Document 0JVddrO0wvq0q04OQa173Ddpm

5) OECD 406-OPPTS 870.2600, Skin sensitization (guinea pig), 132-015 Study Number: 132-015 Page 1 DELAYED CONTACT HYPERSENSITIVITY STUDY OF T-7485 IN HARTLEY GUINEA PIGS (MAXIMIZATION TESTI) STUDY NUMBER: 132-015 SPONSOR 3M Corporate Toxicology 3M Center, Building 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3320 Telephone: 651-733-2501 FAX: 651-737-5506 U V4NITIZED DEC 0 9 2003 SPONSOR'S REPRESENTATIVE TESTING FACILITY Redfield Laboratories Mail: P.O. Box 308 Deliveries: 100 East Boone St. Redfield, AR 72132 Telephone: 501-397-2540 FAX: 501-397-2002 STUDY DATES Study Initiation: December 7,2001 Animal Phase Initiation: December 27, 2001 Animal Phase Completion: April 2,2002 Study Completion: September 20,2002 Study Number: 132-015 Page 2 GLP COMPLIANCE STATEMENT Study Number: 132-015 I certify that this study was performed in compliance with the United States Food and Drug Administration (FDA) Good Laboratory Practice Regulations (21 CFR Part 58) and C(97)186 (Final) (OECD) and that the report accurately reflects the raw data. Study Director Redfield Laboratories Study Number: 132-015 Page 3 QUALITY ASSURANCE STATEMENT Study Number: 132-015 This study has been inspected and audited by the Quality Assurance Unit (QAU) as required by the Good Laboratory Practice (GLP) Regulations (21 CFR Part 58) promulgatedby the U.S. Food and Drug Administration and C(97)186 (Final) (OECD). The followingis a record of the dates that auditslinspectionswere performed and reported by the QAU. I ~ I I I DATEOF DATESREPORTED TO TYPEOF AUDIT/INSPECTION STUDY DIRECTOARND AUDIT/INSPECTION MANAGEMENT 12/07/01 Protocol 12127101 ' Intradermal Dosing Phase 01110/02 Clinical Observation Phase 0111 1/02 Amendment #1 04/20/02, 04/22/0204/25/02 In-Life Raw Data 04/25/02 Formulations 04/30/02,05/02/02 Tables 05113/02 Amendment #2 05116/02 Individual Animal Necropsy Records 06111102-06/12/02 I Draft Report I 09117 / 0 2 Final Report 12/07/01 12/27/01 01/10/02 01/11/02 04/25/02 04/25/02 05/03/02 05/13/02 I 05116/02 06112/02 I 09117 / 0 2 1 The report accurately reflects the raw data. APPROVED BY: I l/yJJJ L 9 +-I I b O / O L Melissa Milligan, B.S., M.T., CLSp(CG) Date Quality Assurance Auditor Redfield Laboratories Study Number: 132-015 Pam 4 TITLE DELAYED CONTACT HYPERSENSITIVITY STUDY OF T-7485 IN HARTLEY GUINEA PIGS (MAXIMIZATION TEST') APPROVED BY: n John Sgng, Ph.D.' Date Study Director . Redfield Laboratories Study Number: 132-015 Page 5 TABLE OF CONTENTS PaRe Cover Page ........................................................................................................................... 1 GLP Compliance Statement................................................................................................. 2 Quality Assurance Statement............................................................................................... 3 Title and Signature Page ...................................................................................................... 4 Table of Contents................................................................................................................. 5 Study Abstract...................................................................................................................... 6 Objective.............................................................................................................................. 8 Materials and Methods......................................................................................................... 8 Archival Statement............................................................................................................. 12 Reference ........................................................................................................................... 12 Results................................................................................................................................ 13 Conclusion ......................................................................................................................... 13 Appendices......................................................................................................................... 14 Appendix 1 - Individual Clinical Observations............................................................. 15 Appendix 2 - Individual Dermal Scores........................................................................ 23 Appendix 3 - Individual Body Weights ........................................................................ 26 Appendix 4 - Summary of Latest Reliable Positive Control Data ................................ 29 Appendix 5 - Certificate of Analysis............................................................................. 32 Appendix 6 - Protocol and Amendments ...................................................................... 38 Appendix 7 - Protocol Devl.ati.on................................................................................... 51 Appendix 8 - Key Personnel ......................................................................................... 53 Study Number: 132-015 Page 6 DELAYED CONTACT HYPERSENSITIVITY STUDY OF T-7485 IN HARTLEY GUINEA PIGS (MAXIMIZATION TEST]) STUDY ABSTRACT The objective of t h s study was to assess the delayed contact hypersensitivity potential of T-7485 in guinea pigs. For the Intradermal Induction Phase on Study Day 1: For Groups 1 and 2, Pair A, Freund's Complete Adjuvant was diluted 1:1 with Sterile Water for Injection, USP to yield a 50% solution. The solution was mixed with a stir bar and magnetic stir plate and covered with foil. For Group 1, Pair B, Sterile Water for Injection, USP was dispensed and covered with foil. For Group 2, Pair Bythe appropriate amount of test article was added to the required amount of Sterile Water for Injection, USP to yield a 125 mg/mL solution. The solution was mixed with a stir bar and magnetic stir plate and covered with foil. For Group I,Pair CySterile Water for Injection, USP was diluted 1:1 with the 50% Freund's Complete AdjuvantKterile Water for Injection, USP solution. For Group 2, Pair C, the appropriate amount of test article was added to the required amount of the 50% Freund's Complete AdjuvantISterile Water for Injection, USP solution to yield a 125 mg/mL solution. The solution was mixed with a stir bar and magnetic stir plate and covered with foil. The Topical Induction phase was conducted on Study Day 7. Approximately 0.5 grams of 10% Lauryl Sulfate (Sodium Dodecyl Sulfate)/Petroleum Jelly was applied to the previous injection sites. Approximately 24 hours later, the Lauryl Sulfate (Sodium Dodecyl Sulfate)/Petroleum Jelly was removed with water and disposable towels or gauze. Following, the Lauryl Sulfate (Sodium Dodecyl Sulfate)/Petroleum Jelly removal, each animal in Group 2 (test article-treated group) received a 0.5-gramapplication of a paste suspension prepared with test article in Petroleum Jelly. The paste was dispensed onto gauze, placed on the treatment sites, and secured with Vetrap@and Dermiform@or Elastoplast tape. The dressing was left in place for an approximate 48-hour period. On Study Day 10, the dressing was removed and the treatment site was washed with water and disposable towels. On Study Day 22, a challenge dose consisting of 0.5 grams of the test article in Petroleum Jelly was administered to the cranial left flank of animals in Groups 1 and 2. The right flank was treated similarly with Petroleum Jelly alone. The exposure period was approximately 24 hours. On Study Day 29, the rechallenge was initiated for the males. The procedure used for the challenge was also used for the rechallenge. Due to a lack of positive results (a dermal score greater than 0), the females were not rechallenged. At approximately 24 and 48 hours after patch removal (Study Days 24 and 25), the treatment sites were scored. The technician scoring the animals was blinded to the identity of the animal. Technician A prepared for scoring outside of the animal room. Study Number: 132-015 Pape 7 Technician B brought the animal to Technician A. Technician A scored the area, and Technician B recorded the scores. At no time was Techcian A aware of the identity of the animal number or group number. Under this experimental design, T-7485 did not produce any adverse effects on body weight or clinical observations. The allergic potential of the test article is considered proven only when the skin reactions to the challenge with the test article clearly outweigh those to the challenge with the vehicle and are greater than the reactions of the controls. In conclusion, no allergic response was produced by T-7485 in guinea pigs using the Maximization Protocol to assess delayed contact hypersensitivity potential. Study Number: 132-015 Page 8 OBJECTIVE The objective of this study was to assess the delayed contact hypersensitivity potential of T-7485 in guinea pigs. MATERIALS AND METHODS TEST ARTICLE AND VEHICLE: The test article and vehicle were identified as follows: Test Article: Lot Number: Date Received: Physical Description: Storage: Amount Received: Lot Number: Date Received: Physical Description: Storage: Amount Received: T-7485 2 December 7,2001 White crystals Room temperature Approximately 200 grams 5 February 28,2002 White crystalline powder Room temperature Approximately 200 grams Other Material: Lot Numbers: Expiration Dates: Physical Description: Storage: Supplier: Sterile Water for Injection, USP JlA402, JlJ585, JlK595 July 2003, January 2004, February 2004 Clear liquid Room temperature B/Braun Medical Inc. Other Material: Lot Numbers: Expiration Dates: Physical Description: Storage: Supplier: Freund's Complete Adjuvant 50K8928, 11K8928,60K8938 March 19,2006, December 27,2006, February 6,2007 Clear yellow liquid with particulates, Clear light yellow liquid 5" f 3C Sigma Other Material: Lot Number: Expiration Date: Physical Description: Storage: Supplier: Lauryl Sulfate (Sodium Dodecyl Sulfate) 37H126 1 June 16,2005 White powder Room temperature Sigma Studv Number: 132-015 Page 9 Other Material: Lot Number: Expiration Date: Physical Description: Storage: Supplier: Petroleum Jelly, White 1261874 19855 November 30,2002 Off white gel solid Room temperature VWR Scientific Products The Sponsor assumed responsibility for characterization (identity, purity, and stability) determinations of the bulk test article. The test article was inventoried when received at Redfield Laboratories, and a record of all test article usage was maintained. The Certificates of Analyses for the test article are presented in Appendix 5. For the Intradermal Induction Phase: For Groups 1 and 2, Pair A, Freund's Complete Adjuvant was diluted 1:1 with Sterile Water for Injection, USP to yield a 50% solution. The solution was mixed with a stir bar and magnetic stir plate and covered with foil. For Group 1, Pair BySterile Water for Injection, USP was dispensed and covered with foil. For Group 2, Pair Bythe appropriate amount of test article was added to the required amount of Sterile Water for Injection, USP to yield a 125 mg/mL solution. The solution was mixed with a stir bar and magnetic stir plate and covered with foil. For Group 1, Pair C, Sterile Water for Injection, USP was diluted 1:1 with the 50% Freund's Complete Adjuvant/Sterile Water for Injection, USP solution. For Group 2, Pair Cythe appropriate amount of test article was added to the required amount of the 50% Freund's Complete Adjuvant/Sterile Water for Injection, USP solution to yield a 125 mg/mL solution. The solution was mixed with a stir bar and magnetic stir plate and covered with foil. For the Topical Induction Phase: The appropriate amount of Petroleum Jelly was weighed and added to the required amount of Lauryl Sulfate (Sodium Dodecyl Sulfate) to yield a 10% mixture. The components were blended thoroughly using a stainless steel spatula. For the Challenge and Rechallenge Phases: For Group 1, Petroleum Jelly was dispensed for dosing. For Group 2, the appropriate amount of test article was added to the required amount of Petroleum Jelly to yield a 33.33% mixture. TEST ANIMALS: Healthy male and female Crl: (HA)BR-Hartley guinea pigs were received from Charles River Laboratories, Inc. (Kingston, New York) for use on the study. The animals were individually housed in polycarbonate cages. Animal identification consisted of uniquely numbered ear tags and color-coded cage cards. On Study Day 1, the males (including replacement animals) were ten to twelve weeks old and weighed 462 to 679 grams, and the females were approximately nine weeks old and weighed 385 to 462 grams. Teklad Certified Guinea Pig Diet #7006 ( M i l l d a t e h t number: 6/1/01 MA, 11/14/01 MA, and 1/9/02MA) and filtered tap water were provided ad libitum. The feed and water were routinely analyzed for contaminants. There were no known contaminants Study Number: 132-015 Pane 10 in the feed or water that would be expected to affect the results of the study. The results of these analyses are on file at Redfield Laboratories. Environmental controls were set to maintain temperatures of 18" to 26C (64"to 79F) with a relative humidity of 30% to 70%. These parameters were recorded at least once daily. A 12:12 hour 1ight:dark cycle was maintained in the animal room. GROUP DESIGNATION AND TREATMENT: Due to equivocal results, the female portion of the study was repeated. The data for the first 15 females is maintained in the raw data but is not included in this report. The males were acclimated for 23 days prior to Study Day 1 and the females were acclimated for 2 1 days prior to Study Day 1. All animals were examined by the Staff Veterinarian prior to being released for use on the study. Fifteen males and fifteen females were randomly assigned to groups by a computer generated weight-ordered distribution such that group mean body weights did not exceed *lo% of the overall mean weight for each sex. The study design was as follows: Group Group Number of Animals Number Desimation Males I Females* 1 Control 5 I I 10 I 2 Treated 10 20 *The number represents the total number of females used for the study. However, only data for the 15 females used for the repeat portion of the study is included in this report. Intradermal Induction Phase On Study Day -1,the hair on the back and flanks of the animals were clipped. On Study Day 1,three pairs of intradermal injections (0.1 mL per injection) were made in an area approximately 4 cm by 2 cm. Injection Pair A Injection Pair B Injection Pair C Injections for the Control Group (Group 1) were: Pair A: 50% Freund's Complete AdjuventISterile Water for Injection, USP solution Pair B: Sterile Water for Injection, USP Pair C: Sterile Water for Injection, USP formulated 1:1 with the 50% Freund's Complete AdjuventISterileWater for Injection, USP solution Injections for the Treated Group (Group 2) were: Pair A: 50% Freund's Complete AdjuvenVSterile Water for Injection, USP solution Pair B: 125 mg/mL test article in Sterile Water for Injection, USP Pair C: 125 mg/mL test article in Sterile Water for Injection, USP formulated 1:1 with the 50% Freund's Complete AdjuventISterile Water for Injection, USP solution Study Number: 132-015 Page 11 Topical Induction Phase On Study Day 7, the treatment area was clipped and approximately 0.5 grams of 10% Lauryl Sulfate (Sodium Dodecyl Sulfate)/Petroleum Jelly was applied to the previous injection sites. Approximately 24 hours later, the Lauryl Sulfate (Sodium Dodecyl Sulfate)/Petroleum Jelly was removed with water and disposable towels or gauze. Following, the Lauryl Sulfate (Sodium Dodecyl Sulfate)/Petroleum Jelly removal, each animal in Group 2 (test article-treated group) received a 0.5-gramapplication of a paste suspension prepared with test article in Petroleum Jelly. The paste was dispensed onto gauze, placed on the treatment sites, and secured with Vetrap@and Dermiform@or Elastoplast tape. The dressing was left in place for an approximate 48-hour period. On Study Day 10, the dressing was removed and the treatment site was washed with water and disposable towels. Challenge and Rechallenge Phases On Study Day 22, the hair was clipped and a challenge dose consisting of 0.5 grams of the 33.33% test article/Petroleum Jelly was administered to the cranial left flank of the animals in Groups 1 and 2. The right flank of the animals was treated with 0.5 grams of Petroleum Jelly only. The materials were applied to a Hill Top Chamber@,placed on the treatment site and secured with Elastoplast tape. The animals were exposed for 24 hours and on Study Day 23, the dressing was removed. On Study Day 29, the rechallenge was initiated for the males. The procedure used for the challenge was also used for the rechallenge. Due to a lack of positive results (a score greater than 0), the females were not rechallenged. JUSTIFICATION FOR SPECIES SELECTION, ROUTE OF ADMINISTRATION, AND DOSE LEVEL: Guinea pigs are the preferred animal model for determining imtation, sensitization, and allergenicity under OECD guideline #406. The dermal route is a potential route of exposure to humans. The amounts of test article applied in this study are the maximum recommended concentrations. CLINICAL OBSERVATIONS: Observations for mortality and moribundity were recorded twice daily (a.m. and pm.).Clinical observations were recorded pretest and once daily. DERMAL SCORING: At approximately 24 and 48 hours after patch removal (Study Days 24 and 25, as well as Study Days 31 and 32 for males), the treatment sites were scored. The technician scoring the animals was blinded to the identity of the animal. Technician A prepared for scoring outside of the animal room. Technician B brought the animal to Technician A. Technician A scored the area, and Technician B recorded the scores. At no time was Technician A aware of the identity of the animal number or group number. Study Number: 132-015 Pane 12 For the evaluation of skin reactions, a four-point scale was used: 0 = No visible change 1 = Discrete or patchy erythema 2 = Moderate and confluent erythema 3 = Intense erythema and swelling The allergic potential of the test article is considered proven only when the s h n reactions to the challenge with the test article clearly outweigh those to the challenge with the vehicle and are greater than the reactions of the controls. BODY WEIGHTS: Body weights were recorded pretest, on Study Days 1 and at termination (Study Days 27 and 36 for males and Study Day 29 for females). The pretest body weights and weights taken on Study Days 25 and 33 are not included in this report but are located in the raw data. Necropsies were scheduled for Study Days 25 and 33 but were postponed due to rechallenging. TERMINATION: Animals that did not survive until the next scheduled observation were weighed, euthanized, and necropsied (no tissues were saved). On Study Day 29, the females were humanely euthanized with an overdose of carbon dioxide and discarded without necropsy examination. On Study Day 36, the males were humanely euthanized in the same manner. HISTOPATHOLOGY: No histopathological examinations were performed. STATISTICS: No statistical analyses were performed. ARCHIVAL STATEMENT All original data and the original final report will be retained at Redfield Laboratories' archives, located at 100 East Boone Street, Redfield, Arkansas, for a period of five years after issuance of the final report. After this time, the Sponsor will be contacted for disposition instructions. REFERENCE I Klecak, G., Test Methods For Allergic Contact Dermatitis In Animals, The Guinea Pig Maximization Test, Dermatotoxicolom fifth edition, Marzulli, F.N. and Maibach, H.I.: 438-441, 1996. Study Number: 132-015 PaPe 13 RESULTS CLINICAL OBSERVATIONS: The individual clinical observations are in Appendix 1. There were no mortalities associated with treatment of test article. There were early deaths but these animals were replaced by amendment to the protocol. Clinical observations were consistent with tissue damage and healing caused by the administration of Freund's Complete Adjuvant. The progression of events consisted primarily of raised area, multiple scabbing, erythema, and skin thickening. There were no clear clinical observations associated with the administration of test article. DERMAL SCORING: The individual dermal scores are in Appendix 2. For either control or test article treated animals, dermal scores did not exceed 1 (discrete or patchy erythema) in males for either the challenge or re-challenge phase. Additionally, scores of 0 (zero) were observed in females. The allergic potential of the test article is considered proven only when the skin reactions to the challenge with the test article clearly outweigh those to the challenge with the vehicle and are greater than the reactions of the controls. As the dermal scores indicate, the test article is not considered an allergen. BODY WEIGHTS: The individual body weights are in Appendix 3. There were no apparent adverse effects of test article-treatment on either male or female body weights. CONCLUSION In conclusion, no allergic response was produced by T-7485in guinea pigs using the Maximization Protocol to assess delayed contact hypersensitivity potential. Study Number: 132-015 APPENDICES Page 14 Study Number: 132-015 Page 15 APPENDIX 1 INDIVIDUAL CLINICAL OBSERVATIONS 1245 1247 1219 1221 1249 SD( 2- 8 ) SD( 6- 8 ) SD( 8 1 SD( 10- 12) SD( 10- 12) SD( 10- 25) SD( 15- 25) SD( 15- 25) SD( 2- 8) SD( 5- 8 ) SD( 8 1 SD( 10 ) SD( 10- 12) SD( 10- 13) SD( 13- 25) SO( 14- 25) SD( 15- 25) SD( 2- 8 ) SD( 8 ) SD( 9 1 SD( 10 ) SO( 10- 36) SD( 15- 36) SD( 19- 36) SD( 21- 27) SD( 28- 36) SD( 2- 8) SD( 8 1 SD( 10- 36) SD( 15- 36) SD( 19- 36) SD( 20- 28) SD( 2- 8) SD( 8 1 SD( 8 1 SD( 10- 12) SD( 10- 12) SD( 10- 25) SD( 14- 25) SD( 15- 25) RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA. DOSE SITES RAISED AREA, RED, DOSE SITES ERYTHEMA, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES SKIN DISCOLORED,BLUE RAISED AREA, RED, W S E SITES ERYTHEMA, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES SKIN THICKENED, DOSE SITES LOCALIZED ALOPECIA, W S E SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES EXCESSIVE LACRIMATION, BOTH IMPAIRED LIMB FUNCTION, HIND LIMBS MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, W S E SITES SKIN THICKENED, DOSE SITES MULTIPLE SCABBED AREAS, UNDERSIDE LOCALIZED ALOPECIA, UNDERSIDE RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES MULTIPLE SCABBED AREAS, UNDERSIDE RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA, RED, W S E SITES ERYTHEMA, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES SKIN THICKENED. DOSE SITES LOCALIZED ALOPECIA, DOSE SITES 1225 1227 1229 1231 1233 SD( 2- 6) SD( 2- e ) SD( 7- 8 ) SD( 10 ) SD( 10- 36) SD( 15- 36) SD( 19- 36) SD( 27- 2e) SD( 28- 36) SD( 2- 6) SD( 2- 8 ) SD( 7- 8 ) SD( 10 SD( 10- 36) SD( 15- 36) SD( 19- 36) SD( 23 ) SD( 2- 5 ) SD( 2- 7) SD( 5- 8 ) SD( e ) SD( 10- 31) SD( 15- 36) SD( 19- 36) SD( 23 ) SD( 30- 36) SD( 2- 5 ) SD( 2- 6) SD( 7- 8 ) SD( 8 ) SD( 10 ) SD( 10- 36) SD( 17- 36) SD( 19- 36) SD( 2- 5) SD( 2- 6) SD( 7- 8 ) SD( 8 ) SD( 10 ) RAISED AREA, WHITE, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES IMPAIRED LIMB FUNCTION, HIND LIMBS MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES MULTIPLE SCABBED AREAS, UNDERSIDE LOCALIZED ALOPECIA, UNDERSIDE RAISED AREA, WHITE, W S E SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES IMPAIRED LIMB FVNCTION, HIND LIMBS MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES ULCER, DOSE SITES, MEDIUM RAISED AREA, RED, DOSE SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES ULCER, DOSE SITES, MEDIUM MULTIPLE SCABBED AREAS, UNDERSIDE RAISED AREA, RED, DOSE SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES RAISED AREA, RED, DOSE SITES IMPAIRED LIMB PUNCTION, HIND LIMBS MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, W S E SITES SKIN THICKENED, DOSE SITES RAISED AREA, RED, DOSE SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES RAISED AREA, RED, DOSE SITES IMPAIRED LIMB FUNCTION, HIND LIMBS 1233 1235 1251 1239 1241 SD( 10- 36) SD( 15- 36) SD( 19- 36) SD( 2- 5 ) SD( 2- 5 ) SD( 6- 8 ) SD( 7- 8 ) SD( 10 ) SD( 10- 36) SD( 15- 36) SD( 19- 36) SD( 23 SD( 2- 6) SD( 2- 8 ) SD( 4- 8 ) SD( 7- 8 ) SD( e SD( 10- 12) SD( 10- 13) SD( 10- 25) SD( 14- 25) SD( 15- 25) SUI 2 - 5 ) SD( 2- 8 ) SD( 6- 8 ) SD( 10 ) SD( 10- 36) SD( 15- 36) SD( 19- 36) SD( 23 ) SD( 2- 5) SD( 2- 8 ) SD( 6- 8 ) SD( 10 ) SD( 10- 36) SD( 15- 36) SD( 19- 36) SD( 23 ) MULTIPLE SCABBED AREAS, W S E SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES RAISED AREA. RED, W S E SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES RAISED AREA, RED, W S E SITES IMPAIRED LIMB FUNCTION, HIND LIMBS MULTIPLE SCABBED AREAS, W S E SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES ULCER, DOSE SITES, MEDIUM RAISED AREA, WHITE, DOSE SITES RAISED AREA, RED, W S E SITES MULTIPLE SCABBED AREAS, DOSE SITES SKIN THICKENED, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA, RED, DOSE SITES ERYTHEMA, DOSE SITES MULTIPLE SCABBED AREAS, W S E SITES SKIN THICKENED, DOSE SITES LOCALIZED ALOPECIA. DOSE SITES RAISED AREA, WHITE, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, W S E SITES IMPAIRED LIMB FUNCTION, HIND LIMBS MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES ULCER, DOSE SITES, MEDIUM RAISED AREA, WHITE, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES IMPAIRED LIMB FUNCTION, HIND LIMBS MULTIPLE SCABBED AREAS, WSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES ULCER, DOSE SITES, MEDIUM 1295 1296 1297 1298 1299 SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( SD( 2- 8 ) 3- 6 ) 6- 8 ) 8 ) 10- 27) 10- 27) 11- 27) 15- 27) 2- 8 ) 3- 6 ) 6- 8) 8 ) 10- 27) 10- 27) 11- 27) 15- 27) 2- 6 ) 2- 8 ) 7- 8) 8 1 10- 27) 10- 271 12- 27) 13- 27) 2- 8 ) 3- 5) 7- 8) 8 ) 10- 27) 10- 27) 13- 27) 24- 27) 2- 8) 3- 6 ) 6- 8 ) 8 ) 10- 27) 10- 27) 10- 27) 14- 27) RAISED AREA, RED, DOSE SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA, RED, W S E SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES RAISED AREA, RED, DOSE SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA, RED, W S E SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES RAISED AREA, WHITE, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES RAISED AREA, RED, W S E SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, M)SE SITES SKIN THICKENED, DOSE SITES RAISED AREA, RED, DOSE SITES RAISED AREA, WHITE, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES LOCALIZED ALOPECIA, DOSE SITES SKIN THICKENED, DOSE SITES PROTOCOL 132-015: DELAYED CONTACT HYPERSENSITIVITY STUDY OF T-7485 IN HARTLEY GUINEA PIGS (MAXIMIZATION TEST') APPENDIX 1: CLINICAL OBSERVATIONS - INDIVIDUAL - FEMALE GUINEA PIGS ________________________L_______________-------------------------------------------------------------------------------------------- D. O. S. E. . G. R. .O.U.P. .2. . . . . . . . . . . . . . . .T.R.E.A.T.E.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G.U. I. N. E. .A. .P.I.G. .#. . . . . . . . . . . . . . . . D. E. S. .C.R.I.P.T.I.O.N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300 SD( 2- 6) RAISED AREA, WHITE, DOSE SITES SD( 2- 8) RAISED AREA, RED, W S E SITES SD( 4- 8) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 1 SD( 10- 14) ERYTHEMA, DOSE SITES RAISED AREA, RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 11- 27) LOCALIZED ALOPECIA, DOSE SITES SD( 12- 27) SKIN THICKENED, DOSE SITES 1301 SD( 2- 6) RAISED AREA, WHITE, DOSE SITES SD( 2- 8 ) RAISED AREA, RED, DOSE SITES SD( 4- 8 ) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 ) SD( 10- 14) ERYTHEMA, DOSE SITES RAISED AREA, RED, W S E SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 10- 27) LOCALIZED ALOPECIA, DOSE SITES SD( 12- 27) SKIN THICKENED, DOSE SITES 1302 SD( 2- 8) RAISED AREA, RED, W S E SITES SD( 2- 8 ) RAISED AREA, WHITE, DOSE SITES SD( 7- 8) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 ) ERYTHEMA, DOSE SITES SD( 10- 15) RAISED AREA, RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 10- 27) LOCAGIZED ALOPECIA, DOSE SITES SD( 12- 27) SKIN THICKENED, DOSE SITES 1303 SD( 2- 8) RAISED AREA, RED, DOSE SITES SD( 2- 8) RAISED AREA, WHITE, DOSE SITES SD( 6- 8) SD( 8 1 SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES ERYTHEMA, DOSE SITES RAISED AREA. RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 11- 27) LOCALIZED ALOPECIA, DOSE SITES SD( 12- 27) SKIN THICKENED, W S E SITES 1304 SD( 2- 6) RAISED AREA, WHITE, W S E SITES SD( 2- 8) RAISED AREA, RED, DOSE SITES SD( 4- 8 ) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 ) ERYTHEMA, DOSE SITES SD( 10- 27) SD( 10- 27) RAISED AREA, RED, DOSE SITES MULTIPLE SCABBED AREAS, DOSE SITES SD( 10- 27) LOCALIZED ALOPECIA, DOSE SITES . . . . . . . . SD( .... .1.3. - . . 2.7.). . . . . . . . . S. .K.I.N. T. .H.I.C.K.E.N.E.D.,. .D.O.S.E. .S.I.T.E.S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SD = STUDY DAY Due t o the fact that all dressings were left in place for SD 9, all clinical observations were called as normal. G.U. I. .N.E.A. .P.I. G. . .#. . . . . . . . . . . .D.E.S.C.R.I.P.T. I. O. N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 SD( 2- e) RAISED AREA, RED, DOSE SITES SD( 2- 8) RAISED AREA, WHITE, DOSE SITES SD( 4- 8 ) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 ) ERYTHEMA, DOSE SITES SD( 10- 14) RAISED AREA, RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 11- 27) LOCALIZED ALOPECIA, DOSE SITES SD( 12- 27) SKIN THICKENED, DOSE SITES 1306 SD( 2- 8 ) RAISED AREA, RED, DOSE SITES SD( 2- 8) RAISED AREA, WHITE, DOSE SITES SD( 4- 8 ) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 ) ERYTHEMA, DOSE SITES SD( 10- 14) RAISED AREA, RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 12- 27) LOCALIZED ALOPECIA, DOSE SITES SD( 15- 27) SKIN THICKENED, DOSE SITES 1307 SD( 2- 6 ) RAISED AREA, WHITE, DOSE SITES SD( 2- 8) RAISED AREA, RED, DOSE SITES SD( 4- 8 ) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 ) ERYTHEMA, DOSE SITES SD( 10- 20) RAISED AREA, RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 10- 27) LOCALIZED ALOPECIA, DOSE SITES SD( 13- 27) SKIN THICKENED, DOSE SITES 1308 SD( 2- 6 ) RAISED AREA, WHITE, DOSE SITES SD( 2- 8) RAISED AREA, RED, DOSE SITES SD( 5- 8) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 1 ERYTHEMA, DOSE SITES SD( 10- 27) RAISED AREA, RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS. DOSE SITES SD( 10- 27) LOCALIZED ALOPECIA, W S E SITES SD( 15- 27) SKIN THICKENED, DOSE SITES 1309 SD( 2- 6) RAISED AREA, WHITE, W S E SITES SD( 2- 8 ) RAISED AREA, RED, W S E SITES SD( 5- 8) MULTIPLE SCABBED AREAS, DOSE SITES SD( 8 1 ERYTHEMA, DOSE SITES SD( 10- 27) RAISED AREA, RED, DOSE SITES SD( 10- 27) MULTIPLE SCABBED AREAS, DOSE SITES SD( 10- 27) LOCALIZED ALOPECIA, DOSE SITES . . . . . . . . .S.D.(. .1.2.-. .2.7.). . . . . . . . . S. K. .I.N. T. .H.I.C.K.E.N.E.D.,. .D.O.S.E. .S.I.T.E.S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SD = STUDY DAY Due to t h e fact that all dressings were left in place for SD 9, all clinical observations were called as normal. Study Number: 132-015 ~_______ ~ Page 23 APPENDIX 2 INDIVIDUAL DERMAL SCORES APPENDIX 2 INDIVIDUAL DERMAL SCORES GrouD 1 - Control I I I I Male I Animal I Study Day 24 24 Hours Postdose Left I Riaht Study Day 25 48 Hours Postdose I Left 1 Right I Study Day 31 24 Hours Postdose Left I Right Study Day 32 48 Hours Postdose I . Left Right Grow 2 - Treated Study Day 24 Study Day 25 I I I Male 24 Hours Postdose I 48 Hours Postdose I I I Animal I Left Right I Left I Right Study Day 31 24 Hours Postdose Left I Right Study Day 32 48 Hours Postdose Left I Right b = Replacement animal for animal #I217 which was found dead on Study Day 11. c = Replacement animal for animal #I223 which was found dead on Study Day 9. d = Replacement animal for animal #I237 which was found dead on Study Day 10. e = Scheduled sacrifice. Group 1 - Control APPENDIX 2 INDIVIDUAL DERMAL SCORES GrouD 2 - Treated Study Number: 132-015 Page 26 APPENDIX 3 INDIVIDUAL BODY WEIGHTS 1300 413. 520. 1301 457. 542. 1302 423. 521. 1303 403. 464. 1304 432. 525. 1305 454. 539. 1306 430. 509. 1307 425. 400. 1308 462. 583. 1. 3. .0.9. . . . . . . . . . 4. .4.7... . . . . .5.2.7... . . . . . . . . . . . SD = STUDY DAY All weights were reported in grams (GI. Study Number: 132-015 Page 29 APPENDIX 4 SUMMARY OF LATEST RELIABLE POSITIVE CONTROL DATA . Study Number: 132-015 Page 30 SUMMARY OF LATEST RELIABLE POSITIVE CONTROL DATA (Redfield Study Number 003-021) The objective of this study was to assess the delayed contact hypersensitivity potential of Hexyl Cinnamic Aldehyde (HCA) and 2-Mercaptobenzothiazole (MBT) in guinea pigs. Healthy male and female guinea pigs were individually housed in polycarbonate cages. On Study Day 1,the animals were approximately five weeks old. The males weighed 257 to 325 grams, and the females weighed 256 to 298 grams. The study design was as follows: Group Number 1 2 3 Group Designation Control Positive Control 1 (HCA) Control - Number of Animals Males Females 5 5 5 5 5 5 Observations for mortality and moribundity were recorded twice daily (a.m. and p.m.). Clinical observations were recorded pretest, once daily, and when a change was noted. On Study Days 24 and 25, the test sites were scored. On Study Days 3 1 and 32, the test sites were scored again, in the same manner as done so for the challenge phase (Study Days 24 and 25). Body weights were recorded pretest, on Study Day 1, and at termination on Study Day 32. The Study Day 1 intradermal induction phase for Groups 1 and 3 consisted of paired injections of the following: Freund's Complete Adjuvanuwater, corn oil, and corn oil formulated 1:1 with Freund's Complete Adjuvanuwater. The intradermal induction phase for Group 2 consisted of the following paired injections: Freund's Complete Adjuvantlwater, 0.3% Hexyl Cinnamic Aldehyde in corn oil, and 0.3% Hexyl Cinnamic Aldehyde in corn oil formulated 1:1 with Freund's Complete Adjuvantlwater. The intradermal induction phase for Group 4 consisted of the following paired injections: Freund's Complete Adjuvanvwater, 3.O% 2-Mercaptobenzothiazole in corn oil, and 3.O% 2-Mercaptobenzothiazole in corn oil formulated 1:1 with Freund's Complete Adjuvant/water. On Study Day 8 (topical induction phase), each animal in Groups 1 and 3 received 0.5 mL of neat corn oil; Group 2 received 0.5 mL of neat Hexyl Cinnamic Aldehyde; and Group 4 received 0.5 mL of 75% 2-Mercaptobenzothiazolein corn oil. All positive control and control articles were applied to a gauze dressing, placed on the previous injection sites, and secured. The dressing remained in place for an approximate 48-hour exposure period. On Study Day 10, the dressings were removed and the area was rinsed with water. Study Number: 132-015 Page 3 1 On Study Day 22, a challenge dose of 0.5 mL of neat corn oil was administered to the right flank of all animals. Animals in Groups 1 and 2 received 0.5 mL of neat Hexyl Cinnamic Aldehyde on the left flank,and animals in Groups 3 and 4 received 0.5 mL of 10% 2-Mercaptobenzothiazole in corn oil on the left flank. Positive control and control articles were applied to a Hill Top Chamber@and secured with elastoplast tape for an approximate 24-hour exposure period. On Study Day 23, the dressings were removed. On Study Day 29, a rechallenge dose was administered in the same manner as the challenge dose. Clinical observations were similar between positive control and control article treated male and female guinea pigs. The observations establish a pattern of Freund's Complete Adjuvant induced trauma followed by healing as indicated by the onset of the observations. There were no apparent adverse effects on body weights. Treatment with Hexyl Cinnamic Aldehyde failed to produce a clear allergic response when compared to corresponding control male and female guinea pigs. In contrast, 2-Mercaptobenzothiazole allergic response was established in the challenge phase and potentiated in the rechallenge phase resulting in moderate and confluent erythema as well as the common intense erythema and swelling in both male and female guinea pigs. In conclusion, the allergenic potential of the positive control agent 2-Mercaptobenzothiazoleis clearly established when compared with the corresponding control agent. Under this study design, the allergenic potential of Hexyl Cinnamic Aldehyde could not be established. 1 Klecak, G., Test Methods For Allergic Contact Dermatitis In Animals, The Guinea Pig Maximization Test, Dermatotoxicology fifth edition, Marzulli, F.N. and Maibach, H.I.: 438-441, 1996. Study Number: 132-015 Pave 32 APPENDIX 5 CERTIFICATE OF ANALYSIS Study Number: 132-015 Page 33 CEntrE Anahftiial bboratorks. hrc. 3048 Rasearch D h State College, PA 18801 www.cenh'elab.com hone: (814) 231-8032 Fax (814) 231-1253 Or(814) 231-1580 RVTERTM CERTIFICQIE OF ANAL-YSIS Revision I Centre Analytical Lnboratoriu COA Reference #: 023-032 3M Product: PF'BS, Lot 2 Test Control Reference #: TCR-OUO17-'71 Purity: 973% Purity' Test Name Appcarancc Identification I speci6cauo~ I Result I 973% I I 1 I White, crystalline solid I COnfOrmS 2. 0.001 W t J W t o m 3. 0.768 w t J I. 2. 3. 4. Elemental Analysis': 1. Carbon 2. Hydrogen 3. Nitrogen 4. sulfur 5 . Fluorine COA023032-7 1.doc 4. 4).009vSthvt% Tbeorehd vduc = 14.2% Thmrctical vaiuc = 0.0% ?heoretical Value = 0.0% Thcorctical:Value = 9.5% 1 Theoretical Value = 50.6% I 1. a . 1 wthvt.% 2. a 1 wt/wt% 3. G.1Wwt% 4. <o.z wt/wt% I 1. 11.0 wtJwt.% 2. 0282wt/wt% 3. 0.465 WtiWtY. I 4. 11.2 M Y 0 5 . 48.2 wtJwt.%m Page I of3 Study Number: 132-015 Page 34 INTERLM CERTTFICATE OF ANALYSIS Revision 1 Centre Analytical Laboratories COA Reference #: 023-032 3M Product: PFBS, Lot 2 Test Control Reference #: TCR-00017-71 Date ofLast Analysis: 01117/01 Expiration Date: 01/17/02 Storage Conditions: e -10-C Re-assessmutt Datc: 01/17/02 - - 'Purity 5 100% (Total N M R impllritic~2,.07% + TOMLC/MS impurities, (5.56% 4.99%*) + Total Metal impurities, 0.006% + Total inorganic anions, 0.016%) Total impurity from all tests =2.662% Purity 100% - 2.662% 97.3% *I .93% ofthe N M R total impurity value is due to the presence ofthe This impurity is also ObSCrVed in the Lc/Ms analysis at 4.99K.I T h e impurity value is taken from the NMR analysis as it is deemed the mom quantitative technique in the absence of standards(See impurity profiic on page 3). 'Potassium and sodium are present as counterions and thus are excluded from the purity calculation. 'Theoretical value calculations based on the empirical formulq C+F9SO~K(+Mw--338.2) Page 2 of 3 Study Number: 132-015 Page 35 CEntw Analytical laboratorks, Inc. 3048 Research Drive State College, PA 16801 www.centrelahcom e: (814) 231-6032 Far (814) 231-1253 M (814) 231~1580 INTERIM CERllFICATE OFANALYSIS Revision I Centre Analytical Laboratories COA Reference #: 023-032 3M Product: PFBS,Lot 2 TesK Control Reference #: TCR-00017-71 Lc/Ms Purity Profile: Peak # 1 Retention Time (min) I Mass(s) I Identity 1 Area I YOImpurity I I I 2 - 3. Total 9.508 I 298.9 I PFBS 1 308351Oo I I - I - I 32651678 1 4un..92c97 .. J 5.56 N M R purity Profile: Component * I I CF+ZF~CFZ-CF~-SO~-, Linear Product I I MolecuhrWeight 298.9 I Relative Weight Percent I I 97.9 0.05 I I 0.13 I Total Impurity I I 0.01 . .. 2.07 +All componentsare assumed to be associated with K" cation. Note: N M R detected impurities in boldface type. This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Reviewal By: =?b/v Date Laboratory Manager, Centre Analytxal Laboratories COA023032-7I.doc Page 3 of 3 Study Number: 132-015 Page 36 Test Name I Appearance I Identification NMR Metals (ICP/MS) 1. calcium 2. h-lagncsirnn 3. Sodiulil' 4. Potassium' 5. Nickel 6. Iron 7. Manganese Total % I m p ~ t y("R) Total % Impurity (L-1 Toohi % Impurity 1-( Residual Solvents TGA Punty by Dsc InorganicAnions (.IC.) i. chlorik 2. Fluoride 3. Bmmidc 4. Nitrate 5. Nitrite 6. Phosphate I. sulfate Organic Acids ' (IC) 1. 2. 3. 4. Elemental Analysis': 1. Carbon 2. Hydrogen 3. Nibgcn 4. sulfur 5. Fluorine Specifications White, crystallinesolid Theoretical Value = 14.2% Theoretical Value = 0.0% Theoretical Value = 0% neantical value = 9.5% Theoretical Value = 50.6% Rmult I I I Conforms Positive 1. 0.002Ww&% 2. <0.001 wt/wt% 3. 1.142wt/wt./. 4. 8.442 Wwy. 5. ~ 0 . 0 0 1wwty. 6. 0.002 WwtYm 7. a O . O O 1 w t / w t O / . 0 . 9 W ~ Y O. 0.774 w t h t X None Quantified 0.02 wt/wt% 99.69% 1. <0.015 wtfwt% 2. 0.101 wt./wt.yI 3. co.o4owthvt% 4. a.O09wtlwt% 5. cO.006wuwt% 6. C0.006 wtJwt-0~ 7. c0.078 wtlwt% 1. <O.l Wt/wt.% 2. C0.I wtlwt% 3. a . 1 wt/wt% 4. a . 2 5 wtM% 1. 10.4 wtJwt% 2. 0.31 1 wtJwt% 3. 3.94 wtlwt% 4. 9.46 w t l w t H 5. 38.1 w t h t x COA023-061 Page I of2 Study Number: 132-015 PaEe 37 Centre Anahtical laboratoric=c;,mCr L P h o n e : (814) 231-8032 ~ Fiuc (814) 231-1253 OC (814) 231-1&- CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Rtference #: 023-067 Date ofLast Analysis: OW31/01 Expiration Date!: OW3 1/03 Storage Conditions: Frozen SlQC Re-assessment Date: 08/3 1/03 'Purity= lodk - (Total N M R implrritits, 0.90% + Total LC/MS implaitiea, 0.774% + ' Total Mctal impurities, 0.004% +- Total Anion Impurities,0.101 +Residual Solvents TGA, 0.02%) - Total impurity fium all tests = 1.799% Purity = 100% 1.799%=9820/. 'Potassium and Sodium are prcscnt 89 counterions and thus arc excluded h r n the purity calculation. %retical value calculationsbased on the empirical fotmula, C+F&O,K+ (Mw-338.2) Prepared By: Scientist, Centre! Analytical Laboratories \\a\- Date Reviewed By: ,Ad> 'Laboratory Manager, Centre Analytical Date Laboratories COA023-067 Page 2 of 2 Study Number: 132-015 Page 38 APPENDIX 6 PROTOCOL AND AMENDMENTS Study Number: 132-015 Page 39 Redfield Laboratories Protocol Prolocol Number: 132-OI5 DELAYED CONTACT HYPERSENSITIVITY STUDY OF T-7485 IN HART-LEY 9 GUINEA PIGS (MAXIMIZATION TEST') PROTOCOL NUMBER: 132-015 OBJECTIVE: To assess the delayed contact hypersensitivity potential of T-7485 in guinea pigs. LOCATION OF STUDY AND CONDITIONS OF TESTING: It is the intention of 3M Corporate Toxicology, in sponsoring this study to generate animal safety data, which may be submitted to regulatory authorities. Redfield Laboratories, 100 East Boone Street, Redfield, Arkansas, 72 132 is accredited by AAALAC and licensed by the United States Department of Agriculture to conduct research in laboratory animals. All the conditions of testing will conform to the Animal Welfare Act (9 CFR) and its amendments. Redfield Laboratories will follow all requirements specified in this approved protocol and all applicable governmental regulations regarding Good Laboratory Practices as well as Redfield Laboratories' Standard Operating Procedures. Changes in the protocol may be made by consultation with and approval from 3M Corporate Toxicology followed by written verification of the change. 3M Corporate Toxicology reserves the right to inspect facilities and procedures used in this study by means of announced or unannounced site visits. Redfield Laboratories will notify 3M Corporate Toxicology promptly by telephone prior to release of any data for review. The submission of false data to the federal government is a crime under 18 U.S.C. 1001. SPONSOR: SPONSOR'S REPRESENTATIVE: TESTING FACILITY: 3M Corporate Toxicology 3M Center, Building 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3320 Telephone: 65 1-733-2501 FAX: 651-737-5506 Redfield Laboratories Mail: P.O. Box 308 Deliveries: 100 East Boone St. Redfield, AR 721 32 Telephone: 501-397-2540 FAX: 501-397-2002 PERSONNEL: Study Director: John E. Seng, Ph.D. Principle Investigator: Amy Babb, B.S., RLAT Study Coordinator: Karen Tranter, B.A., LATG Head Technician: Patricia Shores Veterinarian: Allan Manus, D.V.M., M.Sc., ACLAM Page I of8 1 U7/0I Study Number: 132-015 Page 40 Redfield Laboratories Protocol Protocol Number: 132-015 PROPOSED STUDY DATES: Experimental Start Date: December 12,2001 Experimental Termination Date: January 12,2002 Draft Report Date: February22,2002 1. REGULATORY COMPLIANCE This study will be conducted in accordance with the following testing guidelines: 21 CFR58 El C(97)186 (Final) (OECD) The Quality Assurance Unit in accordance with the Redfield Laboratories' Standard Operating Procedures (SOPS)will audit the protocol, study conduct, and the final report. 2. A N I M A L C A R E C O M M I n E E REVrEW The protocol will be reviewed and approvedby Redfield Laboratories' Institutional Animal Care and Use Committee (IACUC) for compliance with regulationsprior to study initiation. In the opinion of the Sponsor, indicated by signature of this protocol, the study does not unnecessarily duplicate any previous work. 3. MATERIALS TESTARTICLE T-7485 (Pdassium perfluorobutanesulfonate, PFBS) VEHICLE 1% carboxymethylcellulose(aqueous, medium viscosity) IDENTIFICATION The lot numbers and physical descriptions will be listed in the raw data and final report. STORAGECONDITIONS The bulk test article and vehicle will be stored in their original containersat room temperature. OTHEMRATERIALUS SEDM DOSING Sterile water for injection (HzO),Freund's Complete Adjuvant (FCA), sodium lauryl sulfate (SLS), and petrolatum. Lot numbers will be recorded in the raw data. ANALYTICALCHEMISTRY The Sponsor assumes responsibility for characterization (identity, purity and stability) of the bulk material (including under test conditions). The Sponsor will hold information on the composition and method of synthesis of the test article. The dose will be calculated based on the total unit weight of the test article. Page 2 of 8 12/7/01 Study Number: 132-015 Page 41 Redfield Laboratories Protocol ANALYSIS,PREPARATIOANN,D USEOF DOSINMG m m Protocol Number: 132-015 For the intradermal induction phase, the test article will be suspended in 1% carboxymethylcelluloseat the concentrationof test article (w/w) that can be injected intradermally. This concentration will be documented in the raw data and will be used in the Intradermal Induction Phase. (Page 5 ) . For the topical induction phase, the test article will be blended with petrolatum. The FCA for the Intradermal Induction Phase will be prepared as directed on the package insert and mixed 5050 with sterile water. The SLS will be blended at a concentrationof 10% (w/w) with petrolaturn. INVENTORY The test article container(s) will be weighed when received at the testing laboratory, and a record of all test article use will be maintained. Test article will .be stored in original containers unless otherwisedirected by the Sponsor. fiTENT1ON SAMPLES AND TESTARTICLSEHIPPING Unused test article will be returned to the Sponsor or Sponsor's designee at the termination of the study or retained by the testing facility for future studies. The person receiving the material will be notified in advance of shipping. The material will be accompanied by a transmittal letter (receipt signature required) describingthe material contained in the shipment,and the material will be packed in a container suitable to maintain the temperatureconditions specified by Sponsor. SAFETY PRECA UTIONS General safety precautions as required by Redfield Laboratories' policies and procedures will be followed. The sponsor will supplybasic toxicity data on the test article to be used in this study. The Sponsor's representative will be notified of any personnel exposures requiring a physician's examination or care. 4. ANIMALT Species: StraidSource: Guinea Pig Hartley-derived, Charles River Laboratoriesor other reputable source. Age on Receipt: Number and Sex: Animals will be at least six weeks of age when received and no less than 250 grams in weight on Study Day 1. 15 males and 15 females (females nulliparous and non- Identification: pregnant) Ear tag and correspondingcage card. ANIMAHL USBANDRY HOUSING The animals will be individually housed in polycarbonate cages with contact bedding. The cages conform to standards set forth in the Animal Welfare Act (with all amendments)and the Guide for the Care and Use of LaboratoryAnimals, National Academy Press, Washington, D.C.,1996. Page 3 of 8 1 21710 1 Study Number: 132-015 Page 42 Redfield Laboratories Protocol Protocol Number: 132-015 FEED Teklad Certified Guinea Pig Diet #7006 will be provided ad libitum. This diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Results of the manufacturers' analyses are on file at Redfield Laboratories. WATER Tap water will be provided ad libitum via an automatic watering system. Samples of the water are analyzed for total dissolved solids, hardness, and specified microbiological content and for environmental contaminants. Results of these analyses are on file at Redfield Laboratories. CONTAMINANTS There are no known contaminants in the feed or water that would be expected to interfere with this study. ENVIRONMENT Environmental controls are set to maintain temperatures of 64" to 79"F,(18" to 26" C) with a relative humidity of 30% to 70%. These parameters are recorded at least once daily. A 12:12 hour 1ight:darkcycle is maintained. ACCLIMATION Animals will be acclimated for a minimum of seven days prior to Study Day 1 in the room where the experiment will take place. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. Animals will be examined by the Staff Veterinarian prior to release of that shipment of animals for use on the study, JUSTIFICTAIONFOR SPECIESELECTION Guinea pigs are the preferred animal model for determining irritation, sensitization, and allergenicity under OECD guideline #406. 5. EXPERJMENTMAELTHODS SWDY DESIGN Group Number 1 2 Group Designation Control Treated I N u m b e r of Animals Males Females I 5 5 10 10 Page 4 of 8 I 21710 1 Study Number: 132-015 Page 43 Redfield Laboratories Protocol PREPARATIOOFNEXPOSURAEREA Protocol Number: 132-015 On Study Day - I , the hair will be clipped from the back and flanks of each animal. The treatment sites will be inspected for interfering lesions, imtation, or defects that would preclude the use of any of the animals. Only animals with healthy, intact skin will be used. INTRADERMIANLDUCTIOPNHASE On Study Day 1, three pairs of intradermal injections (0.1 mL per injection) will be made in an area approximately 4 cm x 2 cm as follows: Injections for the Control Group (Group 1) are: 1. Pair A: 2. Pair B: 3. Pair C: FCA / H20 1% carboxymethylcellulose 1% carboxymethylcellulose formulated 1:1 with FCA / H 2 0 Injections for the Treated Group (Group 2) are: 1. PairA: 2. Pair B: 3. Pair C: FCA/H20 Test Article in 1% carboxymethylcellulose Test Article in 1% carboxymethylcelluloseformulated 1:l with FCA / H20 TOPICAILNDUCTIOPNHASE On Study Day 7, the treatment area will be clipped and approximately 0.5 grams of a 10% SLS/petrolatum mixture will be applied to the previous injection sites. Approximately 24 hours later, on Study Day 8, the SLS will be removed with water and disposable towels or gauze. Following the SLS removal, each animal in Group 2 (treated group) will receive a paste of 0.5 grams of a suspension of test article in petrolatum. On Study Day 8, the control animals (Group 1) will receive a paste of 0.5 grams ofpetrolatum without the test article. The paste will be dispensed onto a Hill Top Chamber@' and covered and secured with Vetrap@and Dermiform' tape. The occlusive dressing will be left in place during an approximate 48-hour exposure period. On Study Day 10, dressings and petrolatu~dtesatrticle mixtures will be removed with water and disposable towels. Study Number: 132-015 Page 44 Redfield Laboratories Protocol Protocol Number: `132-015 CHALLENGE AND RECHALLENGE PHASES On Study Day 22, the hair will be clipped from the back and flanks of each animal, and a challenge dose consisting of 0.5 grams of the test article in petrolatum will be administered to the cranial left flank of animals in Groups I and 2. The right flank will be treated similarly with petrolatum alone. The materials will be applied to Hill Top Chambers@and covered with Vetrap@and Dermiformmtape. The exposure period will be approximately 24 hours. If equivocal responses are observed in the initial challenge application, the test article animals may be rechallenged at the discretion of the Study Director and after consultation with the Sponsor`srepresentative. If performed, rechallenge will occur one week after the initial test article challenge. DERMASLCORING At approximately 24 and 48 hours after patch removal (Study Days 24 and 25), the test sites will be scored. If necessary, the area may be clipped approximately three hours before the 24-hour scoring. The technician scoring the animals will be blinded to the identity of the animal. For evaluation of skin reactions, a four-point scale will be used 0 = No Visible Change 1 = . Discrete or Patchy Erythema 2 = Moderate and Confluent Erythema 3 = Intense Erythema and Swelling The allergic potential of the test article is considered proven only when the skin reactions to the challenge with the test article clearly outweigh those to the challenge with the vehicle and are greater than the reactions of the controls. CLJNIUL OBSERVATIONS Observations will be performed and recorded twice daily (a.m. and pm.) for moribundity and mortality. Clinical observations will be performed according to Redfield Laboratories'SOPS and recorded pretest, once daily, and when a change is noted. BODY WEIGH^ Body weights will be recorded pretest, on Study Day 1, and at study termination. NECROPSY/PATHOLOGY All animals dying on test, or those sacrificed in extremis, will be subjected to a gross necropsy examination and discarded. Any abnormalities will be recorded, but no tissues will be saved. All animals that survive to the termination of the study will be euthanized by an overexposure to carbon dioxide and discarded without necropsy. Page 6 of 8 I2/7/0I Study Number: 132-015 Page 45 Redfield Laboratories Protocol 6. REPORT Protocol Number: 132-015 A draft report will be sent to the Sponsor. Revisions to the initial draft report will be provided to the Sponsor by mail or fax transmission as printed copies of the corrected pages only. Additional revisions or complete copies of the revised draft reports will be provided at additional expense to the Sponsor. Three copies of the final report will be submitted to the Sponsor approximately two weeks after approval of the draft report. The final report will include all elements requiredrecommended by the regulations and guidelines. The report will include, but is not limited to, those items listed below: a Descriptive text of the study objective b summary a Test article identification (Certificate of Analysis, stability data, and analytical analysis if any are performed) Methods Results and conclusions Body weights and body weight changes Individual dermal scores Necropsy observations (if applicable) A copy of the protocol, all protocol amendments, and all protocol deviations that may affect the integrity of the study 7. MAINTENANCOEF R.4 w DATAAND RECORDS Original data or copies thereof, will be available at Redfield Laboratories to facilitate auditing the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data and the original final report will be retained in the archives of the laboratory for at least five years. Wet tissues, slides, and blocks (if generated) will be retained at Redfield Laboratories for one year. After one year, the storage of the wet tissues, slides, and blocks will be negotiated with the Sponsor. After five years, the storage of the paper data will be negotiated with the Sponsor. The Sponsor will be notified prior to disposal of any original study data. 8. QUALITYASSURINCE REMEW This is to certify that this protocol has been reviewed by Quality Assurance. Frances Pattillo, M.S., RQAP-GLP Manager of Quality Assurance Redfield Laboratories (317I h , 1 Dhe Page 7 of 8 12/7/01 Study Number: 132-015 Page 46 Redfield Laboratories Protocol 9. IA CuC RE MEW Protocol Number: 132-015 This is to certify that this protocol has been approved by the Redfield Laboratories' . Institutional Animal Care and Use Committee. Representative Date Institutional Animal Care and Use Committee Redfield Laboratories IO. FACILITYMANAGEMENTREMEW This is to certify that Redfield Laboratories facility management will provide resources to conduct this study. Date 11. APPROVEDBY: Sponsor's Representative 3M Corporate Toxicology /2////0, Date J o d E. Seng, Ph.D. 0r / Study Director Redfield Laboratories /2 7/6f Date 12. REFERENCE ' Klecak, G., Test Methods For Allergic Contact Dermatitis In Animals, The Guinea Pig Maximization Test, Dermatotoxicology fifth edition, Martulli, F.N. and Maibach, H.I.: 438-441, 1996. Page 8 of 8 12/7/01 StudvNumber: 132-015 Page 47 Redfield Laboratories Protocol Amendment Protocol Number: 132-015 STUDY NUMBER: 132-015 AMENDMENT: 1 STUDY TITLE: DATE ISSUED: DELAYED CONTACT HYPERSENSITIVITY STUDY OF T-7485IN HARTLEY GUINEA PIGS(MAXIMIZATION TEST') January 11,2002 AMENDMENT # 1 ITEM 1: ADD: REASON: . APPROVALS: Page 4, Section 4, Acclimation At the request of the Study Director, excess animals may be used as replacement animals. Due to early deaths, which are not considered related to administration of test article, the Sponsor has requested the replacement of animals. Date Sponsor's Representative 3M Corporate Toxicology John E./Seng, Ph.D. u Date Study Director Redfield Laboratories 1 Study Number: 132-015 Page 48 Redfield Laboratories Protocol Amendmenr Protocol Number. I J t - O l S STUDY NLTMBER: 132-0 15 AMENDMENT: STUDY JITLE: 2 DELAYED CONTACTHYPERSRVSITiVITYSTUDY OF T-7485M HARTLEY GUWEA PfGS (MAXIMlUkION TEST') DATE ISSUED: May 13,2002 AMENDMENT # 2 Eficriw h i e of Amendmenr I/3 1/02 1TEM 1: Page 3,Section 4, &ti& CHANGED FROM: Nurnba and Sex: 15 males and 15 females (fcmales nulliparousand non-pregnant) CHANGED TO: Numbn: and Sex: 15 d e s and 30 females ( d e raulliparous & non-Pregnanr) 9' REASON: Due IO quivocal results. the female portioh o f he study was and additional snimals were ordmd. Eflecrive &re ofAmendmenr 1/31/02 Croup Group Number of A~irrUlr Number Dnimadon M a l a I Femalca 1 I Control I 5 1 5 2 TreaFcd IO 10 ' Croup , Numbtr 1 2 Group Deshadoa Control Treated - . NumberbfAnimals Mala Fsmdrr . 5 10 ID 20 REASON: Due EO equivccal rrsuh, the f w e poniori of tbc study wns rcpcatcd anQ additional animals WCR ordeed. 1 Study Number: 132-015 Page 49 Redfield Laboratories Protocol Amendment Prwocol Number: 131-015 STUDY NUMBER: 132415 AMENDMW: 2 S W Y TITLE: DELAYED CONTACT HYPERSENYS$``lIJ STUDY OF T-748SIN HARTLEY GUINEA PIGS (MAXIM TiON TEST') DATE ISSUED: - May 13,2002 AMENDMENTff2 CHANGED TO: Injectionsfor rht C O ~ UbO u~ p (Group 1) IUC; 1. PairA: 2. Pair 8: 3. Pair C: FCAIhO Srcrilc water forbjsrion 9raile water for injection 1 : I wirh FCA / HzO IqjccGons for the Trmed Group(Omup 2) are: 1. PairA: 2. Pair 8: 3. Pair C: FCA/&O TCU'A~~CIiCn sterile water for injettion Tes Arricle in sterile water for injccrion 1:1 with FCA I H D REA90N: Merhod of formulating trst article wa9 revi$edIO maimaiaconrinuirywirh the control group. 2 Study Number: ' 132-015 Page 50 Redfield Laboratories Protocol Amendment Prorocol Number: i3Z-OiS AMENDMENT: STUDY TITLE: DATE ISSUED: 2 DELAYED CONTACT HYPERSENSfiNITY STUDY OF T-7485IN HARTLEY OUTNEA PIGS (MAIIMIZATIONTEST') May 13,2002 AMENDMENT rl2 Sponsor's Reprewracive 3M Corpoxate Toxicology ' 3 Study Number: 132-015 APPENDIX 7 PROTOCOL DEVIATION Page 51 Study Number: 132-015 Page 52 PROTOCOL DEVIATION 1. Protocol. Section 5. Challenge and Rechallenge Phases. The protocol required that the Hill Top Chambers@be secured with Vetrap@and Demiform@ tape. However, on Study Day 22, Elastoplast tape was used at the request of the Study Director. Thls deviation did not affect the integrity of the study. Study Number: 132-015 APPENDIX 8 KEY PERSONNEL Page 53 - - Study Number: 132-015 Page 54 KEY PERSONNEL Study Number: 132-015 Study Director:......................................... J o E.~Seng, Ph.D. Principle Investigator:..............................Amy Babb, B.S. Study Coordinator:................................... Head Technician: ..................................... Veterinarian: ............................................ Quality Assurance:................................... Report Coordination: ............................... Report Preparation: ................................. Karen Tranter, B.A., LATG Patricia Shores, ALAT Allan Manus, D.V.M., M.Sc., ACLAM Melissa Milligan, B.S., M.T., CLSp(CG) Fran Pattillo, M.S., RQAP-GLP Meredith Joheim, M.S. .Tammy Jones Dana Rose, ALAT