialisable. Salutations. Oat**- MORfiAU Jean-Franois. popie MR GARCIA G. (CAL) MR GUILLAD JL/MR FREMV MR DRUAL P. n 0^cO 5WS a ^,^ e - c S CIT/Study No. 12407 TS< If Atochem 2. Diet formula yj.D ocane' 0"**11 C o s tip a San CIT/Study No. 12407 TS< ilf Atochem 24 Ref: 106 COMPLETE DIET GUINEA-PIG MAINTENANCE DIET Appearance: 4.5 mm diameter granules Conditioning: bags o f 25 kgs Daily portion: Guinea-pigs 35-50 g, water ad libitum . FORMULA % MINERALS (calculated in mg/kg) Nat. CMV C ereals............................................ 42 vai. val. Total Grain biproducts andlegumes...... 46 Vegetable protein (soya bean meal, yeast) ............................ 9 Vitamin and mineral m ixture................3 AVERAGE ANALYSIS % Calorific value (K C al/kg).......... 2600 Moisture ......................................... 10 Proteins........................................... 17 L ip id s............................................... 3 Carbohydrates (N.F.E.)............... 49. p ............. Ca ........... K .... ........ Na .......... M g.. ........ M n .......... 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 Fibre ................................................ 13 Minerals (ash) ........................... 8 VITAMINS (calculated per kg) AMINO ACID VALUES (calculated in mg/kg) Nat. CMV val. val. Total A rginine..............*......................... 8500 Vitamin A Vitamin D3 C ystine......................................... 2500 Vitamin B 1 L y sin e .......................................... 7200 _ Vitamin B2 M ethionine................................... 2100 ^ Vitamin B3 3 5 0 0 IU 3 0 IU 6 mg 5 mg 22 mg Tryptophan .................................. 2000 Vitamin B6 0.7 mg Glycine ........................................ 6000 Vitamin B12 0.003 mg Vitamin C 0 mg FATTY ACID VALUES Vitamin E 15 mg (calculated in mg/kg) Vitamin K3 Vitamin PP 5 mg 97 mg Palmitic a c id ................................ 3600 Palmitoleic acid ........................... 0 Stearic acid................................... 700 Oleic a c id ..................................... 5900 L inoleicacid.................................11200 Folic acid 2.2 mg P.A.B. acid 0 mg Biotin 0.02 mg Choline 1010 mg Meso-Inositol 0 mg Linolenic a c id .............................. 3000 7500 IU 2000 IU 6.4 mg 6.4 mg 26 mg 2.7 mg 0.012 mg 400 mg 60 mg 12.6 mg 14.5 mg1.3 mg 2.5 mg 0.06 mg 60 mg 62.5 mg 11000IU 2030 IU 12.4 mg 11.4 mg 48 mg 3.4 mg 0.015 mg 400 mg 75 mg 17.6 mg 111.5 mg 3.5 mg 2.5 mg 0.08 mg 1070 mg 62.5 mg This food is supplemented with stabilized coated vitamin C, avoiding the need of other food substances (greenery, ascorbic acid) if used within 4 months of date of manufacture. U.A.R., 7 me Gallini, 91360 Villemoisson - Tel: 69.04.03.57 - Fax : 69 04 81 97 (Ref. Doc. UAR: 1992) . ~ s c A CBl pass*n0\ c a n ta i 1 goimpan Saniti*6** - c rr/stu d y No. 12407 TS> 0 ilf Atochem 25 ')) Individual body weight values GoIBP8*^ tain * CIT/StudyNo. 12407 TS' If Atochem 26 Groups Sex INDIVIDUAL BODY WEIGHT VALUES (g) Animals -1 Days ' 1 (1) 8 (1) 15 (1) 25 1 Male 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 Female 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 2 18 2 Male 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 68 517 123 336 537 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 Female 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 (1) = Body weight gain M = Mean on _ n, Company Sanilizsd. Doss not contain TSC CBS CIT/StudyNo. 12407 TS If Atochem 27 4. Individual observation of cutaneous reactions Do^nolcorA ainlSC A C Si * Sanded. CompsftV San r LCrr/StudyNo. 12407 TS' If Atochem MACROSCOPIC EXAMINATION OF CUTANEOUS REACTIONS Challenge application 28 Group Sex Animals Control Male 1 Female Treated Male 2 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 Day 24 scoring period (after 24 hours) Erythema Oedema LF RF LF RF 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 0 00 00 00 00 00 00 00 00 00 0 -0 00 00 00 00 00 00 Day 25 scoring period (after 48 hours) Erythema Oedema LF RF LF RF 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Doss n o i contain TSCA CB1 CIT/Study No. 12407 TS' Ilf Atochem 29 5. Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs i illp Company Sanitized. Doss not contain TSCA CBI CIT/Study No. 12407 TS< ilf Atochem 30 Purpose: check the sensitivity of Dunkin-Hartley Guinea-pigs (Centre d 'levage Lebeau) to a positive control test article Method Magnusson and Kligman' Test substance DINTTRO 2.4 CHLOROBENZENE C.I.T. Study - Date December 1994 (CIT/Study No. 12437 TSG) Number of animals 20 females Induction 0. 1% 5% intradermal route day 1 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 sensi tization reactions in 95% of the guinea-pigs. Group Treated INDIVIDUAL REACTIONS: CHALLENGE PHASE MACROSCOPIC FINDINGS Sex Animals Female - ^sll ' 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 24-hour scoring period Erythema Oedema LF RF LF RF 02 02 01 03 02 04 03 02 02 03 03 3 02 03 02 03 02 03 03 03 00 00 00 02 02 02 02 00 00 02 02 02 00 02 00 02 0'0 02 02 02 48-hour scoring period Erythema Oedema LF RF LF RF Conclusion LF RF 0 2/S 0 0 2/S/A 0 0 1/S 0 0 3/S 0 0 2/S 0 04 0 0 1/S 0 0 2/S 0 0 2/S 0 0 3/S 0 0 3/S 0 0 3/S - 0 0 1/S 0 0 2/S 0 0 2/S 0 02 0 0 1/S 0 02 0 0 3/S 0 0 3/S 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 2 2 -+ - +/-+ -+ -+ -+ -+ -+ - -+ - 4-+ -+ - -+ -+ - : negative + : hypersensitizing reaction + /-: borderline reactions S : dryness of the skin A : crust L F : left flank R F : right flank ^ contain TSCACB1 .Does Company SanKL