Document NNkpwvr7oBE5X4NowmzOJoYR8

M z a _ 2 < U f-0 H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TRADE SECRET STUDY TITLE H-25134: Dermal Sensitization Test - Buehler Method LABORATORY PROJECT IDENTIFICATION DuPont-9303 AUTHOR George E. Moore, B.S. STUDY COMPLETED ON April 5,2002 PERFORMING LABORATORY Product Safety Labs 2394 Route 130 Dayton, New Jersey, 08810 SUBMITTER DuPont Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Page 1 o f 25 Company Sanitized Does not contain TSCA H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 QUALITY ASSURANCE STATEMENT The Quality Assurance Unit randomly selects intervals for QA inspections prior to study initiation. Records o f the findings o f these inspections are kept on file. The summary below provides verification o f statements made in the final report section that addresses Quality Assurance audits. Inspections were made of: DATE PROCEDURE INSPECTED 1/22/02 Test substance dispensing records 1/22/02 Test substance preparation 2/8/02 48 hour scoring (Induction # 3) 3/18/02 Raw data 3/18/02 Draft report y /s V 0 2 - Final report Findings reported to: Study Director 3/18/02 Management' 3/18/02 ____ Annamarie LaPorte Quality Assurance Auditor Page 2 o f 25 Company Sami.^ou. u m s hoi contain ISC A CBl H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 CERTIFICATION We the undersigned declare that the methods, results and data contained in this report faithfully reflect the procedures used and raw data collected during the study. George E. Moore, B.S. Study Director Gary Wnorowski, B.A. Laboratory Director ______f . t L . A . i C ' i i h i l L ^ y Carol Finlay, B.A. f * Study Monitor Date Date Date Page 3 of 25 Company S an ili! Do m noi contain TSCA CBI H-25134: Dermal Sensitization Test - Buehler Method Substance Tested: Svnonvms/Codes: STUDY INFORMATION Submitter's Notebook Numberfsi: Haskell Number: CAS Registry Number Composition: Puritv: Known Impurities: 25134 DuPont-9303 Physical Characteristics: Stability: Sponsor: Study Initiated/Completed: In-Life Initiated/Comnleted: Dark brown solid The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. January 21,2002 / (see report cover page) January 23,2002 / February 23,2002 Page 4 o f 25 C o m p ari Sanitized. D oes not contain TSCA CBI H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE OF CONTENTS QUALITY ASSURANCE STATEMENT.............................................................................................................. 2 CERTIFICATION.................................................................................................................................................. 3 TABLE OF CONTENTS................... 4 STUDY INFORMATION........................................................ 4 H-25134: DERMAL SENSITIZATION TEST - BUEHLER METHOD................................................................. 6 1. PURPOSE.....................................................................................................................................................6 2. SUMMARY................................................................................... 6 3. MATERIALS..................................................................................................................................................7 4. METHODS....................................................................................................................................................8 5. PROCEDURE.............................................................................................................................................. 9 6. EVALUATION................................................................................................................................... *.......10 7. HISTORICAL POSITIVE CONTROL VALIDATION STUDY................................................................... 10 8. STUDY CONDUCT.................................................................................................................................... 11 9. REFERENCES............................................................................................................................................11 10. QUALITY ASSURANCE............................................................................................................................11 11. DEVIATION FROM THE FINAL PROTOCOL...................................................................................... ...11 12. FINAL REPORT AND RECORDS RETENTION..................................................................................... 11 13. RESULTS.................................................................................................................................................... 12 14. CONCLUSION............................................................................................................................................13 TABLE 1: PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC (Test Substance)........................................................................................................................................................ 14 TABLE 1 (cont.): PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC (Positive Control)............................ 15 TABLE 2: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g) (Test Substance)..........................................16 TABLE 2 (cont.): INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g) (Test Substance).............................. 17 TABLE 3: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g) (Positive Control).............................. .......... 18 TABLE 4: INDUCTION PHASE SKIN REACTION SCORES (Test Substance)........................................... 19 TABLE 4 (cont.): INDUCTION PHASE SKIN REACTION SCORES (Test Substance)............................... 20 TABLE 5: INDUCTION PHASE SKIN REACTION SCORES (Positive Control)...........................................21 TABLE 6: CHALLENGE PHASE SKIN REACTION SCORES (Test Substance).........................................22 TABLE 6 (cont.): CHALLENGE PHASE SKIN REACTION SCORES (Test Substance)............................. 23 TABLE 7: CHALLENGE PHASE SKIN REACTION SCORES (Positive Control).........................................24 APPENDIX A: FEED AND WATER ANALYSES..............................................................................................25 Page 5 o f 25 Company Sanitized. Does not contain TSCA CBI H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 H-25134: DERMAL SENSITIZATION TEST - BUEHLER METHOD PROTOCOL NO.: PSL STUDY NUMBER: SPONSOR: SUBMITTER: TEST SUBSTANCE IDENTIFICATION: TEST SUBSTANCE DESCRIPTION: DATE RECEIVED: PSL REFERENCE NO.: DATE OF PROTOCOL APPROVAL: EXPERIMENTAL INITIATION DATE: EXPERIMENTAL COMPLETION DATE: STUDY COMPLETION DATE: P328 DUP 11716 E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. DuPont Haskell Laboratoiy for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, DE 19714-0050 Dark brown solid January 15,2002 020115-2D January 21,2002 January 23,2002 February 23,2002 1. PURPOSE To assess the sensitization potential of H-25134 after repeated topical applications. 2. SUMMARY A dermal sensitization test was conducted with guinea pigs to determine the potential for H-25134 to produce sensitization after repeated topical applications. Page 6 o f 25 .j H-25134: Dennal Sensitization Test - Buehler Method DuPont-9303 An 80%12w/w mixture of the test substance in mineral oil was topically applied for six hours to 20 healthy test guinea pigs, once each week for a three week induction period. A test vehicle control group (ten animals) was maintained under the same environmental conditions and treated with m ineral oil, 100%, for the induction phase. Thirty days (See Section 11) after the first induction dose, a challenge dose of the test substance (HNIC or Highest Non-Irritating Concentration, determined in the preliminary irritation screen to be an 80%' w/w mixture in mineral oil) and vehicle (mineral oil, 100%) were applied to a naive site on each of the test and test substance irritation control guinea pig. Approximately 24 and 48 hours after each induction and challenge dose, the test and test irritation control animals were scored for erythema. A table summarizing the incidence and severity of the sensitization response noted after challenge are found below. 80%l w/w in mineral oil Mineral oil Incidence with Skin Reactions Test Animals Test Irritation Control Animals Hours 24 48 24 48 0/20 0/20 0/10 0/10 0/20 0/20 0/10 0/10 80%1w/w in mineral oil Mineral oil Severity3 Test Animals Test Irritation Control Animals Hours 24 48 24 48 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Based on the results of this study, the test substance is considered not to be a contact sensitizer. The positive response observed in the historical positive control validation study with ahexylcinnamaldehyde, technical grade, 85% validates the test system used in this study (See Section 7). 3. MATERIALS A. Test Substance The test substance identified as H-25134 was received on January 15, 2002 and was further identified with PSL Reference Number 020115-2D. The test substance was stored at room temperature. The sample was a dark brown solid. Prior to use, the test substance was heated in a 60-68C water bath, producing a thick liquid, which was stirred and then separated into 1The test substance, as received, was a solid. To enhance skin contact, the prepared test substance was crushed with a mortar and pestle, then moistened with mineral oil prior to application. 2Animals with scores greater than 0.5 3Sum o f the erythema scores divided by the number o f animals evaluated. Page 7 o f 25 Company Sami*. uw** cuua.o i SCA CBI H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 containers. On each day o f dosing, one container was heated in a 60-68C water bath until the test substance was liquefied. The liquefied test substance was then stirred, and the amount for the dosing mixture was removed. Information about the test substance provided to Product Safety Labs by the Sponsor was: Stability: The test substance is expected to be stable for the duration of testing Expiration Date: Not applicable B. Animals 3.B. 1 Number of Animals: 34 3.B.2 Number of Groups: 3 3.B.3 Number of Animals per Group: Preliminary Irritation Testing: 4 Test Group: 20 Test Vehicle Control Group: 10 3 3 .4 Sex: Male 3 3 .5 Species/Strain: Guinea pigs/Hartley albino 3 3 .6 Age/Body weight: Preliminary Irritation Group; Young adult Test and Test Vehicle Control Groups: Young adult/342-399 grams at experimental start 3 3 .7 Source: Received from Elm Hill Breeding Labs, Chelmsford, MA on January 16, 2002. 4. METHODS A. Husbandry 4.A.1 Housing: The animals were group housed in suspended stainless steel with mesh floors or plastic perforated bottom caging which conform to the size recommendations in the most recent Guide fo r the Care 'and Use o f Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week. 4.A.2 Animal Room Temperature and Relative Humidity Ranges: 17-22 C and 32-65% 4.A.3 Photoperiod: 12 hour light/dark cycle 4.A.4 Acclimation Period: 7 days 4A.5 Food: Pelleted Purina Guinea Pig Chow #5025 4A.6 Water: Filtered tap water was supplied ad libitum by automatic water dispensing system. Page 8 o f 25 Company Sauiiu.w u. uotsa ui conia.! i O CA CBi. H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 4.A.7 Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results o f this study. Analyses of the food and water are conducted at least once a year and the records are kept on file at Product Safety Labs. The dates of the most recent analyses are presented in Appendix A. B. Identification 4.B. 1 Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal. 4.B.2 Animal: Each guinea pig was marked with a color code and given a sequential animal number assigned to study #11716, which constituted unique identification. 5. PROCEDURE A. Preparation and Selection of Animals Within 24 hours prior to each application, the fur of each guinea pig assigned to test and test vehicle control groups was removed by clipping the dorsal area and flanks. Care was taken to avoid abrading the skin. Prior to study initiation, the animals were weighed and the skin was checked for any abnormalities. Only healthy animals without pre-existing skin irritation were selected for test. B. Induction Phase Once each week for three weeks, 0.4 g of an 80%' w/w mixture of the test substance in mineral oil was applied to the left side of each test animal for six hours using an occlusive 25 mm Hill Top Chamber. The chambers were secured in place and wrapped with non-allergenic DuraporeTM adhesive tape to avoid dislocation o f the chambers and to minimize loss of the test substance. After the six hour exposure period, the chambers were removed and the test sites were gently wiped with ethanol then water, using a clean towel to remove any residual test substance. Approximately 24 and 48 hours after each induction application, readings were made of local reactions (erythema) according to the scoring system described in Section 5.D. Four-tenths o f a milliliter of the vehicle (mineral oil, 100%) was used for the induction of the test vehicle control animals and scored as above. C. Challenge Phase Thirty days (See Section 11) after the first induction dose, 0.4 g o f an 80%' w/w mixture o f the test substance (HNIC) in mineral oil was applied to a naive site on the right rear flank o f each animal using the procedures described above. Four-tenths o f a milliliter of the vehicle (mineral oil, 100%) was applied to the right front flank of each test animal for the challenge phase. The test substance irritation control group was also treated with the test substance and vehicle for the challenge phase. These sites were evaluated for a sensitization response (erythema) approximately 24 and 48 hours after the challenge application according to the system described in Section 5.D.1 1The test substance, as received, was a solid. To enhance skin contact, the prepared test substance was crushed with a mortar and pestle, then moistened with mineral oil prior to application. Page 9 o f 25 , Does not contam I tfwH V tii Company Saniti H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 D. Scoring System 0 - no erythema 0.5 - very faint erythema, usually non-confluent 1 - faint erythema, usually confluent 2 - moderate erythema 3 - severe erythema with or without edema E. Body Weights Individual body weights of the animals were recorded prior to the first induction, weekly and again on the day after challenge. The mean and standard deviation were calculated for all body weights and body weight gains. The overall mean body weight gains for test and test vehicle control animals were evaluated statistically by an unpaired t test (INSTAT Biostatistics, created by GraphPad Software, San Diego, and CA.). F. Clinical Observations The animals were observed daily. If any unusual clinical signs were observed, they were recorded. 6. EVALUATION In order to evaluate the sensitization response noted during the challenge phase, two indices were used; one for incidence and one for severity. The incidence index was calculated to evaluate the incidence of erythema (sensitization response) approximately 24 and 48 hours after challenge according to the following: Scores of 1 or greater in the test group are required to be indicative o f sensitization. If scores of one (1) or greater are seen on the control animals, then the reactions of the test substance group animals that exceed the most severe control reactions are considered to be positive scores. Incidence is reported as the number of positive animals in each group divided by the total number of animals tested in that group. Severity is reported as the sum o f the test grades divided by the total number o f animals tested in a given group determined for both 24 and 48 hours. All average grades are to be rounded off to the nearest tenth of a unit. 7. HISTORICAL POSITIVE CONTROL VALIDATION STUDY The procedures used in this study were validated using a-hexylcinnamaldehyde (HCA), technical grade, 85% (purity) as a positive control substance. The most recent validation, PSL Study #11737, was performed by Product Safety Labs and completed on February 2 2 ,2 002. The raw ata and report fo ^ h is study are archived in Product Safety Labs Historical D a t i j H H ^ H H B A Company Sanitized. Does not contain TSCA CBI Page 10 o f 25 H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 This test was conducted at the Dayton Facility with HCA using Hartley strain albino guinea pigs from Elm Hill Breeding Labs following induction and challenge procedures similar to those described in Section 5. The data from this positive control study is summarized below. Positive Control Animals Positive Vehicle Control Animals Sensitization Response Indices Incidence o f Positive Response1 Severity2 Hours 24 48 24 48 5/10 3/10 0.75 0.65 0/5 0/5 0.30 0.20 8. STUDY CONDUCT This study was conducted at Product Safety Labs, 2394 Route 130, Dayton, New Jersey, 08810. The procedures as described in the protocol are based on the following testing guideline: U.S. EPA Health Effects Test Guidelines, OPPTS 870.2600, August, 1998 The primary technician for this study was Rolland Colis, B.S. 9. REFERENCES Robinson, M., Nusair, T., Fletcher, E., and Ritz, H., A Review of the Buehler Guinea Pig Skin Sensitization Test And Its Use in a Risk Assessment Process for Human Skin Sensitization in Toxicology, 61, 91-107,1990. Ritz, H., and Buehler, E., Planning, conduct, and interpretation o f guinea pig sensitization patch tests, in Current Concepts in Cutaneous Toxicity, V.A. Drill and P. Lazar (Eds.), Academic Press, New York, 25, 1980. 10. QUALITY ASSURANCE The final report was audited for agreement with the raw data records and for compliance with the protocol and Product Safety Labs Standard Operating Procedures. Dates o f inspections and audits performed during the study, and the dates o f reporting of the inspection and audit findings to the Study Director and Facility Management are presented in the Quality Assurance Statement. 11. DEVIATION FROM THE FINAL PROTOCOL The protocol states that approximately four weeks (27-28 days) after the first dose, a primary challenge of the HNIC o f the test substance will be applied to a naive site on the right flank of ' each guinea pig using a Hill Top Chamber. Due to a technician oversight, the challenge was performed on Day 30 o f the study. This deviation had no impact on the validity of the study. 12. FINAL REPORT A*1D RECORDS RETENTION A copy of the signed report, copies of all raw data generated at Product Safety Labs and the original signed protocol, will be maintained in the Product Safety Labs archives. 1Animals with scores greater than 0.5. Company Sanitized. Does not contain IS C A CBI 2Sum o f the erythema scores divided by the number of animals evaluated. Page 11 o f 25 H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 Laboratory-specific or site-specific raw data, such as personnel files and equipment records will be retained by the facility where the work was done. The original raw data, the original final report and a copy o f the protocol will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. 13. RESULTS Preliminary irritation testing scores for the test substance and historical positive control animals are presented in Table 1. Individual body weights and body weight gain for test substance and test substance vehicle control, and historical positive control animals are presented in Tables 2 and 3, respectively. Induction and Challenge Phase skin reaction scores for test substance and test substance vehicle control, and historical positive control animals are presented in Tables 4 through 7. All test and vehicle control animals survived and appeared normal throughout the study. There were no statistically significant differences between the overall body weight gain of the test and vehicle control animals. Induction Phase Test Animals (test substance applied as an 80%1 w/w mixture in mineral oil): No dermal irritation was noted for any test site during the induction phase. Test Vehicle Control Animals (mineral oil, 100%): No dermal irritation was noted for any test vehicle control site during the induction phase. Historical Positive Control Animals (HCA, as received): Very faint to faint erythema (0.5-1) was noted for all positive control sites during the induction phase. Challenge Phase Test Animals (test substance applied as an 80%' w/w mixture in mineral oil): No dermal irritation was noted for any test site following the challenge phase. Test Substance Irritation Control Animals (test substance applied as an 80%' w/w mixture in mineral oil): No dermal irritation was noted for any test substance irritation control site following the challenge phase. Test Animals (mineral oil, 100%): No dermal irritation was noted for any test site following the challenge phase. Test Substance Irritation Control Animals .(mineral oil, 100%): No dermal irritation was noted for any test substance irritation control site following the challenge phase. 1The test substance, as received, was a solid. To enhance skin contact, the prepared test substance was crushed with a mortar and pestle, then moistened with mineral oil prior to application. Page 12 of 25 Company Sanitized. Does not contain TSCA CBI H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 Historical Positive Control Animals (applied as a 50% w/w mixture of HCA (as received) in mineral oil): Five of ten positive control animals exhibited signs of a sensitization response (faint erythema [1]) 24 hours after challenge. Similar indications persisted at three o f these sites with very faint erythema (0.5) noted for all other sites through 48 hours. Historical Positive Vehicle Control Animals (applied as a 50% w/w mixture of HCA (as received) in mineral oil): Very faint erythema (0.5) was noted for three of five positive vehicle control sites 24 hours after challenge. Irritation persisted at two of these sites through 48 hours. 14. CONCLUSION Based on these findings and on the evaluation system used, H-25134 is considered not to be a contact sensitizer. The positive response observed in the historical positive control validation study with ahexylcinnamaldehyde, technical grade, 85% (as received) validates the test system used in this study (See Section 7). Company Sanitized, uoes nui umu. i ova cbi Page 13 of25 H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 1: PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1(TEST SUBSTANCE) Animal No. Sex 4166 4167 4168 4169 M M M M 803 24 48 00 00 00 00 Concentration i%)21 75 50 Hours after Patch Removal 24 48 24 48 0000 0000 0000 0000 25 24 48 00 00 00 00 1HNIC - Highest Non-Irritating Concentration 2 Four-tenths o f a gram or milliliter o f the crushed test substance was applied as w/w mixtures in mineral oil using an occlusive 25 mm Hill Top Chamber. 3 The test substance, as received, was a solid. To enhance skin contact, the prepared test substance was crushed with a mortar and pestle, then moistened with mineral oil prior to application. Page 14 of 25 Company Sanitized. Does not contain TSCA CBI H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 1 (cont): PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1(POSITIVE CONTROL-HCA) Historical Positive Control Validation Study2 Animal No. Sex 4170 4171 4172 4173 M M M M 100 24 48 11 Concentration (%)3 75 50 Hours after Patch Removal 24 48 24 48 0.5 0.5 0 0 0.5 0.5 0.5 0 0 0 0.5 0 0.5 0 0 0 0.5 0.5 0.5 0 0.5 0 25 24 48 00 00 00 00 1HNIC - Highest Non-Irritating Concentration ` PSL Study #11737, performed by PSL and completed on 2/22/02. J HCA was applied (0.4 ml) as received and as w/w mixtures in mineral oil. Page 15 of 25 Company SnitizcL Q&a uutcwuutiu. i CB1 H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 2: INDIVIDUAL BODY WEK3HTS/WEIGHT GAIN (g) Test Substance Group Total Animal No. Sex Initial Week 2 Gain Week Gain Week Gain (0 to wk 2) 3 (wk 2 to 3) 4 (wk 3 to 4) Day After Challenge Gain (wk 4 to 5) Weight Gain (wk 0 to 5) 4204 M 342 385 43 427 42 473 46 540 67 198 4205 M 387 432 45 490 58 578 88 640 62 253 4206 M 351 400 49 452 52 524 72 617 93 266 4207 M 352 390 38 418 28 488 70 520 32 168 4208 M 358 410 52 449 39 498 49 543 45 185 4209 M 359 414 55 456 42 512 56 535 23 176 4210 M 373 439 66 484 45 569 85 593 24 220 4211 M 369 436 67 484 48 553 69 635 82 266 4212 M 353 418 65 460 42 523 63 548 25 195 4213 M 362 426 64 479 53 550 71 622 72 260 4214 M 343 381 38 441 60 508 67 565 57 222 4215 M 348 390 42 446 56 505 59 560 55 212 4216 M 353 406 53 464 58 508 44 541 33 188 4217 M 346 392 46 467 75 534 67 571 37 225 4218 M 365 421 56 494 73 588 94 627 39 262 4219 M 346 395 49 444 49 517 73 530 13 184 4220 M 387 466 79 544 78 617 73 687 70 300 4221. M 367 410 43 475 65 549 74 . 589 40 222 4222 M 381 469 88 530 61 639 109 700 61 319 4223 M 351 403 52 464 61 523 59 564 41 213 M ean 359.7 414.2 54.5 468.4 54.3 537.8 69.4 586.4 48.6 226.7 Standard Deviation ! 14.0 24.8 13.4 31.0 12.9 142.9 16.0 52.2 21.6 41.9 Page 16 of 25 not contain TSCACBI Company Sanitize*1-Does H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABUE 2 (cont): INDIVIDUAL BODY WEIGHTS/WEK3HT GAIN (g) Test Vehicle Control Group - Challenge Phase Animal No. Sex Initial W eek 2 Gain (0 to wk 2) W eek 3 Gain (wk 2 to 3) W eek 4 Gain (w k 3 to 4) Day After C h a llen g e G ain (wk 4 to 5) Total W eight G ain (wk 0 to 5) 4224 M 352 401 49 456 55 532 76 572 40 220 4225 M 399 439 40 509 70 564 55 622 58 223 4226 M 356 404 48 458 54 533 75 558 25 202 4227 M 392 459 67 523 64 607 84 663 56 271 4228 M 384 445 61 509 64 598 89 647 49 263 4229 M 377 413 36 450 37 505 55 572 67 195 4230 M 374 402 28 441 39 489 48 558 69 184 4231 M 380 421 41 488 67 563 75 622 59 242 4232 M 364 403 39 441 38 530 89 558 28 194 4233 M 380 436 56 469 33 605 136 660 55 280 Mean 375.8 422.3 46.5 474.4 52.1 552.6 78.2 Standard Deviation 14.9 21.1 12.1 30.6 14.1 41.7 25.0 603.2 44.1 50.6 227.4 15.1 34.8 Company aiH titeu. uos uV) contain i s c a n p j Page 17 of 25 H -25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 3: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g) Historical Positive Control Validation Study1 Anim al Sex No. Initial W eek 2 G ain (0 to wk 2) W eek 3 Gain (wk 2 to 3) W eek 4 G ain (wk 3 to 4) Day After C h allen ge 4234 M 392 447 55 504 57 585 81 635 G ain (wk 4 to 5) Total W eight G ain (wk 0 to 5) 50 243 4235 M 426 485 59 543 58 616 73 674 58 248 4236 M 422 477 55 511 34 579 68 599 20 177 4237 M 391 431 40 503 72 572 69 632 60 241 4238 M 425 500 75 560 60 627 67 679 52 254 4239 M 434 501 67 561 60 633 72 699 66 265 4240 M 417 490 73 545 55 613 68 673 60 256 4241 M 405 456 51 497 41 554 57 589 35 184 4242 M 415 472 57 519 47 560 41 598 38 183 4243 M 419 481 62 544 63 639 95 680 41 261 M ean 414.6 474.0 59.4 528.7 54.7 597.8 69.1 Standard Deviation 14.4 23.0 10.5 24.5 11.1 31.4 14.1 Vehicle Control 4244 M 429 492 63 581 89 652 71 645.8 40.3 695 48.0 231.2 14.3 35.2 43 266 4225 M 427 495 68 572 77 606 34 641 35 214 4246 M 430 508 78 584 76 665 81 697 32 267 4247 M 455 499 44 596 97 678 82 719 41 264 4248 M 411 448 37 523 75 592 69 632 40 221 M ean 430.4 488.4 58.0 571.2 82.8 638.6 67.4 Standard Deviation 15.8 23.4 17.0 28.3 9.8 37.6 19.6 676.8 38.1 38.2 246.4 4.5 26.5 PSL Study #11737, performed by PSL and completed on 2/22/02. Page 18 o f 25 Sanitized. Does not contain TSCA CBI Company H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 4: INDUCTION PHASE SKIN REACTION SCORES Test Substance Group1 Induction Number Hours2* Animal No. 4204 4205 4206 4207 4208 4209 4210 4211 4212 4213 4214 4215 4216 4217 4218 4219 4220 4221 4222 4223 1 24 48 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 2 24 48 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 -0 0 3 24 48 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 'Four-tenths o f a gram o f the crushed test substance was applied as an 80% w/w mixture in mineral oil, using occlusive 25 mm Hill Top Chamber. 2Hours after induction dose. Page 19 o f 25 Company San.i.t.ized Does not contaui jSGACBl H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 4 (cont): INDUCTION PHASE SKIN REACTION SCORES Test Vehicle Control Group1 Induction Number H o u r s 12 Animal No. 4224 4225 4226 4227 4228 4229 4230 4231 4232 4233 1 24 48 00 00 00 00 00 00 00 00 00 00 2 24 48 00 00 00 00 00 00 00 00 00 00 3 24 48 00 00 00 00 00 00 00 00 00 00 1Four-tenths o f a milliliter o f the vehicle (mineral oil, 100%), was applied using an occlusive 25 mm Hill Top Chamber. 2Hours after induction dose. Page 20 of 25 Company Sanitized. Does not contain TSCA CBJ H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 5: INDUCTION PHASE SKIN REACTION SCORES Historical Positive Control Validation Study1 Positive Control Group2 Induction Number 1 Hours3 24 | 48 Animal No. 4234 1 0.5 4235 1 0.5 4236 0.5 0 4237 0.5 0.5 4238 0.5 0 4239 0.5 0.5 4240 1 0.5 4241 0.5 0.5 4242 1 0.5 4243 1 0.5 2 24 ! 48 1 0.5 11 1 0.5 1 0.5 0.5 0.5 1 0.5 11 1 0.5 11 11 3 24 48 10 1 0.5 1 0.5 0.5 0 0.5 0.5 1 0.5 1 0.5 0.5 0 11 1 0.5 1PSL Study #11737, performed by PSL and completed on 2/22/02. 2 Four-tenths o f a milliliter o f HCA was applied as received using an occlusive 25 mm Hill Top Chamber. J Hours after induction dose. Page 21 of 25 JpM^M^SinizaiL th v A C y H -25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 6: CHALLENGE PHASE SKIN REACTION SCORES Test Substance Group1 Animal No. 4204 4205 4206 4207 4208 4209 4210 4211 4212 4213 4214 4215 4216 4217 4218 4219 4220 4221 4222 4223 80%2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Hours after Dosing 24 Mineral oil (100%) 0 80%J 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 48 Mineral oil (100%) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1Four-tenths o f a gram o f the crushed test substance was applied as an 80% w/w mixture in mineral oil, using an occlusive 25 mm Hill Top Chamber to the right rear flank and four-tenths o f a milliliter of the vehicle (mineral oil, 100%) was applied to the right front flank. 2The test substance, as received, was a solid. To enhance skin contact, the prepared test substance was crushed with a mortar and pestle, then moistened with mineral oil prior to application. Page 22 of 25 Company Santuzeu. uOfcS IOvMOBI H -25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 6 (cont): CHALLENGE PHASE SKIN REACTION SCORES Test Substance Irritation Control Group1 Animal No. 4224 4225 4226 4227 4228 4229 4230 4231 4232 4233 80%2 0 0 0 0 0 0 0 0 0 0 Hours after Dosing 24 M ineral oil (100%) 0 80%2 0 00 00 00 00 00 00 00 00 00 48 M ineral oil (100%) 0 0 0 0 0 0 0 0 0 0 1Four-tenths o f a gram o f the crushed test substance was applied as an 80% w/w mixture in mineral oil, using an occlusive 25 mm Hill Top Chamber to the right rear flank and four-tenths o f a milliliter of the vehicle (mineral oil, 100%) was applied to the right front flank. The test substance, as received, was a solid. To enhance skin contact, the prepared test substance was crushed with a mortar and pestle, then moistened with mineral oil prior to application. Page 23 o f 25 Company Sanitized, Does noi couwut iu va CBl H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 TABLE 7: CHALLENGE PHASE SKIN REACTION SCORES1 Historical Positive Control Validation Study2 Animal No. Positive Control 4234 4235 4236 4237 4238 4239 4240 4241 4242 4243 Vehicle Control 4244 4245 4246 4247 4248 H o u r s *23 24 48 0.5 0.5 0.5 0.5 1 0.5 1 0.5 l1 0.5 0.5 0.5 0.5 0.5 0.5 11 11 0.5 0.5 0.5 0 00 00 0.5 0.5 Four-tenths o f a milliliter o f a 50% w/w mixture o f HCA (as received) in mineral oil was applied using an occlusive 25 mm Hill Top Chamber. 2 PSL Study #11737, performed by PSL and completed on 2/22/02. 3Hours after challenge dose. Page 24 of 25 Sanitized. Does not contain TSCACB! Company H-25134: Dermal Sensitization Test - Buehler Method DuPont-9303 APPENDIX A: FEED AND WATER ANALYSES On January 3, 2002 animal feed was analyzed for the presence of the following contaminants: Aldrin BHC-A (Alpha-Hexachlorocyclohexane) BHC-B (Beta-Hexachlorocyclohexane) BHC-D (Delta-Hexachlorocyclohexane) BHC-G (Lindane) Captan Chlordane Chlorpyrifos-Methyl Chlorpyrifos (Dursban) 4,4 DDD 4,4 DDE 4,4 DDT Dieldrin Dylox (Trichlorfon) Endosulfan I & n Endsolfan Sulfate Endrin Endrin Aldehyde Esfenvalerate Fenvalerate Heptachlor Heptachlor Epoxide Mavrik (Tau-Fluvalinate) Methoxychlor Mirex Quintozene None o f the above compounds were present above the limit o f detection (0.005 ppm) LABORATORY: FOOD PRODUCTS LABORATORY, INC. 12003 N.E. Ainsworth Circle Suite 105 Portland, OR 97220 On December 14, 2001, water was analyzed for NJDEPE Safe Drinking Water Act parameters. LABORATORIES: NEW JERSEY LABORATORIES NJDEPE LAB I.D. #15001 A.A. Labs Division 222 Easton Avenue New Brunswick, NJ 08901 SILLIKER LABORATORIES OF NEW JERSEY, INC. 400 South Avenue Garwood, NJ 07027 Results o f water analysis for possible contaminants were acceptable within regulatory standards. Page 25 o f 25 Sanitized- D oes noi contain TSCA CBI Company