Document 376xK3rXqpRmG0Ejd9RLMxwO

DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown, Pennsylvania 18901 Telephone: (215) 348-3868 AR226-3055 (215) ______________________Fax: 348-5081_____________________ Study Title Guinea Pig Dermal Sensitization - Modified Buehler Method with H-21891 Laboratory Project ID Haskell Laboratory Project ID 504-96 Data Requirement EEC 92/69 - Annex V - Part B.6 (1992) - 93/21 (1993), OECD Guidelines for Testing of Chemicals, No. 406, Skin Sensitization, adopted July 17, 1992 U.S. EPA Pesticide Assessment Guidelines Subdivision F, Hazard Evaluation: Human and Domestic Animals, Series 81-6, 1982; EPA Health Effects Testing Guidelines - Subpart E Specific Organ/Tissue Toxicity, September 1985, 40 CFR Part 798, and MAFF Japan, Testing Guidelines for Toxicology Studies, Dermal Sensitization Study, 59 NohSan No. 4200, 1985 Study Completed On 20 September 1996 Performing Laboratory White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown, Pennsylvania 18901 George E. Moore, B.S. for E.I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714 Study Monitor Gregory S. Ladies, Ph.D. Medical Research No. White Eagle Project No. Page 1 of 87 Company Sanitized. Does not contain TSCA CBI White Eagle Project Number: |^^^^R DuPont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 _Guinea-Eig-Dermal-Sensitization--^Modified-Buehler-Method------------------------------------ THIS PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS PagC2>mpariy Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road Doylestown, Pennsylvania 18901 (215) _______________TelephFaox:n(e2:15) 348-3504881-3868_____________________ GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT White Eagle ^^^^ Sponsor: Test Article: E. I. du Font de Nemours and Company H-21891 Test: Protocol Number: Guinea Pig Dermal Sensitization - Modified Buehler Method ^------^(Rev. 04/96) This study was carried out in compliance with the Principles of Good Laboratory Practice (GLP) as promulgated by the following agencies: U.S. Environmental Protection Agency FIFRA as stated in the Federal Register/ 40 CFR, Part 160, August 17, 1989. U.S. Environmental Protection Agency TSCA as stated in the Federal Register, 40 CFR, Part 792, August 17, 1989. OECD Principles of Good Laboratory Practice (c(81)30(Final), Annex 2) MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850) The study was conducted in compliance with the following exception: Treatment solutions were not analyzed for concentration, uniformity or stability of the test and control articles. The procedures used by trained personnel to prepare the treatment solutions insured: a) The accuracy of concentration because the test article diluent (vehicle) and positive control diluents were accurately measured with graduated devices. The test article was weighed on a balance accurate to at least 2 places. The positive control article was weighed on an analytical balance accurate to at least 3 places. b) Uniformity because all solutions were mixed prior to administration to the test system; and c) Stability because treatment solutions were prepared just prior to addition to the test system. ^ ^ { . ^ George E.^Moore, B.S. . 9/^/^ Date Study Director Sponsor: DuPont Specialty Chemicals E.I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, DE 19714 Page 3 Company Sanitized. Does not contain TSCA CBI White Eagle Project Number:! E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method GENERAL INFORMATION Test Material Substance Tested: H-21891 DuPont HLO 504-96 Synonyms/Other Codes: CAS Registry Number: Haskell No.: Composition: 21891 Purity: Major Impurities: Stability: . Physical Form: Sponsor: The test substance appears to be stable under the conditions of the study; no evidence of instability was observed. Tan liquid DuPont Specialty Chemicals E.I. du Font de Nemours and Company Barley Mill Plaza Wilmington, Delaware 19880-0038 WHITE EAGLE TOXICOLOGY PagcAmpany Sanitized. Does not contain LABORATORIES DOYLESTOWN, PENNSYLVANIA 18901 TSCA C--B--I - White Eagle Project Number:! E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 GENERAL INFORMATION Positive Control Material Material Tested: Purity: Synonyms: Other Codes: CAS Registry No.: Stability: l-chloro-2,4-dinitrobenzene Approximately 100% (supplied by Eastman Kodak Co.) DNCB Eastman Kodak Company/ Lot #A11R 97-00-7 (supplied by Eastman Kodak Co.) In the absence of visible evidence to the contrary, the test material was assumed to be stable under the conditions of administration. Study Initiated/Completed: 06/03/96 - 09/20/96 In-Life Phase Start - Completion: 06/18/96 - 07/19/96 WHITC EAGLE TOXICOLOGY LABORATO^^O^^O^^SYL^A^^^^'" TSCA CBI White Eagle Project Number: | J ^ E. I. du Font de Nemours and Company - H-21891 DuPont HLO 504-96 Guinea Pig Dermal Sensitization - Modified Buehler Method___________________ Guinea Pig Dermal Sensitization - Modified Buehler Method with H-21891 Summary The potential of H-21891 to produce contact dermal Sensitization in guinea pigs was assessed by the Modified Buehler Method. Twenty (20) test article animals were induced and challenged by occluded application of the test article to clipped intact guinea pig skin. The same procedures were carried out on contemporaneous control groups, except that the test article was replaced by the vehicle (saline) or positive control (l-chloro-2,4dinitrobenzene). No reaction to very faint redness was observed on the test article animals during the induction phase of the study. No reaction to very faint redness was observed at the test article naive site (right side) approximately twenty-four and forty-eight hours after the challenge application. No reaction was observed on any of the vehicle control animals after any of the induction applications. Twenty-four and forty-eight hours after the challenge application, no reaction was observed at either the vehicle or test article sites. No reaction to faint redness was observed for the positive control animals during the induction phase of the study. No reaction to faint redness was observed at 24 hours after the challenge and no redness to moderate redness at 48 hours after the challenge. No reaction was observed for the positive control naive animals at twenty-four and fortyeight hours after the challenge application of the positive control article. It was concluded therefore, that, under the study conditions utilized, repeated administration of H-21891 did not produce delayed contact hypersensitivity in guinea pigs. It was also concluded that the DNCB, when induced at 0.1% w/v in 50% ethanol: 0.9% saline and challenged at 0.07% w/v in acetone, produced delayed contact hypersensitivity in albino guinea pigs. On the basis of the nature of effects observed in this study and according to the guide for the labeling of dangerous substances published in the Official Journal of the European Communities (EEC Directive 93/21), H-21891 is not a skin sensitizer. PagaOfcmpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: ^ff^ E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 Guinea Pig Dermal Sensitization - Modified Buehler Method with H-21891 Summary (continued) ^y^t^c^ xy ^ ,, ^fteNL. George EAloore, B.S/ Study Director 9-gio ^ Date Reviewed/Audited by: (,^B.S. _ Quality Assurance Manager ^-2o-q(- Date pa p'6^7 WHITH EAGLE TOXICOLOGY LABORATOR^^'SSffiaT^^^As'ft^SSiaPl^ftBntainTSCA CB1 White Eagle Project Number: E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Bjiehler_Method_ DuPont HLO 504-96 White Eagle Project Number: Sponsor Study Number: Test Article: Study Initiation: 504-96 H-21891 06/03/96 Inspections Protocol Procedures Raw Data Draft Report Final Report Quality Assurance Statement Dates 06/05/96 06/05/96 08/07/96 08/21/96 09/20/96 06/27/96 08/08/96 08/22/96 07/17/96 08/16/96 09/05/96 07/19/96 09/05/96 09/06/96 Reports to Study Director and Management 06/14/96 08/16/96 08/27/96 09/06/96 09/20/96 This review was completed on 09/20/96 and released by: n-Tcook, B.S. Quality Assurance Manager Pag^&mpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number^^^HlF E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 TABLE OF CONTENTS Page Title Page .................................................................................................. 1 Good Laboratory Practice Compliance Statement ............................................ 3 General Information .................................................................................... 4 Summary 6 .................................................................................................. Quality Assurance Statement 8 ...................................................................".... Table of Contents 9 ....................................................................................... Objective of Test ....................................................................................... 12 Testing Guidelines .....................................................................................12 Good Laboratory Practice ........................................................................... 12 Justification for Test System ........................................................................ 12 Justification for Route of Administration ....................................................... 12 IACUC Review ......................................................................................... 12 Animals ................................................................................................... 12 Husbandry Conditions ............................................................................... 13 Dose Range-finding .............................................:..................................... 13 Induction Stage ......................................................................................... 13 Challenge Stage 15 ........................................................................................ Statistical Analysis ..................................................................................... 16 Mortality, Morbidity and Termination .......................................................... 16 Data Retention .......................................................................................... 16 Personnel ................................................................................................. 16 Test Article and Positive Control Characterization ......................................... 17 Interpretation of Results ............................................................................. 17 Table I - Scale for Evaluation of Dermal Irritation .......................................... 17 WHITE EAGLE TOXICOLOGY LABORATOR ?ntain TSCA CBI White Eagle Project Number: flJHI DuPont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method__________________ TABLE OF CONTENTS .(continued) Page Results 18 .................................................................................................... Conclusion ............................................................................................... 20 Signatures ................................................................................................ 20 Table 2 - Dose Range-finding Scores ............................................................ 21 Table 3 - Guinea Pig Dermal Irritation/Sensitization Scores - Test Article 23 .......... Table 4 - Guinea Pig Dermal Irritation/Sensitization Scores - Vehicle Control 25 ..... Table 5 - Guinea Pig Dermal Irritation/Sensitization Scores - Positive Control and Positive Control Naive 26 ........................... Table 6 - Daily Observations ................................--.................................... 27 Table 7 - Individual Body Weights ............................................................... 28 Appendix A - Copy of Protocol Cover Sheet & Protocol 30 ................................. Appendix B - Deviations from Protocol ........................................................ 42 Appendix C - Guinea Pig Dermal Sensitization - Modified Buehler Method with Positive Control (a-hexylcinnamaldehyde, tech, 85 %) 44 ............ Pagediftnpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 1890T WHITE DuPont HLO 504-96 EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown, Pennsylvania 18901 _Telephone:_(215).348-3868_ Fax: (215) 348-5081 Protocol Number: White Eagle Project Number: Testing Facility: Sponsor: Test Article: (Rev. 04/96) White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown, PA 18901 E. I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714 H-21891 Test Article Description: Tan liquid Test Article Received: 05/29/96 Vehicle Control Article: Vehicle Control Description: Normal Saline Solution (Vedco Inc., St. Joseph, MO 64504 Lot #5080419, Exp. 8/98) Clear liquid Positive Control Article: Positive Control Description: l-chloro-2, 4-dinitrobenzene (Eastman Kodak Co., Rochester, N.Y. Lot #A11R, Exp. 12/26/98) Yellow crystal Positive Control Vehicles: Ethanol (95%)/Acetone (99.7% pure) Positive Control Vehicles: Description: Clear liquids Dose-Rangefinding Initiated: 06/05/96 In-Life Starting Date: In-life 06/18/96 Completion Date: . 07/19/96 Test: Guinea Pig Dermal Sensitization - Modified Buehler Method* * Rite, H.L. and Buehler, E.V., Planning, Conduct, and Interpretation of Guinea Kg Sensitization Patch Tests, current Concepts in Cutaneous Toxidty, Drill & Lazar editors. Academic Press 1980. WHITE EAGLE TOXICOLOGY IABORATO^^(W?SE^(^t?^S^Ja^9^P"tain TSCA CBI White Eagle Project Numbeq DuPont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 _G.uiriea Pig Dermal Sensitization - Modified Buehler Method Objective of Test: To assess the contact dermal sensitization potential of the test article. Guidelines: Good Laboratory Practice: Justification for Test System: Justification for Route of Administration: The methods employed in the testing of this test article -were similar to those described in the EEC 92/69 Annex V, Part B.6 (1992) 93/21 (1993); the OECD Guidelines for Testing of Chemicals, No. 406, Skin Sensitization, adopted July 17, 1992; U.S. EPA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human & Domestic Animals, Series 81-6, 1982; EPA Health Effects Testing Guidelines Subpart E - Specific Organ/Tissue Toxidty, September 1985, 40 CFR Part 798, and the MAFF Japan Testing Guidelines for Toxicology Studies, Dermal Sensitization Study, 59 NohSan No. 4200, 1985. This study was conducted in compliance with the U.S. EPA FIFRA Good Laboratory Practice Standards (40 CFR 160), U.S. EPA TSCA Good Laboratory Practice Standards (40 CFR 792), OECD Principles of Good Laboratory Practice (c(81)30(Final), Annex 2), and MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850). The laboratory albino guinea pig was the system of choice since it is required in the EPA, OECD, EEC and MAFF Japan guidelines for this type of study and has a long history of use in dermal sensitization studies. The specific route of administration (dermal) is required in the testing guidelines used for this study. IACUC Review: The Protocol for this study was reviewed by White Eagle's Institutional Animal Care and Use Committee (IACUC), and complies with acceptable standards for animal welfare and humane care. Animals: The adult male Hartley guinea pig, weighing 457 to 557 grams, was used for this study. The animals were obtained from Ace Animals Inc., Boyertown, PA 19512, (U.S.D.A. License #23-B-009). Screen animals weighed between 366 and 465 grams. Animals (except screen animals) were weighed prior to initiation, weekly (6-8 days), and after the final dermal evaluation. The animals (except for screens) were randomized into groups using computer generated random numbers. The initial body weights (Day -1) were statistically evaluated by Analysis of Variance (ANOVA) using CSS: Statistica, 3.1 copyright, Statsoft Inc. 1991. The animals were housed and maintained in accordance with standards set forth in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23). The guinea pigs were acclimated to the laboratory for at least 7 days prior to the initial induction application. WHITE EAGLE TOXICOLOGY LABORATC P^iaq|9"ta'" TSCA CBI White Eagle Project Number:! DuPont HLO 504-96 E. I. du Font 'de Nemours and Company - H-21891 _Gumea-Eig-Dermal-Sensitization^Modified-Buehler-Method- Animals: (continued) Husbandry Conditions: The animals were individually identified by color coding with solvent- resistant markers (Blue, Red, White and Green). Each cage was identified with a cage card displaying the project number, date initiated, animal numbers, group, sex and responsible technician's initials. Clinical observations were performed daily during the study. All test animals were euthanized with an intraperitoneal injection of sodium pentobarbital at the conclusion of the study. Temperature: 18C - 27C (65F - 80F) Relative Humidity, 42 - 71 % Light: 12 hour light/dark cycle Diet: Purina Certified Guinea Pig Diet #5026 and tap water were provided ad libitum. Based on our current knowledge, no contaminants are known to be in this diet or water which might be expected to interfere with the objectives of the study. Dose Range-finding: Caging: Stainless steel cages with elevated wire mesh flooring, 1 guinea pig/cage. Prior to initiation of the study, the irritation potential was determined. Two groups of four unexposed animals were exposed to two concentrations of the test material by the patching technique described in the induction stage. In this test, both sides of the animal were closely clipped and one group was exposed to the test article as supplied (100%) and at a 75% concentration in 0.9% saline and a second group was ' exposed to concentrations of 50% and 25% in 0.9% saline. The test site was wiped with deionized water following the removal of the patch, examined and scored for irritation according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section) at 24 and 48 hours from the initial patch application. Method of Study Performance: No reaction was observed at the 100% (as supplied), 75%, 50% or 25% sites. The induction and challenge applications were performed using the test article, as supplied. Test Article .Group Induction Stage: A group of twenty (20) guinea pigs was closely clipped over the induction site (approximately 1.5 X 1.5 inches) on their left flank during the day prior to the day of initiation and repeated as necessary. Based on the range-finding results, the induction applications were performed using the test article, as supplied. A 0.5 mL portion of the test article, was applied to a 25 mm HillTop ChamberTM (HillTop Research Inc., Cincinnati, OH 45242) on a piece of dental dam (approximately 2 inches x 2 inches). After positioning the chamber and occlusive dental dam onto the prepared skin test site, plastic wrap was wrapped snugly around the trunk of the animal and overwrapped with ElastikonTM. WHITE EAGLE TOXICOLOGY LABORATOB ?ntain TSCA CBI White Eagle Project Number: ^^fl^ E. I. du Font de Nemours and Company - H-21891 DuPont HLO 504-96 -Gumea-Eig-Dermal-Sensmzation^Modlfied.Buehler Method Method of Study Performance: (continued) Induction Stage: (continued) Test Article Group (continued) Approximately six hours later, the chamber was removed, and the test site was wiped with deionized water. The test site was examined following removal of the patch and scored for irritation according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section) at approximately 24 and 48 hours from the initial patch application. The procedure was performed once a week for three weeks (7-day intervals), for a total of three, six-hour applications with the test article. Vehicle Control Group A group of ten (10) guinea pigs was closely clipped over the induction site (approximately 1.5 X 1.5 inches) on their left flank during the day prior to the day of initiation and repeated as necessary. A 0.5 mL portion of 0.9% saline was applied to a 25 mm HillTop ChamberTM on a piece of dental dam (approximately 2 inches x 2 inches). After positioning the. chamber and occlusive dental dam onto the prepared skin test site, plastic wrap was wrapped snugly around the trunk of the animal and overwrapped with ElastikonTM. Approximately six hours later, the chamber was removed, and the test site was wiped with deionized water. The treated site was scored for irritation according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section) at approximately 24 and 48 hours from the initial patch application. The procedure was performed once a week for three weeks (7-day intervals), for a total of three, six-hour applications with the vehicle. Positive Control A group of five (5) guinea pigs was closely dipped over the induction site (approximately 1.5 X 1.5 inches) on their left flank during the day prior to the day of initiation and repeated as necessary. A 0.5 mL portion of 0.1% l-chloro-2,4-dinitrobenzene (DNCB) w/v in 50% ethanol: 0.9% saline was applied to a 25 mm HillTop ChamberTM on a piece of dental dam (approximately 2 inches x 2 inches). After positioning the chamber and ocdusive dental dam onto the prepared skin test site, plastic wrap was wrapped snugly around the trunk of the animal and overwrapped with ElastikonTM. WHITE EAGLE TOXICOLOGY ^%^d%^o^lti':E^?9^fl^fe?ntain LABORAT01 "^CA CBI White Eagle Project Number:! DuPont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method Method of Study Performance: (continued) Induction Stage: (continued) Positive Control (continued) Approximately six hours later, the chamber was removed, and the test site was wiped with ethanol and then with deionized water. The test site was examined following removal of the patch and scored for irritation according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section) at approximately 24 and 48 hours after the initial patch application. The procedure was performed once a week for three weeks (7-day intervals), for a total of three, six-hour applications with DNCB. Challenge Stage: Test Article Group After the third induction application, the animals were rested for approximately 15 days. At the termination of the rest period, a challenge application, using the procedures described previously, was applied to a naive challenge site (on the dipped right flank). Based on the rangefinding results, the challenge application was performed using the test article, as supplied. The challenge application remained on for approximately 6 hours. Upon removal of the challenge application, the contact area was wiped with deionized water. Approximately 24 and 48 hours after the challenge application, the sites were examined for dermal irritation and signs of elicited sensitization, according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section). Vehicle Control Group After the third induction application, the animals were rested for approximately 15 days. At the termination of the rest period, using the procedures described previously, the vehicle control group was challenged with 0.9% saline on the clipped caudal left flank. The test article, as supplied, was placed on the naive clipped right flank. The challenge application remained on for approximately 6 hours. Upon removal of the challenge application, the contact areas were wiped with deionized water. Approximately 24 and 48 hours after the challenge application, the sites were examined for dermal irritation and signs of elicited sensitization, according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section). Page,J.5_ WHITE EAGLE TOXICOLOGY LABORATOR i TSCA CBI White Eagle Project Number: j E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method Method of Study Performance: (continued) Challenge Stage: (continued) Positive Control Group DuPont HLO 504-96 Statistical Analysis: Mortality. Morbidity and Termination: Data Retention: Personnel: After the third induction application, the animals were rested for approximately 15 days. At the termination of the rest period, a challenge application, 0.07% DNCB in acetone, using the procedures described previously, was applied to a naive challenge site (on the clipped right flank). At the time of challenge, a group of five naive animals was also treated with 0.07% DNCB in acetone,, using procedures described previously. The challenge application for these groups remained on for approximately 6 hours. Upon removal of the challenge application, the contact area was wiped with acetone and then with deionized water. Approximately 24 and 48 hours after the challenge application, the sites were examined for dermal irritation and signs of elicited sensitization, according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section). Individual body weight percentage gain was statistically evaluated by Analysis of Variance (ANOVA), with the Neuman Keuls test applied in the event of a significant finding (P<0.05) for the data as a whole. Statistical analysis was performed using CSS: Statistica, 3.1 copyright, Statsoftlnc. 1991. No deaths occurred during the study. All animals were euthanized with an intraperitoneal injection of sodium pentobarbital at the conclusion of the study. All raw data generated, all records accumulated during the performance of this study and a copy of the final report will be retained at White Eagle Toxicology Laboratories for a period of ten (10) years, after which time the data will be shipped to the Sponsor or, discarded upon written request from the Sponsor. The following is a list of personnel for this study: George E. Moore, Study Director Shashi P. Logani Richard J. Hershman Stanley Sekelewski Lisa Wilkoski Paged6npany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number:! DuPont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 Guinea Fig Dermal Sensitization - Modified Buehler Method Test Article and Positive Control Characterization: The Sponsor was responsible for test article characterization and stability determination prior to initiation of the study. Details regarding the identity, strength/ stability, purity and composition of the test article are in the possession of the -Sponsor. Information supplied to White Eagle Toxicology Laboratories regarding characterization of the test article will be filed in the archives of White Eagle Toxicology Laboratories, Doylestown, Pennsylvania. Details regarding the identity, strength, stability, purity, and composition of the positive control are provided by the Manufacturer and are on file at White Eagle Toxicology Laboratories/ Doylestown, Pennsylvania. The positive control has been examined and has been found to be soluble in ethanol and acetone. Mixtures or dilutions were not analyzed by White Eagle or the Sponsor. Interpretation of Results: All non-eschar and non-necrotic test sites were scored on a scale of 0 to 3 as indicated in Table I. TABLE I Scale for Evaluation of Dermal Irritation 0 = no reaction 0.5 = very faint redness (usually nonconfluent) 1 = faint redness (usually confluent) 2 = moderate redness 3 == severe redness with/without swelling Note: 0.5 scores were recorded in the raw data as a + Scores of 1 or greater in the test group are required to be indicative of sensitization. If scores of one (1) or greater are seen on the vehicle control animals, then the reactions of the test article group animals that exceed the most severe vehicle 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 of the test grades divided by the total number of animals tested in a given group determined for both 24 and 48 hours. Grades of + are equal to 0.5 for calculation of severity indices. All average grades are to be rounded off to the nearest tenth of a unit. A minimum of two out of five positive control animals must be shown to have elicited a positive reaction (Scores .>. 1) to me positive control material to validate the test system for this particular sample. WHTTE EAGLE TOXICOLOGY LABORATOR O^E^ma0^3'" TSCA CB1 White Eagle Project Number: DuPont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method Interpretation of Results: (continued) If there is no evidence of sensitization in the study, it is concluded that the risk of sensitizing humans is minimal. If results are seen which indicate animals in the test group show significant responsiveness compared to the vehicle control group, it is concluded that subsequent exposure of humans to the material may involve substantial risk of sensitization. This rating scale is not fixed rigidly. Scientific evaluation of the data and judgement will determine whether the material will be classified as a sensitizer or not. Tables 2 through 7 Dose Range-finding During the dose range-finding portion of the study, the test article, when applied dermally as supplied and at 75%, 50% and 25% concentrations in 0.9% saline did not produce any dermal irritation at 24 and 48 hours post application. The induction and challenge phases of the study were performed using 0.5 mL of the test article, as supplied. Test Article Group - Induction and Challenge All test animals appeared to be normal throughout the study. All animals exhibited an overall gain in body weight during the study (-1 through 32 days). One of twenty animals lost weight between Days 28 and 32. The water valve was functional and the animal had feed. A statistically significant difference was identified between the individual body weight percentage gains of the test article group and each of the other three groups of animals (Analysis of Variance). No reaction to very faint redness was observed during'the induction phase of the study. No reaction to very faint redness was observed at the test article naive site (right side) approximately twenty-four and forty- eight hours after the challenge application. The incidence of sensitization was 0/20. WHITE EAGLE TOXICOLOGY LABORATr0^1 ^yl^^^E^sft^iffi^efl^'" "^A CBI White Eagle Project Number:! DuPont HLO 504-96 E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method Results: (continued) Vehicle Control Group All vehicle control animals appeared normal and exhibited an overall gain in body weight during the observation period (-1 through 32 days). A statistically significant difference was identified between the individual body weight percentage gains of the vehicle control group and the test article group. There were no statistically significant differences identified between the individual body weight percentage gains of the vehicle control group of animals and any of the other two groups of animals (Analysis of Variance). No reaction was observed on any animals after any of the induction applications as well as approximately 24 and 48 hours after the challenge application at either the vehicle or test article sites. Saline is not a sensitizer. Positive Control Group All positive control animals appeared normal and exhibited an overall gain in body weight during the observation period (-1 through 32 days). A statistically significant difference was identified between the individual body weight percentage gains of the positive control group and the test article group. There were no statistically significant differences identified between the individual body weight percentage gains of the positive control group of animals and any of the other two groups of animals (Analysis of Variance). No reaction to faint redness was observed during the induction phase of the study. No reaction to faint redness was observed at 24 hours after the challenge and no redness to moderate redness 48 hours after the challenge. The incidence of sensitization was 3/5 and severity was 0.4 at 24 hours and 0.9 at 48 hours. The positive control (l-chloro-2,4-dinitrobenzene), is a dermal sensitizer in this study. Naive Positive Control Group All positive control naive animals appeared normal and exhibited an overall gain in body weight during the observation period (-1 through 32 days). One of five animals lost weight between Days 28 and 32. The water valve was functional and the animal had feed. A statistically significant difference was identified between the individual body weight percentage gains of the positive control naive group and the test article group. There were no statistically significant differences identified between the individual body weight percentage gains of the positive control naive group of animals and any of the other groups of animals (Analysis of Variance). WHITE EAGLE TOXICOLOGY LABORAT^01^%?Yl^^ti^sft^AQfeeflntain TSCA CBI White Eagle Project Number:' DuFont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 _G.umea_Eig_Dermal-Sensitization^Modified-Buehler-Method- Results: (continued) Naive Positive Control Group (continued) No reaction was observed at twenty-four and forty-eight hours after the challenge application of the positive control article. Positive Control - a-hexylcinnamaldehyde. tech. 85% Conclusion: A second positive control was run at White Eagle Labs (Appendix D) in order to document the effect of a weak sensitizer in this test system. The test article, H-21891, when induced and challenged at a dose level of 0.5 mL of test article, as supplied, is not a dermal sensitizer in albino guinea pigs. The positive control, l-chloro-2/4-dinitrobenzene, when induced at a dose level of 0.1% w/v concentration in a 50% ethanol: 0.9% saline solution and challenged at a dose level of 0.07% w/v concentration in acetone is a dermal sensitizer in albino guinea pigs. Signatures: Submitted by White Eagle Toxicology Laboratories: .^<^^^^^ George lyMoore, B.S^ ^Lg-^ Study Director 9^-^ Date Reviewed/Audited by: 9-^o-SL Date WHITE EAGLE TOXICOLOGY Page(2ftnpany Sanitized. Does notcontain LABORATTOORRIES DOYLESTOWN, PENNSYLVANIA 1B90I TlOSCOAM^CDBlI --DI White Eagle Project Number:^ E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 2 Dose Range-finding Scores DuPont HLO 504-96 Temporary Animal Number 142 143 144 145 Screen #1 - Left Side 0.5 mL of H-21891 24 Hours 0 48 Hours 0 Temporary Animal Number 142 143 144 145 Screen #2 - Right Side 75% concentration of H-21891 in 0.9% saline 24 Hours 0 48 Hours 0 Body Weights (g) Temporary Animal #142 393 Temporary Animal #143 380 Temporary Animal #144 380 Temporary Animal #145 366 Page21 WHITE EAGLE TOXICOLOGY LABORATOREg-'^WBeEaiT&Wt'^ESris'^CifiHIBQtgBSDntain TSCA CBI White Eagle Project Number:----------! E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 2 fcontinued) Dose Range-finding Scores DuPont HLO 504-96 Temporary Animal Number 146 147 148 149 Screen #3 - Left Side 50% concentration of H-21891 in 0.9% saline 24 Hours 0 0 0 0 48 Hours 0 0 0 0 Temporary Animal Number 146 147 148 149 Screen #4 - Right Side 25% concentration of H-21891 in 0.9% saline 24 Hours 0 48 Hours 0 0 0 0 0 0 0 Body Weights (g) Temporary Animal #146 375 Temporary Animal #147 382 Temporary Animal #148 418 Temporary Animal #149 465 Pager WHTTE EAGLE TOXICOLOGY LABORATOK y.^As^^lBql^nta'n TSCA CB1 White EagIe Project Numi)er:^H| DuPont HLO 504-96 E. I. du PC)nt de Nemours and Corn.pany - H-21.891 Guinea Pi}; Dermal Sensitiization - Nlodined Butihler Me1fchod TABL E3 Guniea PigJDiirmal Irritation/Sensi tization Scor'PS rrest Article - H-2189:1 Indue)tton Study Anwal Number 1 Applii;ation 1 24Hrs 48Hrs 0 0 Application 2 24Hrs 48Hrs 0 0 Applic ation 3 24Hrs 48Hrs 0 0 2 0 0 0 + + 0 3 0 0 + 0 0 0 4 0 0 0 0 0 0 5 0 0 + 0 0 0 6 0 0 0 0 0 0 7 0 0 0 0 0 + 8 0 0 + 0 0 0 9 0 0 + 0 0 0 10 0 0 0 0 + + 11 0 0 0 0 0 0 12 0 0 0 0 0 0 13 0 0 + + + + 14 0 0 0 0 0 0 15 0 0 0 0 0 0 16 0 0 + 0 0 0 17 0 0. 0 0 0 0 18 0 0 0 0 + + 19 0 0 0 0 0 0 . 20 0 0 +" 0 0 0 Note: All induction applications were performed with 0.5 mL of the test article, as supplied. WHITE EAGLE TOXICOLOGY LABORATO^E^RSS^S^^^ft^SSn'yfefiffT"S^C'A" CBI White Eagle Project Number: ------f DuPont HLO 504-96 E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 3(continued) Guinea Pig Dermal Irritation/Sensitization Scores Test Artide - H-21891 Challenge Study Animal Number 24 Hrs 48 Hrs 100 2 00 3 +0 4 0 0 5 0 + 6 7 0000 .8 + 0 9 0 0 10 0 0 11 0 0 12 0 0 13 0 0 14 0 0 15 0 + 16 0 0 17 , 18 19 20 0 + 00 0 0 0 0 Note: The challenge application was performed with 0.5 mL of the test article, as supplied. WHITE EAGLE TOXICOLOGY LABORATOI^^PftE^^'^SAs-R.^SSiAafeStBntTaS'"CA CBI White Eitigle Pn)ject Numbi^rflj DuPont H LO 504-96 E. I. du Pont de;Ne:mours ;and ( ^ompany-H -21891 Guinea I'ig Dermal Sensifcsatiori - Modiifiedl3uehler M(ithod TA BL)E4 Guin eaPij^ Derm.allrritation/Senssitizationi Scores \ 'ehide Control Study Animal Number Hrs: Indviction - 0.9% Salin e 1 24 48 2 24 48 3 24 48 21 0 0 0 0 0 0 Cha:Uenge H-21891 24 48 0 0 Oia1 lenge 0.9% Saline 24 48 0 0 22 0 0 0 0 0 0 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 / 24 0 0 0 0' 0 0 0 0 0 0 25 0 0 0 0 0 0 0 0 0 0 26 0 0 0 0 0 0 0 0 0 0 i 27 0 0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 0 29 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 Note: The challenge application of the test article sites was performed with 0.5 mL of the test article, as supplied. Page-25 WHTTE EAGLE TOXICOLOGY LABORATOIfe^;-CWBeSl^^t^lrts'i@<'^fiMclfe^aPntain TSCA CB1 White Eagle Project Number: E. I. du Font de Nemours anid Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 5 DuPont HLO 504-96 Guinea Pie Dermal Irritation/Sensitization Scores Positive Control (DNCB) and Naive Positive Control Induction Study Animal Number Application 1 24Hrs 48Hrs Application 2 24Hrs 48Hrs Application 3 . 24Hrs 48Hrs Challenge 24Hrs 48Hrs 31 0 + 32 0 0 1+ 0+ 1+11 33 + + 1 1 34 0 0 1111 1111 35 0 0 1 1 1 1 + 0 0 2 + 1 0 + Positive Control Naive 1 Challenge 24Hrs 48Hrs 36 no induction applications 0 0 37 no induction applications 0 0 38 no induction applications 0 0 39 no induction applications 0 0 40 no induction applications Note: All sites stained yellow; however, erythema could be scored. 0 0 Induction: DNCB Preparation = 0.1% DNCB w/v in 50% ethanol (95% Gear Spring Grain Alcohol/ Clearspring Distilling Co., Clearmont and Frankfort, KY and Cincinnati, OH, Lot #10465177, Exp. 6/18/97) and 0.9% Normal Saline Solution (Vedco Inc., St. Joseph, MO 64504, Lot #5080419, Exp. 8/98). Challenge: DNCB Preparation = #020795 Exp. 5/98). 0.07% DNCB in acetone (Allied Signal Inc., Morristown, NJ, Lot WHITE EAGLE TOXICOLOC Paee- StEwswrtaxpntain TSCA CBI White Eagle Project Number: IH^y E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitizarion - Modified Buehler Method TABLE 6 Daily Observations Day -1: Day 1: Day 2: Day 3: Day 4: Day 5: Day 6: Day 7: Day 8: Day 9: Day 10: Day 11: Day 12: Day 13: Day 14: Day 15: Day 16: Day 17: Day 18: Day 19: Day 20: Day 21: Day 22: Day 23: Day 24: Day 25: Day 26: Day 27: Day 28: Day 29: Day 30: Day 31: Day 32: All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. All animals in all groups appeared to be normal. DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATO^^lSa^O^^E^SYe^U??^"^1" "TSGA CBI White Ea gle Project N umber:^ E. I. du I 'ont de Nemc?urs and iCompany' - H-218 91 Guinea P'ig Dermal Sensitizatioln - Modified Buerder Metliiod TABLE 7 DuPont HLO 504-96 Test ArtidIe - H-21891 Individu al Body Weights ^ Study Animal Number Initiarr Day 7 Day 14 Day 21 Day 28 Final Day 32 Change (-1 to 31) 1 530 2 464 3 517 4 496 5 538 6 474 7 507 8 543 9 481 10 525 11 508 12 536 13 473 14 491 15 524 16 518 17 496 1 ' 18 470 19 512 20 541 586 641 694 756 492 531 589 626 558 588 638 658 529 562 611 672 577 611 657 699 504 540 579 626 540 571 623 665 574 609 656 707 518 561 603 629 567 619 669 716 535 597 616 681 567 625 686 743 499 550 601 623 531 571 639 690 554 620 678 714 562 625 687 747 539 591 645 709 505 537 581 634 560 595 647 683 582 627 700 752 771 +241 645 +181 682 +165 673 +177 ' 709 +171 646 +172 679 +172 717 +174 641 +160 739 +214 650 +142 747 +211 634 +161 703 . +212 727 +203 757 +239 717 +221 642 +172 688 +176 766 +225 Mean 507 Std.Dev. 25 544 589 640 687 697 29 34 38 45 45 +189 29 s Vehicle Control - Saline Study Animal Number 21 22 23 24 25 26 27 28 29 30 Ininal+ 501 533 464 557 489 484 521 464 537 506 Day 7 Day 14 Day 21 Day 28 Final Day 32 541 586 640 669 676 577 . 619 667 740 742 526 570 640 718 730 608 663 715 787 788 542 589 640 721 726 535 564 621' 667 674 583 624 689 747 753 512 542 575 623 627 582 626 661 697 715 577 641 722 782 788 Change r-lto321 +175 +209 +266 +231 +237 +190 +232 +163 +178 +282 Mean 506 Std.Dev. 31 558 602 657 715 722 31 38 44 52 51 +216 40 += Body weights taken on Day -1 (the day before the first induction application of test article or vehicle). WHITE EAGLE TOXICOLOGY LABORATS01^^E^OTOa',1!-^^^^^^^?"^'" TSCA CBI White Ea^^le Project'.Number^B E. I. du P ont de Nernours and ( ^ompan^' - H-218!31 Guinea Pi.g Dermal'iensitizatiori - Modified Buehler Meth od TABLE 7fcontinned) Individu alBody1Weights^fR) Positive Ccmtrol fDinitrochlorobenzene) Study Animal Number 31 32 33 34 35 Initial+ 499 542 528 491 457 Mean 503 Std.Dev. 33 Day 7 547 547 581 553 525 551 20 Day 14 598 631 642 603 615 Day 21 636 694 696 661 685 Day 28 697 734 768 738 745 Final Day 32 703 753 769 743 756 618 674 736 745 19 26 26 25 DuPont HLO 504-96 Change (-1 to 32) +204 +211 +241 +252 +299 +241 38 Naive Positive Control (Binitrochlorobenzenel Study Animal Number Initialt Day 7 Day 14 Day 21 Day 28 Final Day 32 Change (-1 tp 32) 514 37 486 38 522 39 537 567 635 703 764 770 508 585 637 703 699 565 622 684 745 761 618 687 734 792 792 +256 +213 +239 +255 40 477 535 577 634 677 684 +207 Mean 507 Std.Dev. 25 559 621 678 736 741 41 44 43 46 47 +234 23 + = Body weights taken on Day -1 WHITE EAGLE TOXICOLOGY LAB ^QWte^ffiQgLBQtaiontain TSCA CBI White Eagle Project Number.^f^ff E. I. du Font de Nemours and Company - H-21891. Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 APPENDIX A COPY OF PROTOCOL COVER SHEET & PROTOCOL pa p-p ^0 WHITE EAGLE TOXICOLOGY LABORATOiaE^93%EB^iWJ^ESrts-ewe9lfiq6?8TpSnCtAainCBI White Eagle Project Number I DuPont HLO 504-96 -ERO-T-OCOLJ (Rev.-04/96) Guinea Pig Dermal Sensitizalion - Modified Buehler Method' White Testing Fadlity: Eagle Toxicology Laboratories 2003 Lower State Road j^^L Doylestown, PA 18901 -7 / OO/ Sponsor E.I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine f. Newark, DE 19714 TpstArHrip. Vn -\~---?r5^?-HT-phr4b^^-- --------C^--TT--O(--)LL--o--oL--L'--..^--^^--',,--r.r--<>--J\QSAli----!-------------------------------------- White Eagle Project Number: Test Article Description: Test Article Receipt Date: Proposed In-Life Starting Dale: Proposed In-Life Completion Date: Tfl^ li-,ui^ ^9/9^ ^/V/-?^ VlWt -itlO/QC. To be Completed by White Eagle REGULATORY COMPLIANCE INFORMATION The Sponsor is responsible for test article characterization prior to initiation of the study: 1. The identity, strength, composition & purity of the Test Article has been documented by the Sponsor as of s / n /^ly . 2. The Sponsor has labeled the Test Article containers) with the name, expiration date, batch number, and where applicable storage conditions S3 YES D NO 3. The Test Article has been determined by the Sponsor to be stable under specific test and storage conditions as of *5 / n / "^. 4. If applicable, reserve samples of Test Article/Vehicle MIXTURES are to be RETURNED TO SPONSOR FOR ANALYSIS and STABILITY TESTING 0 YES E^NO EPA CONFIDENTIALITY STATEMENT B"AA. Q B. Sponsor will include their own EPA.Confidentiality Statement in the Final Report White Eagle will include standard EPA Confidentiality Statement in the Final Report If "B", select one of the following: D No Data Confidentiality Claims D Data Confidentiality Claims This study does not unnecessarily duplicate previous experiments nor is there an acceptable in-vitro method that can substitute for the use of animals. PROTOCOL AMENDMENTS Testing Facility: Gb.^^7)4^ Study Director PROTOCOL APPROVAL ^-3-^ Date Sponsor; ^- r^-/4 Sponsor Representative SJfJn1l^ Date WHITE EAGLE.TQXICOLOGY LAB ^m^mf)^^^ "'"SCA CBI White Eagle Project Number: '. DuFont HLO 504-96 White Eagle Toxicology Laboratories 2003 Lower State Road Doylcslown, Pennsylvania, 18901 Telephone: (215) 348-3868 Pax: (215) 348-5081 Protocol Numbcn Protocol Name: Guinea Pig Dermal Scnsitization - Modified Bucliler Method This protocol contains information that is proprietary and confidential to White Eagle Toxicology Laboratories. Use of this information is restricted to the parties to wliich While Eagle Toxicology Laboratories has issued it in the course of business. Any duplication or distribution of the contents williout prior approval of White Eagle Toxicology Laboratories is strictly prohibited. r^^^^^^^^^^0^^" WHITE EAGLE TOXICOLOGY LABO.RAT01 CBI White Eagle Project Numbe! DuPont HLO 504-96 'WHITE'EATGLE TOXICOLOGY LABORATORIES 2003 Lower Slate Road Doylestown, Pennsylvania, 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 Protocol Numbrr: ?ev. 04/96) Project Number: {to be completed by While Eagle Toxicology Laboratories} Testing Facility: White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown, PA 18901 Sponsor: E. I. du Font de Ncmours and Company Haskell Laboratory for Toxicology and Industrial Medicine " . Elkton Road, P.O. Box 50 Newark, DE 19714 Test Article: {to be specified} Test Article Description: {to be specified} Positive Control Article: Positive Control Description: Positive" Control Vehicle.^: Positive Con iTol Vehicle Descriptions: Test Article Received: l-cliloro-2, 4-dinitrobenzcne (Easlman Kodak Co., Rochester, N.Y.) Yellow crystal Ethyl Alcohol (95%) Acetone (99.7%) Clear liquids {to be specified} Proposed In-Life Starting Date: Proposed In-Life Completion Dale: Icat: Objective of Test: {to be specified} {to be specified} Guinea Pig Dermal Sensilizalion - Modified Duchler Method* To assess the contact dermal scnsitizalion potential of the test article. Testing Guidelines: The methods employed in the testing of this test article are similar to those described in the U.S. EPA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human & Domestic Animals, Series 81-6, 1982; EPA Health Effects Testing Guidelines - Subpart E - Specific Organ/Tissue Tpxicity, September 1985, 40 CPR Part 798; the OHCD Guidelines for Testing of Chemicals, No. 406, Skin Sensilizalion, adopted July 17, 1992; EEC 92/69 Annex V, Part n.6 (1992) 93/21 (1993); and the MAFF Japan Testing Guidelines for Toxicology Studies, Dermal Sensilizalion Sludy, 59 NohSan No. 4200, 1985. "Ritz, 1-1.L. nnd Cuchlcr, E.V., Planning, Conduct, and Interpretation of Guinea Pig Sensilizalion Patch Tests, current Concepts in Cutaneous Toxicity, Drill & Lnznr editors, Academic Press 1980. WHITE EAGLE TOXICOLOGY LABO.RATrO^] ^^OYL^T&^^sft^fP^antain TSCA CBI White Eagle Project Number: 1 DuFont HLO 504-96 %---- Protocol Number|^^^Hy(Rev. 04/96) Guinea Pig Dermal Sensilizalion - Modified Duchlcr Method Page 2 of 9 Good Laboratory Statement: This study Laboratory Laboratory Laboratory Laboratory is conducted in compliance with the U.S. EPA FIFRA Practice Standards (40 ,CFR 160). U.S. EPA TSCA Practice Standards (40 CFR 792), OECD Principles of Practice (c(81)30(FinaI), Annex 2), and MAFF Japan Practice Standards (59 NoIiSan No. 3850). Good Good Good Good lustificalion for Test System: The laboratory albino guinea pig is the system of choice since it is required in the EPA, EEC, OECD, and MAFF Japan guidelines for this type of study and has a long history of use in dermal sensitization studies. Justification for Route of Administrnli'on: The specific route of administration (dermal) is required in the testing guidelines for this study. Animals: The adult male Hartley guinea pig, weighing 300 to 550 grams, is used for this study. The animals are obtained from Davidson Mill Breeding Labs, Jamcsburg, NJ 08831, U.S.D.A. License #22-A-003, Ace Animals Inc., Uoyertown, PA 19512, U.S.D.A. License #23-0-009, Elm I-Iill Breeding Labs, Chelmsford, MA 01824, U.S.D.A. License //14-B-006 or Camm Research Lab Animals, Wayne, NJ 07470, U.S.D.A. License #22- 005. The source of animals will be documented in the study records. The animals (except for screens) are weighed prior to iniliolion, weekly and upon completion of the study. -Tlie animals are randomized using computer generated random numbers into groups and the initial body weights (Day -1) are statistically evaluated by Analysis of Variance (ANOVA). Statistical analyses and random number generation arc performed using CSS: Stalistica, 3.1 copyright, Slatsoft Inc. 1991. Rechallenge naive vehicle controls, if utilized, arc not weighed prior to initiation of the study, but are weighed prior to re-challenge. The animals arc housed and maintained in accordance witli standards set forth in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23). The guinea pigs are acclimated to the laboratory for at least 7 days prior to the initial induction application. Clinical observations arc performed daily on all animals during the study. JACUC Review: The animals are individually identified by color coding with walerand/or solvent-resistant markers. Each cage is identified with a cage card displaying the project number, date initiated, animal numbers, group, sex, and responsible technician's initials. The Protocol for (his study has been reviewed by While Eagle's Institutional Animal Care and Use Committee (IACUC) and complies with acceptable standards for animal welfare and humane care. WHITE EACLH TOXICOLOGY LABORATORIES - DOYLISTOWN, PENNSYLVANIA ^^te^TOwl?^^^^131" WHITB EAGLE TOXICOLOGY LABORATORIES - TSCA csl White Eagle Project Number:! DuFont HLO 504-96 Protocol Number----^^^^(Rcv. 04/96) Page 3 of 9 Guinea Pig Dermal Sensilizalion - Modified Duehler Method Husbandry Conditions: Temperature: 18C - 26C (64.4F - 78.8F) Relative Humidity, 50 20% Light: 12-hour light/dark cycle Diet: Plirina Certified Guinea Pig Diet #5026 and tap water arc provided nd libihim. Dased on our current knowledge, no contaminants are known to be in this diet or water which might be expected to interfere with the objectives of the study. Selcclion of Vehicle: Dflse R.-mgefinding: Caging: Stainless steel wilh elevated wire mesh flooring, 1-5 guinea pigs/cage. If the test article is a solid, it is ground into a fine powder and the preparation of a homogenous paste, suspension or solution of the lest article suitable for application will be prepared. If the test article is a liquid, the dilutions will be prepared with the appropriate vehicle. Prior to initiation of the study, a series of screens is conducted in order to determine the irritation potential of the test article. Groups of 4 unexposed animals are weighed and exposed to different concentrations of the lest material by the patching technique described in the induction stage. In this test, both sides of the animal are closely clipped and exposed to the various concentrations of the material (one concentration per side). The treated sites are scored for irritation using the scale included in Table I (Interpretation of Results) at approximately 24 and 48 hours. Concentrations at several intervals (if necessary) arc utilized to determine the mildly irritating concentration of the test article and the highest non-irritating concentration. If the test article is a powder, then 0.5 g of test article is moistened (or mixed) with the appropriate vehicle (considered 100%) and applied to a 25 mm Hill Top ChamberTM (Hill Top Research Inc., Cincinnati, OH 45242). All concentrations of ii powdered lest article up to 50% are prepared as a percentage of lest article in 0.5 g or 0.5 mL of solution, suspension, or mixture. Before beginning the study, the Sponsor is contacted and provided with results of Hie irritation screen. The Sponsor will llicn set the induction and challenge concentrations. Method of Study Performance: Test Article Group Induction-Stage: A group of twenty (20) guinea pigs is closely clipped over Ihe induction site on their left Hank (approximately 1.5 X 1.5 inches) during the day prior to the day of initiation and repeated as necessary. If the induction site is moved, an additional area (sufficient for patching) is closely clipped at the new site. WHITE EACI.I; TOXICOLOGY I.ABOIIATORIES - DOY1.ILSTOWN. riiNNSYI.VANIA :'': Pagee WHITE EAGLE TOXICOLOGY LABORATORIES -1 ^i^N^&flfi^^"1 TSCA CBI White Eagle Project Number:! DuFont HLO 504-96 -Protocol-Number: I ^ev.-04/96)- Guinea Pig Dermal Scnsilizalion - Modified Buehler Method _PageJ_of9_ Method of Study Performnnce: Test Article Group (continued) Induction Stage: (continued) . Dased upon the range-finding results, a mildly irritating dose will be used for the induction stage. A 0.5 mL portion of a liquid test article or 0.5 g of a solid test article (moistened with the appropriate vehicle) is applied to a 25 mm Hill Top ChamberTM which is placed onto the test site and covered with a piece of dental-dam (approximately 2 inches X 2 inches). After positioning the chamber and the occlusive dental dam onto the prepared skin test site, plastic material is 'wrapped snugly around the trunk of the animal and covered with clastic tape. Approximately six hours later, the chamber is removed and (he lest site is wiped with an appropriate solvent. The treated site is scored for irritation, using the scale included in Table I, at approximately 24 and 48 hours from the initial application. The procedure is performed once a week for three weeks, for a total of three, six-hour applications wilh Ihc lest material. The interval between induction exposures may vary from 6 lo 8 days. Should pronounced irritation be observed at the induction site, the site may be moved for the next induction application, with its relative location fully documented. Vehicle Conlrol Group A group of ten (10) guinea pigs is closely clipped over the induction site towards the head on their left flank (approximately 1.5 X 1.5 inches) during the day prior to the day of initiation and repealed as necessary. If the induction site is moved, an additional area (sufficient for patching) is closely clipped at (lie new site. A 0.5 mL portion of the vehicle (used for the test article group) is applied to a 25 mm Hill Top Chamber11"' which is placed onto Hie test site and covered with a piece of dental dam (approximately 2 inches X 2 inches). After posilioning the chamber and the occlusive dental dam onto the prepared skin lest site, plastic material is wrapped snugly around the trunk of (lie animal and covered with elastic tape. If the test article does not require a vehicle (liquid dosed as supplied), saline will be used for induction and challenge control. Approximately six hours later, the chamber is removed and the test site is wiped with nn appropriate solvent. The treated site is scored for irritation according to the scale included in Table I at approximately 24 and 48 hours from the initial application. The procedure is performed once a week for three weeks, for a total of three, six-hour applications with the vehicle. The interval between induction exposures may vary from 6 to 8 days. Should pronounced irritation be observed at the induction site, the site moy be moved for the next induction ripplicolion, with its rclalivc location fully documented. WHITE EAG1.E TOXICOLOGY LABORATORIES DOYLESTOWN, PENNSYLVANIA Pagrf56npany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWM, PENNSYLVANIA 18901 White Eagle Project Number: j DuFont HLO 504-96 Protocol Numbcr:^^^^f[Rev. 04/96) Guinea Pig Dermal Scnsilizalion - Modified Buehler Method Page 5 of 9 Pcrformnncc: Positive Control A group of five (5) guinea pigs is closely clipped over the induction site on (heir left flank (approximately 1.5 X 1.5 inches) during the day prior to the day of initiation and repeated as necessary. If the induction site is moved, an additional area (sufficient for patching) is closely clipped at the new site. A 0.5 mL portion of 0.1% l-chloro-2,4-dinitrobenz;cne (DNCD) w/v in 50% ethanol: 0.9% saline is applied to a 25 mm Mill Top ChamberTM which is placed onto the test site and covered with a piece of dental dam (approximately 2" X 2"). After positioning the chamber and the occlusive dental dam onto the prepared skin lest site, plastic material is wrapped snugly around the trunk of the animal and covered with elastic tape. Approximately six hours later, the chamber is removed, and Hie lest site is wiped with the appropriate solvent. TIic test site is scored for irnlnlion according to (lie scale included in Table I at approximately 24 and 48 hours from the initial application. The procedure is performed once a week [or three weeks, for a total of three, six-hour applications with DNCB. The interval bclween induction exposures may vary from 6 to 8 days. Should pronounced irritation be observed at the induction site, the site may be moved for the next induction application, with its relative location fully documented. Challenge Stage: Test Material Group After the third induction application, the animals arc rested for approximately 14 to 16 days. At the termination of the rest period, a challenge application, using the procedures described previously, is applied to a challenge site (on the right flank). Based upon the rangefinding results, the highest non-irritating dose will be used for the challenge phase. The challenge application remains on for approximately 6 hours. Upon removal of the challenge application, the contact area is wiped with an appropriate solvent. In the event of questionable findings. a rechallcnge application of all animals may be performed on a second naive silc no sooner than six days after primary challenge, along with a group of 5 naive animals taken from the colony pool, serving as vehicle controls. Dates of application and. observations are recorded. Approximately 24 and 48 hours after the challenge (and rechallenge if necessary) application, the sites are examined for dermal irritation and/or signs of elicited sensitization, according to the scale included in Table I. WI irn; EAC1.E TOXICOLOGY LAIIORATOIIIES - DOYI.ESTOWN. I'ENNSYl.VANIA Pagegrnpan^San^ WHITE EAGLE TOXICOLOGY LABORATORIES - DC-- ritain TSCA CBI White Eagle Project Number:! 0'p ; DuPont HLO 504'/e Protocol Number:! ?ev. 04/96) Guinea Pig Dermal Sensilization Modified Buchlcr Method Page 6 of 9 Challenge Stage: Vghicle Conlrol Group After the third induction application, the animals are rested for approximately 14 to 16 days. At the termination of the rest period, the vehicle control group is challenged with the vehicle on the left flank (caudal) and the test article on Hie right flank using the procedures described previously. The challenge application remains on for approximately 6 hours. Upon removal of'the challenge application, the contact areas are wiped with an appropriate solvent. Dates of applicalion and observations arc recorded. Approximately 24 and 48 hours after the challenge application, the sites ore examined for dermal irritation and/or signs of elicited scnsilization according to tlic scale included in Table I. Pn';jJ|ive fontrol Group Roch.-illcnge: Data Retention: After the third induction application, the animals are rested for approximately 14 to 16 days. At the termination of the rest period, 0.07% DNCD in acetone, using the procedures described previously, is applied to a challenge site (on the right flank). A group of five. naive animals is also treated using the procedures described previously at the time of challenge. The challenge application for these groups remains on for approximately 6 hours. Upon removal of the challenge application, the contact area is wiped with an appropriate solvent. Dates of application and observations are recorded. Approximately 24 and 48 hours after the challenge application, the sites are examined for dermal irritation and/or signs of elicited sensilization according to the scale included in Table I. The Sponsor is notified of animals that are considered sensitized. Verbal instructions for rechallenge arc given by the Sponsor. All animals considered sensitized can be rechallenged six days after primary challenge, but not before. The positive control group is not rechallenged. Tlic animals are evaluated as described in "Interpretation of Results". All raw data generated, all records accumulated during the performance of this study and a copy of the final report will be retained at White Eagle Toxicology Laboratories for a period of ten (10) years, after which time the data will be shipped to the Sponsor or, discarded upon written request from the Sponsor. Test Article Characterization: The Sponsor will be responsible for test article characterization and stability determination prior to initiation of the study. Details regarding the identity, strength, stability, purity, and composition of the test article will be provided by the Sponsor. Information supplied to While Eagle Toxicology Laboratories regarding characterization of the test article will be filed in the archives of White Eagle Toxicology, Doylestown, Pennsylvania. wnrru UACI.E TOXICOLOGY LAIIORATOIUIB - DOYI.CSTOWN, PENNSYLVANIA PageCSSnpanySanitized; Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATIO-O1RIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: 1 DuFont HLO 504-96 Protocol Numbcr:^^^^^1^ev. 04/96) Guinea Pig Dermal Scnsilization - Modified Buchler Method Page 7 of 9 Positive Control Characterization: Details regarding the identity, strength, stability, purity, and composition of the positive control are provided by the Manufacturer and are on file at White Eagle Toxicology Laboratories, Doylestown, Pennsylvania. The positive control has been examined and has been found to be soluble in ethanol and acetone. Any mixtures or dilutions will not be analyzed by While Eagle or the Sponsor. Statistical Analysis: Mortality. Morbidity and Termination: Protocol Changes: Statistical Methods: Individual body weight percentage gain is statistically evaluated by Analysis of Variance (ANOVA), with the Neuman Kculs test applied in (he event of a significant finding (P<0.05) for (lie data as a whole. Statistical analyses are performed using CSS: Statistica, 3.1 copyright, Statsoft Inc. 1991. All animals found dead during the observation period or euthanized in extremis are necropsied to establish, if possible, the cause of death or illness. All test animals are euthanized by CO inhalation or IP pentobarbital at the completion of the study. t\ All changes in or revisions of the approved protocol and the reasons will be documented, signed by the study director, dated and maintained with the protocol. Records Maintained: Records to be maintained include: - irritation scores for screen animals - irritation scores for redness and swelling (induction and challenge) - daily clinical observations - initial, weekly, and final body weights (including group means, standard deviation, and change between initial and final) - test article preparation - calculations and statistical analyses - randomization scheme Final Report: A final report is written which includes, but is not limited to, the items cited in U.S. EPA FIFRA Good Laboratory Practice Standards (40 CFR Part 160), Subpart J, Records and Reports. U.S. EPA TSCA Good Laboratory Practice Standards (40 CFR 792), Subpart J Records and Reports. OECD Principles of Good Laboratory Practice, c(81)30 Annex 2 (Final), Section II, Part 9.2, Content of the Final Report, and MAFF Japan Good Laboratory Practice Standards, 59 NohSan No. 3850, Chapter VIII, Report and Records. Wl irn'. liACl.li TOXICOLOGY l-AllOHATOlillS - UOY1.USTOWN, rHNNSYl.VANIA WHITE EAGLE/TOXICOLOGY LABORATOR L^^'^sYLmBpteft"^'"TSCA CBI White Eagle Project Number: f 9 .. , ',, . : DuFont HLO 504-96 Protocol Number: fl^^|H^v. 04/96) Page 8 of 9 Guinea Pig Dermal Sensilizalion - Modified Buehler Method Interpretation of ^^^^^^^^^^^^^ ^^^^^^^^^^^ Results: All non-eschar and non-necrolic lest sites are scored on a scale of 0 to 3 as indicated in the following Table I. When edema is present with scores of +, 1, or 2 for erythema, it will be. documented. TABLE I Scale for Evaluation of Dermal Irritation 0 = no reaction 0.5 = very faint redness (usually nonconfluent) 1 = faint redness (usually confluent) 2 = moderate redness 3 = severe redness with/without swelling Note; 0.5 scores will be recorded in (lie raw data as a + Scores of 1 or greater in the test group are required to be indicative of sensilizalion. If scores of one (1) or greater are seen on the vehicle control animals, then the reactions of the test article group animals that exceed the most severe vehicle control reactions are considered to be positive scores. INCIDENCE is reported as the number of positive nnimals in each group divided by [lie total number of animals tested in that group. SEVERITY is reported as the sum of the test grades divided by the total number of animals tested in a given group determined for both 24 and 48 hours. Grades + are equal to 0.5 for calculation of severity indices. All average, grades arc to be rounded off to the nearest tenth of a unit. A minimum of two out of five positive control animals must be shown to have elicited a positive reaction (Scores S. one (1)) lo the positive control material to validate the test system. If there is no evidence ofsensilization in the study, it is concluded that the risk of sensitizing humans is minimal. If results are seen which indicate animals in the test group show significant responsiveness compared to the vehicle control group, it is concluded that subsequent exposure of humans to the material may involve substantial risk of sensitizalion. This rating scale is not fixed rigidly. Scientific evaluation of the data and judgement will be used to decide whether material will be classified as a sensitizer or not. Wlim; UACI.ETOXIC01.0GY LAIIORATORICS - OOY1.ESTOWN, PENNSYLVANIA WHITE EAGLE TOXICOLOGY LABORAT^^B?^^?^^^?^^^'" TSCA CBI White Eagle Project Number:^ DuPont HLO 504-96 Protocol Number: ^^^H^^^ev. 04/96} Guinea Pig Dermal Sensilization - Modified Buehler Method Signatures: Submitted by White Eagle Toxicology Laboratories: Page 9 of 9 U^e^^^)/f ^ George E/Moore, B.S. Study Director Approved by: Sponsor Representative ^-/g-y^ Date f l A Date WHITE EAGLE TOXICOLOGY LABORATORIES - DOYl-ESinwN. PnNNSYI.VANIA PagC^paju; WWTE EAGLE TOXICOLOGY LABORATORIES -DC White Eagle Project Number: f^BF E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 APPENDIX B DEVIATIONS FROM PROTOCOL WHITE EAGLE TOXICOLOGY LABORATrOS] ^^M'^^^S^R.'^n'Wfe^intain TSCA CBI DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown/ Pennsylvania 18901 Telephone: (215) 348-3868 -Fax:-(2I5)-348=508r White Eagle Project Number: Protocol Number: Sponsor: Test Article: Test: GLP Compliance: DEVIATIONS FROM PROTOCOL #1, #2, #3, #4, #5 F(Rev. 04/96) E. I. du Font de Nemours and Company H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method 40CFR160 40CFR792 OECD MAFF Japan 1) The Temperature during the In-Life phase of this study was 65F-80F instead of 64.4F-78.8F as stated in the protocol. 2) The Relative Humidity during the In-Life phase of this study was 46%71% instead of 30%-70% as stated in the protocol. 3) One vehicle control group animal was 7 grams above the body weight range of 300 to 550 g as stated in the protocol. The following are amendments to protocol which were not approved in writing until the completion of the study; therefore, they are presented as deviations. 4) Page 7, Final Report Section Add: A second positive control (a-hexylcinnamaldehyde, tech, 85%) will be reported as an appendix. Reason: Sponsor's Request 5) All positive control animals were wiped with the appropriate vehicle and then deionized water, instead of the appropriate vehicle only. The deionized water was used to remove the residual vehicle. Submitted by: Reviewed by: Approved by: These deviations are not thought to have affected the integrity of this study. .QJL^^CT^L <^C^ ///ff^L^' George E^Ioore, B.S. / Study Director 9~^^ Date ^^^GS^> Quality Assurance Manager ^A?- 3 Sponsor Representative R-<o-C Date ^ ^ \\b Date page dmpany Sanitized. Does not contain TSCA CBI White Eagle Project Number: ^||^y E. I. du Font de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 APPENDIX C Guinea Pig Dermal Sensitization - Modified Buehler Method with Positive Control (a-hexyldnnamaldehyde, tech, 85%) WHITE EAGLE TOXICOLOGY LABORATOI^BlSa'E^M1!^^^^ TSCA CB1 White Eagle Project Number:fU^ DuPont HLO 504-96 DuPont HLO 306-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown, Pennsylvania 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 Study Title Guinea Pig Dermal Sensitization Modified Buehler Method with Positive Control (a-hexyldnnamaldehyde, tech, 85%) Laboratory Project ID Haskell Laboratory Project ID 306-96 Data Requirement EEC 92/69 - Annex V - Part B.6 (1992) - 93/21 (1993), OECD Guidelines for the Testing of Chemicals, No. 406, Skin Sensitization, adopted July 17,1992 U.S. EPA Pesticide Assessment Guidelines Subdivision F, Hazard Evaluation: Human & Domestic Animals, Series 81-6, 1982 EPA Health Effects Testing Guidelines - Subpart E Specific Organ/Tissue Toxicity, September 1985. 40 CFR Part 798, and MAFF Japan Testing Guidelines for Toxicology Studies, Dermal Sensitization Study, 59 NohSan No. 4200, 1985 Study Completed On 26 June 1996 Contracting Laboratory White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown, Pennsylvania 18901 Author George E. Moore, B.S. for E.I. du Font de Nemours and Company Haskell Laboratoiy-for Toxicology and Industrial Medidne Elkton Road, P.O. Box 50 Newark, Delaware 19714 Study Monitor Gregory S. Ladies, Ph.D. Medical Research, No. White Eagle Project No. 'age 1 of 43 WHrrE EAGLE TOXICOLOGY ^BORATO^E^OYL^^'^^^^Ny^fl"^^ TSCA CBI White Eagle Project NumberfP DuPont HLO 504-96 DuPont HLO 306-96 THIS PAGE RESERVED FOR SPEOnC COUNTRY REQUIREMENTS Page 2 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA IW01 WHrrE EAGLE TOXICOLOGY LA;BORAPTaOgI&CS4-6DipOaYnLEYSTSOaWnNit,izj.e--d--. --D--oe^s'-DAypftlc.^oWnltwaiinnTS00CfAt C^BDIl White Eagle Project Numberg^l DuPont HLO 504-96 WHITE-EAGtE-TOXieOLOGY-tABOR^^Qfife3^ 2003 Lower State Road, Doylestown, Pennsylvania 18901 . Telephone: (215) 348-3868 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT White Eagle Project Number: Sponsor: Test Article; Test: Protocol Number: ^^^^^^B^f E. I. du Font de Nemours and Company a-hexyldnnamaldehyde, tech, 85% GuineaPigDermal Sensitization - Modified Buehler Method ^^MHy(Rev. 04/96) This. study was carried out in compliance with the Principles of Good Laboratory Practice (GLP) as promulgated by the following agencies: U.S. Environmental Protection Agency HFRA. as stated in the Federal Register. 40 CFR, Part 160, August 17, 1989. U.S. Environmental Protection Agency TSCA as stated in the Federal Register, 40 CFR, Part 792, August 17, 1989. OECD Principles of Good Laboratory Practice (c(81)30(Final), Annex 2) MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850) The study was conducted in compliance with the following exception:-' Treatment solutions were not analyzed for concentration, uniformity or stability of the test and control articles. The procedures used by trained personnel to prepare the treatment solutions insured: a) The accuracy of concentration because the test article diluent (vehicle) and positive control diluents were accurately measured with graduated devices. The positive control article was weighed on an analytical balance accurate to at least O.OOlg. The test article was weighed on a balance accurate to at least 0.01g. b) Uniformity because all solutions were mixed prior to administration to the test system; and c) Stability because treatment solutions were prepared just prior to addition to the test system. ^Ic^^'^____ Geo'rgeE^Moore, B.S. Study Director (o-a.&-T( Dale Sponsor: DuPont Agricultural Products Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, DE 19714 Page3 WHITE EAGLE TOXICOLOGY LABO^I T^^BYrESTOwff^E^^A^^P^'" TSCA CBI White Eagle Project Number: \ DuPont HLO 504-96 _WhHe.Eagle.Project.Number:1 _DuEont.HL0.306-96_ E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech. 85% Guinea Pig Dermal Sensidzation Modified Buehler Method DuPom HLO 306-W Substance Tested: Synonyms/Other Codes: CAS Registry Number HaskellNo.: Purity: Major Impurities: Siataliir Physical Fbnn: Sponsor: Stmcture: GENERAL INFORMATION Test Material Octanal. 2-(phenylmcthylene>- HCA Hexyl cinnamaldchyde a-Hexylcinnamaldchydc 2-HexylcinnamaIdehyde Hexyl cinnamic aldehyde a-Hexylcinnamic aldehyde a-HexylcinnamyI aldehyde a-N-hexyl-bcta-phcnylacroIein Octanal, 2-(phenylmcthylcnc)Lot Number 10021 HF 101-86-0 21780 85% (Supplied by Aldrich Chemical Co.; Milwaukee. WI 53233); Expiration date: 5/99 None known to be of toxicologies! significance at this time. The test substance appeals to be stable under the conditions of (he study: no evidence of instability was observed. Yellow liquid DuPont Agricultural Products E. L du Font de Nemours and Company Barley Mill Plaza Wilmington. Delaware 19880-0038 CHO Pt-CH==C- (CH2S-" Study Initiated - Completed:. 4/19/96 - 6/26/96 In-Life Phase Initialed - Completed: 4/30/96-5/31/96 .4. WHITE EAGLE TOXICOLOGY LABORATOMES - DOYLESTOWN. PENNSYLVANIA 1B801 Pag(C48ipany Sanitized. Does not contain TSCA CB1 WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: f H ^ DuPont HLO 504-96 White Eagle Project Number: ^------y DuPont HLO 306-96 E. I. du Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Fig Dermal Sensitization - Modified Buehler Method Guinea Pig Dermal Sensitization - Modified Buehler Method with Positive Control (a-hexyldnnamaldehyde, tech, 85%) Summary The potential of a-hexyldnnamaldehyde, tech, 85% (HCA) to produce delayed contact hypersensitivity in guinea pigs was assessed by the Modified Buehler Method. Thirty (30) test article animals were induced and challenged by occluded application of HCA to clipped intact guinea pig skin. The same procedures were carried out on contemporaneous control groups except that the test article was replaced by vehicle. No reaction to severe redness with or without swelling and eschar was observed during the induction phase of the 2.0% HCA group in ethanol. Approximately twenty-four hours and forty-eight hours after the challenge application, no reaction to severe redness, with or without swelling was observed at the test article naive site (right side). The inddence of sensitization after the challenge application of 1.0% HCA in acetone was 8/10 and the severity was 1.4 at 24 hours and 1.1 at 48 hours. No reaction to faint redness was observed during the induction phasb of the 1.0% HCA group in ethanol. Approximately twenty-four hours and forty-eight hours after the challenge application, no reaction to moderate redness was observed at the test artide naive site (right side). The inddence of sensitization after the challenge application of 0.75% HCA in acetone was 2/8 and the severity was 0.8 at 24 hours and^O.l at 48 hours. No reaction to faint redness was observed during the induction phase of the 0.5% HCA group in ethanol. Approximately twenty-four hours after the challenge application, no reaction to very faint redness was observed at the test artide naive site (right side) and no reaction was observed at 48 hours. The inddence of sensitization after the challenge application of 0.25% HCA in acetone was 0/10. No reaction to very faint redness was observed during the induction phase of the 2.0% HCA vehicle group. Very faint redness was noted for 1/10 animals at the test article site, approximately 24 hours after the challenge application. Other than This one test site, no reaction was observed approximately 24 and 48 hours after the challenge application of either the vehide (acetone) or test artide (1.5% HCA in acetone). No reaction to very faint redness was observed during the induction phase of the 1.0% HCA vehicle group. Very faint redness was noted for 1/10 animals at the test artide site, approximately 24 hours after the challenge application. Other than this one test site, no reaction was observed approximately 24 and 48 hours after the challenge application of either the vehide (acetone) or test artide (0.75% HCA in acetone). No reaction was observed' on any 0.5% HCA vehide animals after any of the induction applications as well as approximately 24 and 48 hours after the challenge application of either the vehide (acetone) or test artide (1.0% HCA in acetone) sites. PageS WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA 18901 WHOT EAGLE TOXICOLOGY LABORATOIES-DBYL'^d^^N^&fiS^r^'" TSCA CB' White Eagle Project Number: |^B^ DuPont HLO 504-96 White Eagle Project Number: ^^^B^___________________DuPont HLO 306-96 E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech. 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Guinea Pig Dermal Sensitization - Modified Buehler Method with Positive Control (a-hexyldnnamaldehyde, tech, 85%) Summary (continued) It was concluded therefore, that, under the study conditions utilized, repeated administration of 2.0% and 1.0% n-hexyldnnamaldehyde, tech, 85% (HCA) in ethanol and challenged with 1.50% and 0.5% HCA in acetone, respectively, produced delayed contact hypersensitivity in guinea pigs. On the basis of the nature of effects observed in this study and according to the guide for the labeling of dangerous substances published in the Official Journal of the European Communities (EEC Directive 93/21), a-hexyldnnamaldehyde, tech, 85% is a skin sensitizer. J ^ L ^ ^ ^ ^ George E/Moore. B.S. Study Director c^-% Date Reviewed/Audited by: Quality Assurance Manager 0-2^0 Date Page 6 WHITE EAGLE TOXICOLOGY LAOORATOKTES DOYLESTOWN, PENNSYLVANIA 11901 Pag6&ipany Sanitized: Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number! DuPont HLO 504-96 JVhite_Eagle.Project.NumberJ _D_uF.ont.HLO_306-96_ E. I. du Font de Nemours and Company a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method While Eagle Project Number: Sponsor Study Number: Test Article: Study Initiation: J^U 305% a-hexyldnnamaldehyde, tech, 85% (HCA) 04/19/96 Inspections Protocol Procedures Raw Data Draft Report Final Report Quality Assurance Statement Dales 04/23/96 04/23/96 05/01/96 06/13/96 06/14/96 06/26/96 04/24/96 05/14/96 06/14/96 06/17/96 04/29/96 Reports to Study Director and .Management 06/18/96 06/26/96 This review was completed on 06/26/96 and released by: - Quality Assurance Manager Paageee77 WHITE EAGLE TOXICOLOGY LABORATORIES . DOYLESTOWN, PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATOMES -1 antain TSCA CBI White Eagle Project Number:! DuPont HLO 504-96 _White-EagIe-Eroject-Number:( TABLE OF CONTENTS DuPont HLO 30&-96 Page Title Page .................................................................................................. 1 Good Laboratory Practice Compliance Statement ............................................ 3 General Information.................................................................................... 4 Summary 5 ...................................^.............................................................. Quality Assurance Statement ....................................................................... 7 Table of Contents ....................................................................................... 8 Objective of Test ....................................................................................... 10 Testing Guidelines .................................................................................... 11 Good Laboratory Statement ........................................................................ 11 Justification for Test System ........................................................................ 11 Justification for Route of Administration ....................................................... 11 Animals ................................................................................................... 11 IACUC Review ......................................................................................... 12 Husbandry Conditions .............................................................................. 12 Test Article Vehicle ................................................................................... 12 Dose Rangefinding .................................................................................... 12 Induction Stage ......................................................................................... 13 Challenge Stage ........................................................................................ 14 Statistical Analysis..................................................................................... 14 Mortality, Morbidity and Termination .......................................................... 14 Data Retention .......................................................................................... 14 Personnel ................................................................................................. 15 Positive Control Characterization ................................................................ 15 Interpretation of Results ............................................................................. 15 Table 1 - Scale for Evaluation of Dermal Irritation .......................................... 15. Results 16 ...............,,................................................;..--......................:...... Conclusion ............................................................................................... 18 Signatures ............................................................................................... 19 Page 8 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA IB901 WHITE EAGLE TOXICOLOGY r^^ LABORATORIES o- v^^E^syL^^^^ TSCA CBI White Eagle Project Number: i DuPont HLO 504-96 White Eagle Project Number| DuFont HLO 306-96 TABLE OF CONTENTS (continued) Page Table 2 - Challenge Screens ..................................................................... 20 Table 3 - Guinea Pig Dermal Imtation/Sensitization Scores - Test Article 2.0% HCA ................ 22 Table 4 - Guinea Pig Dermal Imtation/Sensitization Scores - Vehicle Control 2.0% HCA 23 .............. Table 5 - Guinea Pig Dermal Imtation/Sensitization Scores Test Article 1.0% HCA ................ 24 Table 6 - Guinea Fig Dermal Imtation/Sensitization Scores Vehicle Control 1.0% HCA .............. 25 Table 7 - Guinea Pig Dermal Imtation/Sensitization Scores Test Article 0.5% HCA ................ 26 Table 8 - Guinea Pig Dermal Imtation/Sensitization Scores Vehicle Control 0.5% HCA .............. 27- Table 9 - Daily Observations ....................................................................... 28 Table 10 - Individual Body Weights - 2.0% HCA and Vehicle Control .............. 29 Table 11 - Individual Body Weights 1.0% HCA and Vehicle Control ................30 Table 12 - Individual Body Weights - 0.5% HCA and Vehicle Control .............. 31 Appendix A - Copy of Protocol Coversheet and Protocol ................................ 32 Appendix B - Deviations from Protocol ........................................................ 43 Page 9 WHITE EAGLE TOMCOLOOT LABORATORIES DOYLESTOWN. PENNSYLVANIA 18WI WHITE EAGLE TOXICOLOGY LABC ^Sg^^X^^?"^" TSCA CB' White Eagle Project Number: | DuPont HLO 504-96 -DuEont-HLO-306-96- WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown, Pennsylvania 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 Protocol Number: Projprt NumbeiTesting Facility: Sponsor: Test Article: Tpst Article Description: Test Article Received: Vpi.rl. Control Articles: Vehicle Control Descriptions: Screens Initiated: In-Life Study Initiated: lest: Objective of Test: (Rev. 04/96) White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown. PA 18901 E. I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road. P.O. Box 50 Newark, Delaware 19714 a-hexyldnnamaldehyde, tech, 85% (HCA), Lot # 10021 HF, Exp. date 5/99, Supplied by White Eagle Labs. Gear liquid 05/01/94 Ethyl Alcohol (95%) (dearspring Distilling Co., Cleannont & Frankfort, KY and Cincinnati, OH, Lot #09015145, Exp. 10/10/96) Acetone (99.7% pure) (Allied Signal Inc., Morristown, NJ 07962, Lot #020795, Exp. 5/98) Clear liquids 04/23/96 04/30/96 In-life Study Completed: 05/31/96 Guinea Pig Dermal Sensitization - Modified Buehler Method* To validate the test system using a known sensitizer, a-hexyldnnamaldehyde, tech, 85% (HCA). Ritz, H.L. and Buehler, E.V., Planning, Conduct, and Interpretation of Guinea Pig Sensitization Patch Tests, current Concepts in Cutaneous Toxidty, Drill & Lazar editors, Academic Press 1980. Page 10 WHITE EACLE TOXICOLOGY LADORATOMES DOYLESTOWN. PENNSYLVANIA ItWl WHITE EAGLE TOXICOLOGY LABORATOR ^^Asft^&flPfeaff"^1" TSCA CBI White Eagle Project Number:! DuPont HLO 504-96 White Eagle Project Number \ DuPont HLO 306-96 E. I. du Font de Nemours and Company - oi-hexylcinnamaldehyde, tech, 85% Guinea Fig Dermal Sensitization Modified Buehler Method Testing Guidelines: Good Laboratory Statement Justification for Test System: Tustification for Route of Administration: The methods employed in the testing of this test article were similar to those described in the EEC 92/69 Annex V, Part B.6 1992 93/21 (1993); the OECD Guideline for Testing of Chemicals, No. 406, Skin Sensitization. adopted July 17, 1992; U.S. EPA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human & Domestic Animals, Series 81-6, 1982; EPA Health Effects Testing Guidelines - Subpart E - Specific Organ/Tissue Toxidty, September 1985. 40 CFR Part 798, and the MAFF Japan Testing Guidelines for Toxicology Studies, Dermal Sensitization Study, 59 NohSan No. 4200, 1985. This study was conducted in compliance with the U.S. EPA FIFRA Good Laboratory Practice Standards (40 CFR 160), U.S. EPA TSCA Good Laboratory Practice Standards (40 CFR 792), OECD Principles of Good Laboratory Practice (c(81)30(Final), Annex 2), and MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850). The laboratory albino guinea pig was the system of choice since it is required in the EPA, OECD, EEC and MAFF Japan guidelines for this type of study and has a long history of use in dermal Sensitization studies. The specific route of administration (dermal) is required in the testing guidelines used for this study. Animals: The adult male Hartley guinea pig, weighing 373 to 517 grains, was used for this study. The animals were obtained from Ace Animals Inc., Boyertown, PA 19512 U.S.D.A. License #23-B-009. Screen animals were weighed prior to initiation and all others were weighed prior to initiation, weekly (6-8 days), and upon completion of the study. The animals were randomized into groups and the initial body weights (Day -1) were statistically evaluated by Analysis of Variance (ANOVA). Statistical analyses and random number generation were performed using CSS: Statistics, 3.1 copyright, Statsoft Inc. 1991. The animals were housed and maintained in accordance with standards set forth in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23). The guinea pigs were acclimated to the laboratory for at least 7 days prior to the initial induction application. Clinical observations were performed daily on all animals during the study. The animals were individually identified by color coding with water and/or solvent-resistant markers. Each cage was identified with a cage card displaying the project number, date initiated, animal numbers, group, sex and responsible technician's initials. All animals were euthanized with pentobarbital at the conclusion of the study. Page 11 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA IWO) WHITE EAGLE TOXICOLOGY LABORATrO^' ^^i^^o^/'i^^&Wtia^tain TSCA CBI White Eagle Project Number: | DuPont HLO 504-96 White Eagle Project Number:! DuPont HLO 306-96 E. I, du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Fig Dermal Sensitization - Modified Buehler Method IAC.UC Review: Husbandry Conditions: The Protocol for this study was reviewed by White Eagle's Institutional Animal Care and Use Committee (IACUC), and complies with acceptable standards for animal welfare and humane care. Temperature: 18C - 26-C (64F - 78-F) Relative Humidity, %: 24 88 Light: 12-hour light/dark cycle Diet; Purina Certified Guinea Pig Diet #5026 and tap water were provided ad libitum. Based on our current knowledge, no contaminants are known to be in this diet or water which might be expected to interfere with the objectives of the study. Caging: Stainless steel with elevated wire mesh flooring, 4-5 guinea pigs/cage. Test Article Vehicle: Ethyl Alcohol (95%) was used as the test article vehicle for the induction phase of this study and acetone (99.7%) for the challenge phase of the study. Dese Rangefinding: Historically, 5.0% and 2.5% HCA in 95% ethanol have produced no irritation to faint irritation after the first induction application and necrosis after the second or third induction application. Based on this steep dermal response, concentrations of 2.0%, 1.0% and 0.5% HCA in 95% ethanol were used for the induction phase of the study. Challenge concentrations were determined using two groups of 4 unexposed animals. The animals were weighed and exposed to concentrations of 1.50%, 0.75%, 0.50% and 0.25% HCA in acetone by the patching .technique described in the, induction phase. Other than very feint redness noted for 1/4 animals (1.50% in acetone) at 24 hours, no reaction was observed for any of the four concentrations. Challenge concentrations of 1.50%, 0.75% and 0.25% in acetone were chosen for the 10%, 1.0% and 05% in 95% ethanol induction application animals respectively. WHITE EAGLE TOXICOLOGY LABORATOBES DOYLESTOWN, PENNSYLVANIA 18901 Page(56TTpany_Sanit WHITE EAGLE TOXICOLOGY LABORATORIES - r -intain TSCA CBI White Eagle Project Number^ DuPont HLO 504-96 -White-Eagle-Eroject-Number:| -DuPont-HLO-306-96- E. I. du Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Method of Study Performance: Test Article Group Induction Stage: Three groups of ten (10) guinea pigs were closely dipped over the induction site on their left flank (approximately 1.5 X 1.5 inches) during the day prior to the day of initiation and repeated as necessary. Based upon historical data, dose levels of 2.0%, 1.0% and 0.5% w/v of HCA in 95% ethanol, were used for the induction stage. A 0.5 mL portion of the appropriate concentration of HCA in 95% ethanol was applied to a 25 mm Hill Top ChamberTM which was placed onto the test site and covered with a piece of dental dam (approximately 2 inches X t inches). After positioning the chamber and the occlusive dental dam onto the prepared skin test site, plastic wrap was wrapped snugly around the trunk of the animal and overwrapped with ElastikonTM. Approximately six hours later, the chamber was removed and the test site was wiped with ethanol and then deionized water. The treated site was scored for irritation, using the scale induded in Table I, at approximately 24 and 48 hours from the initial application. The procedure was performed once a week for three weeks, for a total of three, six-hour applications with the appropriate concentration of HCA. The interval between induction exposures was 7 days. Vehicle Control Group Three groups of ten (10) guinea pigs. were dosely dipped over the induction site towards the head on their left Hank (approximately 1.5 X 1.5 inches) during the day prior to the day of initiation and repeated as necessary. ; A 0.5 mL portion of the vehide (95% ethanol) was applied to a 25 nun Hill Top ChamberTM which was placed onto the test site and covered with a piece of dental dam (approximately 2 inches X 2 inches). After .' positioning the chamber and the ocdusive dental dam onto the prepared skin test site, plastic wrap was wrapped snugly around the trunk of the animal and overwrapped with ElastikonTM. Approximately six hours later, the chamber was removed and the test site is wiped with deionized water. The treated site was scored for irritation according to the scale induded in Table I at approximately 24 and 48 hours from the initial application. The procedure was performed once a week for three weeks, for a total of three, six-hour applications of 95% ethanol. The interval between induction exposures was 7days. (81 Page 13 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA IRX>1 WHrrBB.GLBW^COLOGV^ORA^5^^ White Eagle Project Number DuPont HLO 504-96 White Eagle Project Number;! -DuPont-HLO 306-96- E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Challenge Stage: Test Material Group After the third induction application, the animals were rested -for 15 days. At the termination of the rest period, a challenge application. using the procedures described previously, was applied to a challenge site (on the right Hank). Based on the challenge dose rangefinding; animals induced with 2.0%, 1.0% or 0.5% HCA in 95% ethanol were challenged with 1.50%, 0.75% and 0.25% HCA in acetone respectively. The challenge application remained on for approximately 6 hours. Upon removal of the challenge application,, the contact area was wiped with acetone and then deionized water. Dates of application and observations were recorded. Approximately 24 and 48 hours after the challenge application, the sites were examined for dermal irritation and/or signs of elicited sensitization, according to the scale included in Interpretation of Results (Table 1). Vehicle Control Group After the third induction application with the vehicle (95% ethanol), the 3 groups of ten animals were rested for 15 days. At the termination of the rest period, each of the 3 groups was challenged with acetone on the left flank (caudal). Each of the three groups also received either 1.50%, 0.75% or 0.25% HCA in acetone on the right Hank. The. challenge application remained on for approximately 6 hours. Upon removal of the challenge application, the contact areas were wiped with acetone and . then deionized water. Dates of application and observations were recorded. Approximately 24 and 48 hours after the challenge application. the sites were examined for dermal irritation and/or signs of elicited sensitization according to the scale included in Interpretation of Results (Table 1). Statistical Analysis: Individual body weight percentage gain was statistically evaluated by Analysis of Variance (ANOVA). with the Neuman Keuls test applied in the event of a significant finding (P<0.05) for the data as a whole. Statistical analyses were performed using CSS: Statistics, 3.1 copyright, Statsoftmc. 1991. Mortality. Morbidity and Termination: Animals #15 and #45 (1.0% HCA test article group) were found dead during the induction phase of the study (Days 14 and 16, respectively). All other animals were euthanized at the completion of the study with IP injection of pentobarbital. Data Retention: All raw data generated, all records accumulated during the performance of this study and a copy of the final report will be retained at White Eagle Toxicology Laboratories for a period of ten (10) years, after which time the data will be shipped to the Sponsor or, discarded upon written request from the Sponsor. Page 14 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN, PENNSYLVANIA IW01 WHITE EAGLE TOXICOLOGY LABORATOR ^Ms^^te"^" TSCA CBI White Eagle Project Number: I DuFont HLO 504-96 White Eagle Project Number; 1 DuPont HLO 306-96 E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal SensiHzation - Modified Buehler Method Personnel: The following is a list of personnel for this study: George E. Moore, Study Director Shashi P. Logani Richard J. Hershman Stanley Sekelewski Lisa Wilkoski Positive Control Characterization: Details regarding the identity, strength, stability, purity, and composition of the positive control are provided by the Manufacturer and are on file at White Eagle Toxicology Laboratories, Doylestown, Pennsylvania. The positive control has been examined and has been found to be soluble in ethanol and acetone. Any mixtures or dilutions were not analyzed by White Eagle or the Sponsor. Interpretatioiuif Results: All non-eschar and non-necrotic test sites were scored on a scale of 0 to 3 as indicated in Table 1. lAELEJ. Scale for Evaluation.of Dermal Irritation 0 = no reaction 0.5 = very faint redness (usually nonconfluent) 1 = feint redness (usually confluent) 2 = moderate redness 3 = severe redness with/without swelling Note: 0.5 scores were recorded in the raw data as a + Scores of 1 or greater in the test group are required to be indicative of sensitization. If scores of one (1) or greater are seen on the vehicle control animals, then the reactions of the test article group animals that exceed the most severe vehicle 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 of the test grades divided by the total number of animals tested in a given group determined for both 24 and 48 hours. Grades of + are equal to O.S for calculation of severity indices. All average grades are to be rounded off to the nearest tenth of a unit. A minimum of two out of ten animals in a group must be shown to have elidted a positive reaction (Scores S. 1) to HCA to validate the test system. Pace 15 WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN. PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATOB ^iBSift"13'" TSCA CB1 White Eagle Project Number| DuPont HLO 504-96 White Eagle Project Number: j -DuEont-HLO-306-96- E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Results: Tables 2 through 12 Test Article Group - Induction and Challenge 2.0% HCA All test animals appeared to be normal throughout the study. All animals exhibited an overall gain in body weight during the study (-1 through 32 days). One of ten animals exhibited a loss of body weight between Days 14 and 21 and 3/10 animals exhibited a loss of-body weight between Days 29 and 32. The water valves were functional and the animals had feed. There were no statistically significant differences identified between the individual body weight percentage gains of the test article group and any of the other five groups of animals (Analysis of Variance). No reaction to severe redness with or without swelling and eschar was observed during the induction phase of the study. Approximately twenty-four hours and forty-eight hours after the challenge application, no reaction to severe redness with or without swelling was observed at the test article naive site (right side). The incidence of sensitization after the challenge application of 1.5% HCA in acetone was 8/10 and the severity was 1.4 at 24 hours and 1.1 at 48 hours. " ' 1.0%HrA Two animals (#15 and #45) were found dead during the induction phase of the study (Days 14 and 16 respectively). All other test animals appeared to be normal throughout the study. All surviving animals exhibited an overall gain in body weight during the study (-1 through 32 days). Three of the eight remaining animals exhibited a loss of body weight between Days 29 and 32. The water valves were functional and the animals had feed. There were no statistically significant differences identified between the individual body weight percentage gains of the test article group and any of the other five groups of animals (Analysis of Variance). No reaction to faint redness was observed during the induction phase of the study. Approximately twenty-four hours after the challenge application, no reaction to moderate redness was observed at the test article naive site (right side) and no reaction to very faint redness was observed at approximately 48 hours. The incidence of sensitization after the challenge application of 0.75% HCA in acetone was 2/8 and the severity was 0.8 at 24 hours and 0.1 at 48 hours. Pace 16 WHITE EACLE TOXICOLOGY LABORATORIES DOYLESTOWN, PENNSYLVANIA 181 -WHITE EAGLE TOXICOLOGY LAB. ORATPOsRgIEQSC- ^DfOfnYpLaEnSyTOSWanNi,tiFzEeNdN. SYDLoVeAsMnJoCTtOcCoInT"t*a--in" T' S--C--A- C~0B1I White Eagle Project J f f ^ Number DuFont HLO 504-96 White Eagle Project NumberJ^B^^^__________________DuPont HLO 306-96_______ E. I. du Font de Nemours and Company - a-hexyldnnamaldehyde< tech, 85% Guinea Pig Dermal Sensitizalion - Modified Buehler Method Results: (continued) Test Article Group Induction and Challenge (continued) {L55LHCA All test animals appeared to be normal throughout the study. All animals exhibited an overall gain in body weight during the study (-1 through 32 days). One of (en animals exhibited a loss of body weight between Days 29 and 32. The water valve was functional and the animal had feed. There were no statistically significant differences identified between the individual body weight percentage gains of the test article group and any of the other five groups of animals (Analysis of Variance). No reaction to faint redness was observed during the induction phase of the study. Approximately twenty-four hours after the challenge application, no reaction to very faint redness was observed at the test article naive site (right side) and no reaction at forty-eight hours. The incidence of sensitization after the challenge application of 0.25% HCA in acetone was 0/10. Vehicle Control Group - Induction and Challenge 2.0% HCA All vehicle control animals appeared normal and exhibited an overall gain in body weight during the observation period (-1 through 32 days). One of ten animals lost weight between Days 29 and 32. The water valve was functional and the animal had feed. There were no statistically significant differences identified between the individual body weight percentage gains of the vehicle control group of animals and any of the other five groups of animals (Analysis of Variance). No reaction to very faint redness was observed during the induction phase of the 2.0% HCA vehicle group. Very faint redness was noted for 1/10 animals at the test article site, approximately 24 hours after the challenge application. Other than this one test site, no reaction was observed approximately 24 and 48 hours after the challenge application of either the vehide (acetone) or test article (1.5% HCA in acetone). 1.0% HCA All vehide control animals appeared normal and exhibited an overall gain in body weight during the observation period (-1 through 32 days). One of ten animals lost weight between Days 2-1 and 29 and 1/10 animals between Days 29 and 32. The water valves were functional and the animals -had feed. There were no statistically significant differences identified between the individual body weight percentage gains of the vehide control group of animals and any of the other five groups of animals (Analysis of Variance). Page 17 WHTTE EAGLE TOXICOLOGY LABORATORIES OOYLESTOWN. PENNSYLVANIA 11901 WHITE EAGLE TOXICOLOGY LABORATPOaRgIECS6- 1DnQpYaLnEySTSOaWnNit, iPzeENdN. SDYLoVeAsNjIiAoitscSaOnl tainTSCA CBI' White Eagle Project Numberj DuPont HLO 504-96 White Eagle Project Number! -DuEont-HLO-306-96- E. I. du Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Results: (continued) Vehicle Control Group - Induction and Challenge (continued) 1.0% HCA (continued) No reaction to very faint redness was observed during the induction phase of the 1.0% HCA vehicle group. Very faint redness was noted for 1/10 animals at the test article site, approximately 24 hours after the challenge application. Other than this one test site, no reaction was observed approximately 24 and 48- hours after the challenge application of either the vehicle (acetone) or test article (0.75% HCA in acetone). 0.5% HCA All vehicle control animals appeared normal and exhibited an overall gain in body weight during the observation period (-1 through 32 days). Qne of ten animals lost weight between Days -1 and 7; The water valve was functional and the animal had feed. There were no statistically significant differences identified between the individual body weight percentage gains of the vehicle control group of animals and any of the other five groups of animals (Analysis of Variance). No reaction was observed on any 0.5% HCA vehicle animals after any of the induction applications as well as approximately 24 and 48 hours after the challenge application of either the vehicle (acetone) or test article (1.0% HCA in acetone) sites. Conclusion: The test article, a-hexyldnnamaldehyde, tech, 85% (HCA), when induced with concentrations of 2.0% or 1.0% in ethanol and challenged with 1.5% and 0.75% HCA in acetone respectively, appears to be a. dermal sensitizer in albino guinea pigs. Page 18 WHITE EACl TOXICOLOGY LABORATORIES-DOTLESrOWN. PENNSYLVANIA 18901 Pag62npany Sanitized; Does not contain TSCA'CBI WHITE EAGLE TOXICOLOGY LABORATrO01MES - DOYLESTOWN, PENNSYLVANIA WOi White Eagle Project J J ^ Number DuPont HLO 504-96 _White_EagIe-Project-Number:^^^^^----------------------------------DuPont-HLO-306-96-- E. I. dii Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% ' Guinea Pig Dermal Sensitizalion - Modified Buehler Method Signatures; Submitted by White Eagle Toxicology Laboratories: Jy^^-^^f^- George Ef Moore, B.S. Study Director G-^-^ Dale Reviewed/Audited by; Quality Assurance Manager C-Zt.-^ Date Page 19 WWTS EAGLE TOXICOLOGY lABORATOHES - DOYIESIOWN. PENNSYLVANIA 1B01 . PageOS&npany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number:) DuPont HLO 504-96 White Eagle Project Number: ( DuPont HLO 306-96 E. I. du Pont de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLH 2 Challenge_Sgeeas Animal Number 61 62 63 64 Screen #1 - Left Side 1.50% concentration in Acetone 2iLHours+ 0 48 Hours f 0 Animal Number 61 62 63 64 Screen #2 - Right Side 0.75% concentration in Acetone 24 Hours 0 48 Hours 0 Body Weights fg) Animal #61 415 Animal #62 393 Animal #63 409 Animal #64 401 + = Site stained light pale yellow; however, erythema could be scored Page 20 WHTre EAGLE TOXICOLOGY LABORATORIES OOYLESTOWN, PENNSYLVANIA lOTOl WHITE EAGLE TOXICOLOGY S LABORAT01 &^oyl^^'As^^te TSCA CBI White Eagle Project Number: | DuPont HLO 504-96 White Eagle Project Number: | DuPont HLO 306-96 E. [. du Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 2 (continued) Challenge Screens Animal Number 65 66 67 68 .'^rgpn ^ . Left Sidp 0.50% concentration in Acetone 24 Hours 0 48 Hours 0 Animal Number 65 66 67 68 Screen #4 - Right Side 0.25% concentration in Acetone 24 Hours 0 0 0 0 ; 48Hqurs 0 0 0 0 Body Weights (g) Animal #65 377 Animal #66 400 Animal #67 380 Animal #68 373 Page 21 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA 18901 " ' FagecSSnDany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 . White Eagle Project Number: |^M^ DuPont HLO 504-96 White Eagle Project Number: ^^BB__________________DuPont-HLO-306-96- E. I. du Font de Nemours and Company - n-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 3 2J1ZJHCA Guinea Pig Dermal Irritalibn/Sensilization Scores Animal Number 17 Reading 1 24 4fi 0 0 2 3 Hours After Application Number 2i S& 24 48 1 1 S S Challenge 24Hrs* 48 Hi"; 3 2 18 + 0 1 1 S S 2 + 27 0 0 0 0 + + 1 1 31 00 00 + + 32 00+033 00 35 00+OSS 41 001033 47 +02+SS 55 22 0010 57 1 + 3 3 1 2 2 1 + 0 + 1 0 0 0 0 1 1 * Readings are performed approximately 24 hours after the challenge application S = Eschar Note: AU 6-hour exposures. Induction applications - 2.0% HCA in 95% ethanol Challenge application 1.5% HCA in acetone Page 22 WHITE EACIE TOMCOIOCT LABORATORIES DOyi-ESTOWN. PENNSYLVANIA 1(901 : PagflC66npany Sanitized. Does not contain TSCACBI WHITE EAGLE TOXICOLOGY LABORATORIES - DO.YLESTOWN, PENNSYLVANIA 18901 White Eagle Project Numbier^^HB DuFont HLO 504-96 White Eagie Project Niunber^^^B D uPont HLO 306-96 E. I. du PC>nt de Nemours and Coin pany - a-hexylcinnariialclehyde, tech, i85% Guinea Pi(; Dermal Serisitization - N lodified Buehler Met 1iod TABLE 4 2.0?E, HCA - Vehicle Cnnltrol (1;uinea Pig Dennal Imtation/Sensiliization Scores Animal Number 3 Reading Hours After Application Number 1challenge 1 2 3 Acetone 24 AS 24 IS 21 4fi ;!4JjITS* 48Hrs 0 0 0 0 0 0 0 0 Challenge L5SLHCA 24Hrs 48Hrs + 0 10 0 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 30 0 0 + 0 0 + 0 0 0 0 34 ' 0 0 0 0 0 0 0 0 ' 0 0 37 0 0 0 0 0 0 0 0 0 0 56 0 0 0 0 0 0 0 0 00 * Readings are performed approximately 24 hours after the challenge application Note: All 6-hour exposures. Induction applications - 95% ethanol Challenge application - 1.5% HCA in acetone Challenge application - Acetone Pagce 23 WHITE EAGLE TOXICOLOGY LABORATORIES . DOYLESTOWN, PENNSYLVANIA 1B901 PWampar /: WHITE EAGLE TOXICOLOGY LABORATORIES - r 31-itain TSCA CBI White Eagle Project ^fHi Number: DuPont HLO 504-96 'WKite'Eagle'ProJect'NumBer'^^B^^^DuPont HLCrSOS^ia' E. I. du Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 5 1-0% HCA Guinea Pig Dermal Imtation/Sensitization Seores Animal Number 4 1 2 3 5 0000 11 7 0 0 12 000000 15 Reading - Hours After Application Number 24 iS 24 IS 24. 48 00 +0 11 00 00 00 00 +0 ++ 0 0 0 0 FD Challenge 24 Hrs* 48 Hrs + 0 + 0 + 0 + 0 ++ 42 45 00 00+0 00 00FD 0 0 48 2 + 0000 ++ 59 2 0 0 0 0 0 0 0 * Readings are performed approximately 24 hours after the challenge application FD = Animal #15 and #45 were found dead on Days 14 and 16 of the study, respectively. Note: All 6-hour exposures. Induction applications -1.0% HCA in 95% ethanol Challenge application - 0.75% HCA in acetone Page 24 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN, PENNSYLVANIA 18901 PageC^SnpanvSanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORTBS - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: DuPont HLO 504-96 White EaglePnjject INlumber;' D uPont HL,0 306-96 E. I. du PC>nt d< Nemours ancK 2ompany - a ;-hexy Icinnarnal dehy de, tech, 185% Guinea Pi(;DermalSfnsitizati or\-tlodified Buehller Met hoid -uIBLE 6 l.fl?&HCA- VFhilde-Con tTt1'1 Animal Number 1 Guinea !PiS^t:nnaLLitaiioli/Senal iza tion ;Scores Rf ading - Hours A ftpr ApplicaJiionNrumber Challenge 1 2 3 Acetone 24 43 24 AS 24 48 24 Hr-;* 4iLUr5 0 0 0 0 0 0 0 0 Chal lenge 0.75^S-HCA 24JEIia 48 Hr^ 0 0 9 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 36 0 0 0 0 0 0 0 0 + 0 43 0 0 0 0 0 0 0 0 0 0 46 0 0 + 0 0 0 0 0 0 0 49 0 0 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 0 51 0 0 0 0 0 0 0 0 0 0 i 54 0 0 0 0 0 0 0 0 0 0 * Readings are performed approximately 24 hours after the challenge application Note: All 6-hour exposures. Induction applications 95% ethanol Challenge application - 0.75% HCA in acetone Challenge application - Acetone WHITE EAGLE TOXICOLOGY LABORATOfflES - DOYLESIOWN. PENNSYLVANIA IW01 " ' '. ' :: Page^ WHITE EAGLE TOXICOLOGY LABORATORIH^-1 ^fe^S^^LRqS^P"13"1 TSCA CBI White Eagle Project Numberf DuPont HLO 504-96 -White-Eagle-Eroject-Number:^^1y------------------------------DuPont-HI.,0-306-96- E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization Modified Buehler Method IAELE-Z (L52jaCA Guinea Pig Dermal Imtation/SensitiMtion Scores Animal Number 2 010020300 13 19 0000++ 33 000011 38 000000 39 000000 00 40 000000 00 44 53 58 Reading - Hours After Application Number 24 4fi 24 48 24 4fi 00 00 ++ Challenge 24 Hrs* 48 Hrs 0 0 0 0 0 0 0 0 0 0 00 00 ++ 00 00 ++ 00 00 00 + 0 0 0 0 0 * Readings are performed approximately 24 hours after the challenge application Note: AU 6-hour exposures. Induction applications - 0.5% HCA in 95% ethanol Challenge application - 0.25% HCA in acetone Page 26 WHITE EAGLE TOXICOIOGV LABORATORIES - DOYLES-TOWN. PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORAT0^08W%ByrWMt^AsftWSRotefiontaTiSnCA CBI White Eagle Project Number:^ DuPont HLO 504-96 White j EaRllePrc)JectNlumber: D uPont-HLO 306-96---------------------------------- E. I. du Po'nt de Nem ours anc1 Cornpany - n -hexyld;nnamalIdehyde, tech,.B5% Guinea Pig[Denmal Sfnsitizali on -Nlodified Buehler Methoid L5^ELO (L1??'HCA-J^hide.SjDatiil\ Guinea 1EigjBi:rmaUrrLtation/Sensitizsitfon Scores Animal Number Rfflding. -Hours After AppiicaJiflrLNurnber Challenge 1 2 a Acetone 24 43 24 48 24 43 24Jjis* 4ajHra Challenge 0.25% HCA 24Js.. 48Hrs 6 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 21 0 0 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 0 0 25 0 0 0 0 0 0 0 0 0 0 , . 26 0 0 0 0 0 0 0 0 0 0 29 0 0 0 0 0 0 0 0 0 0 52 0 0 0 0 0 0 0 0 0 0 i 60 0 0 0 0 0 0 0 0 0 0 * Readings are performed approximately 24 hours after the challenge application Note: AH 6-hour exposures. Induction applications 95% ethanol Challenge application - 0.25% HCA in acetone Challenge application Acetone Page 27 WHITE EAGLE TOXICOLOGY LABORATORIES DOVLESTOWN. PENNSYLVANIA IWOl - - . Pagq^nparyLSanitiz WHITE EAGLE TOXICOLOGY LABORATORIES - DC fi.ol. contain TSCA CBI White Eagle Project Number^ DuPont HLO 504-96 White Eagle Project Number:! DuPont HLO 306-96 E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 9 Daily Observation^ Day -1: All animals in all groups appeared to be normal. Day 1: All animals in all groups appeared to be normal. Day 2: All animals in all groups appeared to be normal. Day 3: All animals in all groups appeared to be normal. Day 4: All animals in all groups appeared to be normal. Day 5: All animals in all groups appeared to be normal. Day 6: Day 7; All animals in all groups appeared to be normal. All animals in all groups appeared to be nonnaL Day 8: All animals in all groups appeared to be normal. Day 9: All animals in all groups appeared to be normal. Day 10: All animals in all groups appeared to be normal. Day 11: All animals in all groups appeared to be normal. Day 12: All animals in all groups appeared to be normal. Day 13: All animals in all groups appeared to be normal. Day 14: Animal #15 (1.0% HCA test article group) was found dead. No gross abnormalities were noted at necropsy. All other Allanimals in all groups appeared to be normal. Day 15: animals in all groups appeared to be normal. Day 16: Animal #45 (1.0% HCA test article group) was found dead. than green liquid in the gastrointestinal tract, no Other animals gross abnormalities were noted at necropsy. All other all groupasppeared to be normal. Day in 17: AHanimals in 18: All animals all grouapps peared to be normal. Day all groupasppeared to be normal. Day in 19: All 20: All 21: All 22: All animals all i n animals all i n animals all i n animals all groupasppeared to be normal. Day grouapps peared to be normal. Day grouappspeared to be normal. Day grouapps peared to be normal. Day in 23: All 24: All 25: Ail 26: All 27: All 28: All 29: All in 30: All in 31: All Paee28 32: All animals all i n animals all i n animals all i n animals all in animals all i n animals all i n animals all animals all animals all iinn animals all grouappspeared to be normal. Day groupasppeared to benormal. Day groupasppeared to be normal. Day grouapps peared to be normal. Day groupasppeared to benormal. Day grouapps peared to benormal. Day grouappspeared to benormal. Day grouappspeared to benormal. Day groupasppeared to benormal. Day grouappspeared to benormal. WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN, PENNSYLVANIA 1B501 PaeeCS Sanitized. Does not contain TSCA CBI EAGLE TOXICOLOGY LABORATOlfes -DOYLE^rOWNPE, NNSYLVANIA 18901 WHITE White Eagle Project Number: DuPont HLO 504-96 White Eagle Project Number:| DuPont HLO 306-96 E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method lAELEJfl Test Article - 2.0% HCA Individual Body Weights Ig) Animal lumber 17 18 27 31 32 35 41 47 55 57 Initialf 456 435 480 460 437 470 432 474 420 483 Day 7 514 494 564 510 499 553 505 571 476 548 Day 14 DayJl Da)L22 564 621 702 550 606 689 628 610 679 575 635 709 552 689 758 622 670 750 571 626 715 645 707 777 522 569 624 618 703 763 Final Day-32 689 689 677 699 763 771 717 790 637 789 Change (-lto32) +233 +254 +197 +239 +326 +301 +285 +316 +217 +306 Mean 455 Std.Dev. 22 523 585 644 717 722 33 41 46 47 53 +267 45 Vehicle Control - Elhanol/Acetone Animal fslumber Initialt Day 7 Day 14 Day 21 Day 29 Final Dav.^2 Change (-1 to 32) 3 476 10 423 16 501 22 429 23 450 28 444 30 498 34 462 37 463 56 469 546 606 661 715 702 456 520 569 645 653 550 622 671 765 777 508 585 649 737 742 525 612 683 770 776 521 569 619 673 673 568 650 710 787 795 549 629 684 741 752 523 573 648 706 711 495 559 617 678 687 +226 +230 +276 +313 +326 +229 +297 +290 +248 +218 Mean 462 Std.Dev. 26 524 593 651 722 727 32 39 41 47 49 +265 40 + = Body weights taken on Day -1 (the day before the first induction application of test article or. vehicle). Page 29 WHITE EAGLE TOXICOLOGY IABORATOBES DOYLESTOWN. FENNSYLVANIA 18501 Page(7(Snpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number^ DuPont HLO 504-96 White Eagle Project Number: | DuPont HLO 306-96 E. I. du Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization ^ Modified Buehler Method TART.Rn TestArliclp-in%HrA Individual Body Weight (g) Animal Number 4 5 7 11 12 15 42 45 48 59 Mean Std.Dev. Inirialt 459 479 442 406 434 469 433 424 517 475 454 32 Day 7 549 538 494 475 502 537498 500 598 570 526 39 Pay 14 Day 21 613 685 629 697 538 568 517 580 559 619 552 596 563 > 648 712 647 711 585 646 50 61 Day 29 741 785 641 639 694 650 763 792 713. 65 Final Day 32 729 811 638 626 716 651 766 807 718 74 Change (-1 to 32) +270 +332 +196 +220 +282- +218 +249. +332 +262 51- Vehicle Control - Ethanol/Acetone Animal Numbpr 1 9 20 36 43 46 49 50 51 54 IniHalt 475 455 426 479 407 465 469 433 480 453 Day? 53.1 501 482 559 462 547 518 468 537 563 Day 14 607 556 540 639 507 614 590 519 614 632 Day 2^ 662 641 599 712 564 590 603 554 677 636 Day 29 716 724 581 776 615 697 666 627 721 718 Bnal Day 32 735 728 582 809 618 721 667 627 731 704 Change f-l.to32) +260 +273 +156 +330 +211 +256 +198 +194 +251 +251 Mean 454 Std.Dev. 25 517 582 624 684 692 37 48 51 60 68 +238 49 + = Body Weights taken on Day -1 (the day before the first induction application of test article or ' vehicle). = Animals #15 and #45 were found dead on Days 14 and 16 respectively Pace 30 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN, PENNSYLVANIA 18901 Paged^npany Saniti2 WHITE EAGLE TOXICOLOGY LABORATORIES -F - -. - . antain TSCA CBI White Eagle Project Number:) DuFont HLO 504-96 White Eagle Project Number -DuPont-HLO 306-96- E. I. du Font de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method IAELEJ2 Test Article - 0.5% HCA IndhivaiduaallBBooddy WWpeiigghhttss f(e^) Animal. Number 2 13 19 33 38 39 40 44 53 58 Initialt 458 385 442 464 451 462 413 481 507 474 Day.? 521 440 527 529 519 521 462 560 580 560 Eay-U 577 482 569 603 595 586 500 630 632 618 Eay-Zl 652 542 656 685 662 654 564 684 708 693 Ea)L22 723 599 714 752 737 709 623 739 754 763 Enal Day-32 731 603 733 788 759 686 639 739 792 770 Change (-1 In 32) +273 +218 +291 +324 +308 +224 +226 +258 +285 +296 Mean 454 Std.Dev. 35 522 579 650 711 724 43 51 55 56 63 +270 37 Vehicle Contml - Ethanol/Acetonc Animal Number Initialt Day 7 Day-14 Pay 21 Day 29 final Day 32 Change (-1 to 32) 6 455 8 411 14 472 533 608 670 754 773 479 549 614 685 701 560 627 701 766 776 +318 +290 +304 21 431 517 590 653 705 716 +285 24 396 490 568 618 705 704 +308 25 421 505 573 628 691 688 +267 26 499 591 680 759 825 845 +346 29 451 519 554 581 631 588 +137 52 516 508 559 610 651 644 +128 60 411 484 547 613 686 687 +276 Mean - 446 Std.Dev. 40 519 586 645 710 712 35 42 53 57 72 +266 74 + o Body weights taken on Day -1 (the day before the first induction application of test article or vehide). Page 31 WHITE EAGLE TOXICOLOGY LADORATOfflES . DOYLESTOWN, PENNSYLVANIA 18901 WHITE EAGLE S^^^^^m^mf^w^TSCA TOXICOLOGY -LABORATORIES - CBI White Eagle Project Number: f DuPont HLO 504-96 -White Eagle Project Number: ^^^y-----------__________DuEont.HLQ.30fr:96_ E. I. du Font de Nemours and Company - n-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method APPENDIX A COPY OF PROTOCOL COVERSHEET AND PROTOCOL Page 32 WHITE EAGLE TOXICOLOGY LABORATORIES . DOYLESTOWN, PENNSYLVANIA 1M01 WHITE EAGLE TOXICOLOGY LABORATORIES-1 . i^E^^gM^^'" TSCA CBI White Eagle Project Number:, DuPont HLO 504-96 _F_1L_E_N_o_. _51_5_0_4/_19__"_96_0_9_:2_3 _1D_:H_(^_SK_E_LL_L_A_B _TO_X_IC_OL_O_GY_/ __3_02_3_6_6 _52_0D7 uPont-HLQ.3PDw6b -%2 - PROTOCOL------------Hi(Rev. 04/96) Guinea Pig DcnBSRniiSiSulon^Moditicd Uuchler Method TcEting Facility; Sponsor: While Eagle Toxicology Laboratories 2003 Lower Slalc Koad ILL ilu Poni do Ncmoura jnd Company Hukall Laboratory lor Toxicology And Doylcttown, PA 18901 InduTrial Medicine Newark. DE 19714 Test Article: ------' H ^^J^^^^^^JL^LSL^^ 'Xejc^.^S^, While Eaetc Project Number. TcH Article DCTcriplion; Tct Article Receipt Dole: Proposed In-Ufe Starting D*te: Proponed In-Ufe Completion Dole: -fl^lHBE------ Cir'lr J''?^^ V 'S' -'S ll}^ .'^-l __ (a _ _ ^Jf-'^ei-- 1C' ^(S-ni'\^M-.^i To be Completed by Whito Eglc EPA CONI;lDENrnALITY STATEMENT .KA,. a n. Sponsor will indudc their own EPA ConddcntialHy Stolcment in the Finol Report While Eoglo will Include alanilafd UPA ConfidcnUality Statement In the rinnl Report If "D", select one of the following: o No Data Confidcniialily Claima o Data Contidcntialily Claims This sludy docs nol unnecessarily duplicate previous expcruncnts nur is thcro an acceptable in-viiro metliod that can subslllulc for the use of animaln. PROTOCOL AMENDMENTS Rl^n. jCtf- .A^L^^ia^. fL- .^m/ ^g^, ^ g 5^ ^t^tAv ^ ^^UsJ. J^^, JS^a/iZcZ ^m^t, - Ckj^ciA^L MHXL^jeLan^^i ^C^nJL,1//,^^ PROTOCOL APPROVAL Tciiling Facility: QI^^T^^ Study Difector * Sponsor: f ^^<r^L H-if-^ Dote Sponsor Rcpcacnialivc y//?/?^ Date ; "t > Pagee 33 WHITE EAGLE TOXICOLOGY LABORATORIES DOVLESTOWN. PENNSYLVANIA 18901 ^ EACLB TOXICOLOCV ^BORA^3^^^ TSCA CB1 White Eagle Project Number: DuPont HLO 504-96 =-^------DuPont-HLO-AUb-W White Eagle Toxicology Laboratories 2003 Lower Slalc Road Doylcslown.rcnnsylvania, 18901 Telephone: (215) 34B-3868 Fax: (Z15) 318-5081 Protocol Number rrolocol Name: Guinea Pig Dermal Sensitization Modified Uuclilcr Method This prolocol contains information (hat is proprietary and confidential to While Eagle Toxicology Laboratories. Use of this Information is restricted to the parlies to which While Eagle Toxicology Laboratories has issued it in lllc course of business. Any duplication or dislribution of the contents without prior approval o( While Eagle Toxicology Laboratories is strictly prohibited. ' Page 34 WHBTE EAGLE TOXICOLOGY LADpRATOMS DOYLESTOWN, PENNSYLVANIA IBfll WHITE EAGLE TOXICOLOGY LABORAT ^^SAisft'^rf?^^"^1" TSCA CBI White Eagle Project Number:! DuPont HLO 504-96 -DuEont-HL0^005==- WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower Slalc Road, Doylcslown, Pennsylvania 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 Protocol Number: |Rcv. 04/96) - Proji-cl Numbpn Tcsline Facility: Sponsor: Test Arlidp: TcaLArlidc Pcscriplion: Tcsl Arlidc licccivcd: {to be complclcd by Wliilc Eagle Toxicology Laboralorics) While Eagle Toxicology Laboralorics 2003 Lower Stale Road Doylcslown, Pennsylvania 18901 E. I. du Font dc Ncmours and Company HaskeII Laboratory for Toxicology and Industrial Medicine Elklon Road, P.O. Box 50 Newark, DC 19714 Positive Control (a-licxylcinnamaldchydc, tcch, 85%) Clear liquid Supplied by Wliilc Eagle Toxicology Laboratories VchidLConlml Ar|iclg.'i: Ycludi^Cflnlifll Dcamplions: Proposed InJJfc Starting Pal.;: Ethyl Alcohol (95%) Acetone (99.7%) Clear liquids {to be specified) Proposed In-r-ifc CQinplclion.Palc: {to be specified) leal; Guinea Pig Dermal Scnsilizalion - Modified Buchler Method* Objpclivp of Tp-il: Testing Guidelines: To validate the (csl system using a known scnsilizcr, a-hcxyldnnamaldchydc (HCA). The mclliods employed in the testing of this lest article arc similar lo Ihosc described in the U.S. ERA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human & Domestic Animals, Series 81-6, 1982; EPA I-lcallh Effects Testing Guidelines Subpnrt E Spcci(ic Organ/Tissue Toxidly, September 1985, 40 CFR Pnrl 798; Ihc OECO Guidelines for Testing of Chemicals, No. 406, Skin Scnsilizalion^ adopted July 17, 1992; EEC 92/69 Annex V, Part 0.6 (1992). 93/21 (1993); and the MAFF Japan Testing Guidelines for Toxicology Studies, Dermal Scnsilizalion Study, 59 NohSan No. 4200. 1985. "Kilz, H.L. and Buchler, E.V., Planning, Conduct, and Inlcrprclalion or Guinea Pig Scnsilizalion rlch Tests, current Conccpis In Culaneous Toxidly,, Drill & Lazar editors, Academic Press 1980. : ; Page 35 ; Whire EAdE TOXICOLOGY LABORATORIES - DOYLESTOWN. rENNSWANIA 18901 WHITE EAGLE TOXICOLOGY LABORATORIES -1 V1^^^^?"^1" TSCA CBI White Eagle Project Number| DuPont HLO 504-96 -DuPont-Ht<3^06-96^-- Prolocol Numbcifl^^^^y (Rev. 04/96) Guinea Pig Dermal Scnsitizalion - Modified Duchlcr Mclhod Pace 2 of 7 Good Laborniofy Statement: This study is conducted in compliance wilh Ihc U.S. EPA F1FRA Laboratory Practice Standards (40.CFR 160), U.S. EPA TSCA Laboratory Practice Sinndards (40 CFR 792), OECD Principles of Laboratory Practice (c(81)30(Final), Annex 2), and MAFF Japan Laboratory Practice Standards (59 NohSan No. 3850). Good Good Good Good Juslificniion for Test System: The laboratory albino guinea pig is the system of choice since it is required in the EPA, EEC. OECD, and MAFF Japan guidelines for this type of study and has a long history of use in dermal scnsilizalion studies. Justification lQI_QUlC_Of Adminislralion: The specific route of administration (dermal) is required in the Icsling guidelines for this study. Anin'inis' The adult male Hartley guinea pig, weighing 300 lo 550 grains, is used for (his study. The animals arc obtained from Davidson Mill Orccding Lobs, Jamcsbiirg. NJ. 08831 U.S.D.A. License iit22-A-003, Ace Animals Inc., Uoycrlown. PA 19512 U.S.D.A. License <?23-B-009, Elm Hill llrccding Labs. Chclmsford, MA 01824 U.S.D.A. License tfl4-D-006 or Camm Research Lab Animals, Waync, NJ 07470 U.S.D.A. License #22005. The source of animals will be documented in (lie study records.* The animals (except for screens) arc wciglicd prior to initiation, weekly and upon completion of the study. The animals arc randomized using computer generated random numbers inio groups and the initial body weights Pay -I) arc statistically evaluated by Analysis of Variance (ANOVA). Statistical analyses and random number generation arc performed using CSS: Slalislica, 3.1 copyright. Sl.ilsofl Inc. 1991. Rc- challcngc naive vehicle controls, if utilized, arc not weighed prior to initiation of the study, but arc weighed prior to re-challenge. The animals are housed and maintained in accordance with standards set forth in (he Guide for the Care and Use of Laboratory Animals (N.itional Institutes of Health Publication No. 86-23). The guinea pigs nrc ncctimnlcd to the laboratory for at least 7 days prior to the initial induction application. Clinical observations arc performed daily on all animals during the study. IACUC Review: The animals arc individually identified by color coding with water* nnd/or solvcni-rcsislnnt markers. Each cage is identified with a cngc card displaying the study number, dale initialed, animal numbcr(s), group, sex, and responsible technician's initials. The Protocol for this study has been reviewed by While Eagle's Institutional Animal Care and Use Committee (1ACUQ and complies with acceptable standards for animal welfare and humane care. WHTTC EACUI TOXICOLOGY LADOKATORIES OOYLCS1WN, PENNSYLVANIA ; Page 36 : WHITE EAGLE TOXICOLOGY LABORATORIES DOTLESIWN. PENNSYLVANIA IBMl WHITE EAGLE TOXICOLOGY LABORATT ^^^^Ms^A^0!^^'^ TSCA CBI White Eagle Project Number: DuPont HLO 504-96 -DuPont-HLO'-306^5==- Prolocol Number: ] i(Rcv. 04/96) Guinea Pig Dermal Scnsilizalion - Modified Buchlcr Mclhod Page 3 of 7 Husbandry Condilions: Temperature: 18-C - 26C (64.4'F - 78.8T) Rclalivc Humidity: 50 20 % Ught: 12-hour light/dark cydc Diet: Purina Ccrlificd Guinea Pig Diet tf5026 and lap walcr are provided ad libilum. Dascd on our current knowledge, no contaminants arc known to be in this diet or water which might be expected to interfere with the objectives of the study. Caging; Stainless slecl wilh elevated wire mesh flooring, 1-5 guinea pigs/cage. Test Article Vehicle: Elhyl Alcohol (95%) will be used as the lest article vehicle for the induction phase of this study and acetone (99.7%) for the challenge phase of this study. Enac Kan^efindiug: Ilisloricnily, 5.0% and 2.5% HCA in 95% cthanol have produced no irritation to faint irritation after the first induction application and necrosis after the second or third induction application. Based on (his steep dermal response, conccniralions of 2.0%, 1.0% and 0.5% will be used for the induction phase of this study. Two groups of 4 uncxposcd animals will be weighed and exposed lo different challenge conccniralions of HCA in acetone by the palcliing technique described in the induction stage. Concentrations of 1.50%. 0.75%, 0.50% and 0.25% in acetone will be utilized to determine non-irritating levels lo be used- as challenge concentrations. Mnhod of Study Performance: Tcsl Article Group InJurlion Stage: Three groups of ten (10) guinea pigs arc closely clipped over the induction site on Ihcir left finnk (approximately 1.5 X 1.5 inches) during the day prior to the day of initiation and repeated as necessary. If the induction site is moved, an additional area (sufficient for patching) is closely dipped at the new site. Based upon historical dala, dose levels of 2.0%, 1.0% and 0.5% w/v of HCA In 95% clhanol. will be used (or the induction stage. A 03 mL portion of the appropriate concentration of HCA in 95% clhanol is applied to a 25 nun Hill Top Chamber1*1 which is placed onto the lest site and covered with a piece of dental dam (approximately 2 inches X 2 inches). After positioning the chamber and the occlusivc dental dam onto the prepared skin lest site, plastic material is wrapped snugly around the trunk of the animal and ovcrwrappcd with clastic tape. wumi EAGLE TOX1COLOCT UUlOllATOlUES DOYUSTOW, reHNSKLVANIA : ; Page 37 WfflTE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN. PENNSYLVANIA IBSal WHITE EAGLE TOXICOLOGY LABORATOB ?ntain TSCACBI White Eagle Project Number:! DuPont HLO 504-96 -DuPont-HLO^Oo-96--'-- Frolocol Numbcr^^^^^B (Rev. 04/96) Guinea Pis Dermal Scnsiliz.ilion - Modified Buchlcr Mclliod Page 4 of 7 Induclion Singe: (conlinucd) Approximately six liours later, (lie cliambcr is removed and Ihc lest silc is wiped wilh an appropriate solvent. The treated silc is scored for irritation, using (lie scale included in Table I, at approximately 24 and 48 hours from the initial application. The procedure is performed once a week for Ilircc weeks, for a tola! of three, six-hour applications with Ilic appropriate conccniralion of I-ICA. Tl>c interval between induction exposures may vary from 6 to 8 days. Should pronounced irritation be observed at the induction site, the site may be moved for the next induction application, with its relative location fully documented. Vehicle Control Croup Three groups of ten (10) guinea pigs arc closely clipped over the induction silc towards Ihc head on their left (lank (approximately 1.5 X 1.5 inches) during the day prior lo Ilic day of initiation and repealed as necessary. If the induction site is moved, an additional area. (sufficient for patching) is closely clipped at (he new site. A 0.5 mL portion of the vehicle (95% clhanol) is applied lo a 25 mm Hill Top Cliambcr111 which is placed onto Ihc lest site and covered with a piece of denial dam (approximately 2 inches X 2 inches): Aflcr positioning Ihc chamber and Ihc occlusivc denial dam onto Ihc prepared skin lest site, pl.-islic malcrial is wrapped snugly around Ilic trunk of Ihc animal and ovcrwrappcd with claslic lapc. Approximately six hours lalcr, Ihc chamber is removed and the lest silc is wiped wilh an opproprialc solvent. The treated silc is scored for irrilalion according to Ihc scale included in Table I at approximately 24 and 48 liours from the initial application. The procedure is performed once a week for three weeks, for a total of three, six-hour applications of 95% clhanol. The interval between induction exposures may vary from 6 to 8 days. Should pronounced irritation be observed at the induction silc, the silc may be moved for the next induction application, with its relative location fully documented. Challenge Stage: Test Material Croup After Ihc Iliird induction application, the animals arc rested for approximately 14 to 16 days. At the termination of the rest period, a cliallcngc application in acetone, using the procedures, described previously, is applied lo a challenge site (on the right flank). The challenge application remains on for approximately 6 liours. Upon removal of the challenge application, the contact area is wiped with an appropriate solvent. In the event of questionable findings, a rcchallcngc application of all animals is performed on a second naive site no sooner than six days after primary challenge, along with a group of 5 naive animals taken from the colony pool, serving as vehicle controls. Dales of application and observations arc recorded. Approximately 24 and 48 hours after the challenge (and rcchallcngc if necessary) application, Ilic sites are examined for dermal irritation and/or signs of clicilcd scnsilizalion, according lo Hie scale included in Table I. wi irro liAcu; TOXICOLOGY I-AIIORATOIUCS . DOYUSTOWN, niNNsn.VANiA .. : Page 38 WHITE EAGLE TOXICOLOGY LADORATOlUES DOYLESTOWN. PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATOR White Eagle Project Number:! DuFont HLO 504-96 -DuPont-HLO;30& rrolocol Numbcn^^^^^y (Rev. 04/96) Pace 5 of 7 Guinea Pig Dcrm.-il Scnsilizniion Modified Duchlcr Method ChallcngcJSlagc; Vehicle Control Croup After Ihc Iliircl induction applicalion, (he animals arc rcslcd for opproximalcly 14 to 1C days. At the Icrmin.ilion of the rest period, the vehicle conlrol croup is challenged with occlonc on the left flank (caudal) and Ihc npproprialc conccniralion of 1-1CA in acetone on Ihc righl flank using Ihe procedures described previously. The challenge applicalion remains on for approximately 6 hours. Upon removal of the challenge application, Ihc contact areas arc wiped with an appropriate solvent. Dales of application and observations arc recorded. Approximately 24 and 48 hours after (he challenge application, the sites arc examined for dermal irritation and/or signs of dialed scnsilizalion according, to the scale included in Table t. Ecchallcngc: If the study is not positive, Ihc Sponsor is notified and the lest animals arc rcchallcngcd 6 to 8 days after the primary challenge. Pain Retention: All raw data generated, all records accumulated during the performance of Illis study and a copy of the fin.-il report will be retained at White Eagle Toxicology Laboratories for a period of ten (10) years, after which lime (lie data will be shipped (o the Sponsor or, discarded upon written request from the Sponsor. Positive Conlrol .qiaraclcdzaliaa: Details regarding the identity, strength, stability, purity.. and composition of the positive control arc provided by the Manufacturer and arc oh file at White. Eagle Toxicology Laboratories, Doylcstown, I'cnnsylvnnia. The positive control has been examined and lias been found to be soluble in clhanol and acclonc. Any mixtures or dilutions will not be analyzed by While Eagle or the Sponsor. Slalislical Analysis: Mortality. Morbidity and Terminnlion: Statistical Methods: Individual body weight percentage gain is statistically evaluated by Analysis of Variance (ANOVA), willi the Neuman Kculs test applied in the event of a significant finding (F<0.05) for the data as a whole. Statistical analyses arc performed using CSS; Slalislicn, 3.1 copyright, Slalsoft Inc. 1991. All animals found dead during the observation period or euthanized in extremis arc nccropsicd to establish, if possible, the cause of death or illness. All lest animals arc euthanized by CO, inhalation or IP pcntotorbilal at (he completion of the study. Protocol Changes: All changes in or revisions of the approved protocol and the reasons will be documented, signed by the study director, dated and maintained with the prolocol. Wlimi CAGU; TOXICOLOGY LAnOHATOlUIS- DOtUSTOWN. riiNNSYLVANIA ' Pscc 39 ' ' ' WHITE EAGLE TOXICOLCCr.LABORATOMES . DOYLCSTOWN. PENNSYLVANIA I8CT1 FaeeCSSnpany Sanitized. Does not contain TSCA CBI WHrrE EAGLE TOXICOLOGY LABORATOMES- - DOYLE^TOWN,PENNSYLVANIA 18901 White Eagle Project Number:) DuPont HLO 504-96 DuPont HLO 306-96- Protocol Number: ^^^^^f(Kcv. 04/96) Guinea Pic Dermal Scnsilizalion - Modified Buchlcr Mclhod Page 6 of 7 Records Mainl.iinpcl- Records to be maintained include: - irrilalion scores for screen animals - irrilalion scores for redness and swelling (induction and challenge) - daily clinical observations - initial, weekly, and final body wcighls (including group standard deviation, and change between initial and final) - HCA preparation means. - calculations and statistical analyses - randomization scheme Finnl Krpnrl: A final report is written which includes, but is not limited to, the items died in U.S. EPA FIFRA Good Laboratory Practice Standards (40 CFR Part 160). Subpart J, Records and Reports. U.S. EPA TSCA Good Laboratory Practice Standards (40 CFR 792), Subpart J Records and Reports, OECD Principles of Good Laboratory Practice, c(81)30 Annex 2 (Final), Section 1L Part 9.2, Content of the Final Krport. nnd MAFF Japan Good Laboratory Practice Standards, 59 NoliSan No. 3850, Chapter VIII, Report and Records. Intcrprclalinn of Results: All non-eschar and non-nccrolic lest sites arc scored on a scale of 0 to 3 as indicated in the following Table 1. When edema is present with scores of +, 1, or 2 for erythema, it will be documented. TABLE I Scale for Evaluation of Dermal Irritation 0 = no reaction 0.5 = very faint redness (usually nonconducnt) 1 <= faint redness (usually confluent) 2 " moderate redness 3 = severe redness with/without swelling Note: 0.5 scores will be recorded in the raw data as a + Scores of 1 or greater in the lest group arc required to be indicative of scnsilizalion. If scores of one (1) or greater arc seen on the vehicle control animals, (hen the reactions of the lest article group nnimnk that exceed the most severe vehicle control reactions tire considered to be positive scores. INCIDENCE is reported as the number of positive animals in cadi group divided by the total number of animals tested in that group. SEVERITY is reported as (he sum of the lest grades divided by the total number of animals tested in a given group determined for both 24 and 48 hours. Grades + arc equal to 0.5 for calculation of severity indices. All average grades arc to be rounded off to the nearest tenth of a unit. A minimum of two out of len positive control animals must be shown to have elicited a positive reaction (Scores ^ one (1)) to (he positive control material to validate Ihc lest system. WHITE CACI.B TOXICOLOGY LADOHATD1U13 . TOVUSTOWN. PENNSYLVANIA fi- ; ; Praagre 40 WHrTE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA 16W1 PageCMripany Sanitized; Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number------j^^ DuPont HLO 504-96 DuPont HLO 306-96 Protocol Number: ^^^^^(Rcv. 04/96) Guinea Pig Dermal Scnsilizalion Modified Buchlcr Method ^B Signatures: Submitted by While Eagle Toxicology Laboratories: Page 7 of 7 .(^L^^^ ^ L ^ George E./Moorc. U.S. ^ Study Director Approved by: ^-/g-^ Dale h^^. J ^ Sponsor Representative ^J^ Dale "Wl *.- wnm; EACUITOUCOLOCr LAlIOnATOnilS DOYUSTOWN. rCNNSYLVANIA ^ 1 : . J Page 41 WtTE EAGLE TOXIC01.0CY LABORATORIES DOYLESIOWN. rETOCVLVANIA 1801 ^ pa?e^?-a-n^s-a-n-i^zed^^^ WHITE EAGLE TOXICOLOGY LABORATORIES -DC White Eagle Project Number: ------^ DuPont HLO 504-96 White Eagle Project Number: Bj^MB^________________DiiPont HLO 306-96 E. I. du Font de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method APPENDIX B DEVIATIONS FROM PROTOCOL Paee42 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA 1890) WHITE EAGLE TOXICOLOGY LABORATO^I^O^^tep^ft^^^ TSCA CBI White Eagle Project Number:! DuPont HLO 504-96 DuPont HLO 306-96 -WHITE-EAGLE-TGXIGOLOGY-LABORA-TeRIES- 2003 Lower State Road, Doylestown, Pennsylvania 18901 Telephone: (215) 348-3868 Fax; (215) 348-5081 White Eagle Project Number: Protocol Number: Sponsor: Test Article: Test: GLP Compliance: DEVIATIONS FROM PROTOCOL #\, #2. #3 '(Rev. 04/96) E. I. du Font de Nemours and Company a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensilization - Modified 40CFR160 40CFR792 OECD Buehler Method MAFF Japan 1) The Relative Humidity during the In-Life phase of this study was 24%88% instead of 30%-70% as stated in the protocol. 2) Due to a Holiday, animal body weights were taken on Day 29 (week 5) instead of Day 28 (week 4). The following is an amendment to protocol which was not approved in writing until the completion of the study; therefore, it is presented as a deviation. 3) Protocol Cover Sheet and Protocol Change From: a-hexylcinnamaldehyde, lech, 85% Change To: a-hexylcinnamaldehyde, tech, 85% These deviations are not thought to have affected the integrity of this study. ' Submitted by: j^^t^C n/ffr^. _- George E. Moore, B.S. Study Director G^-W Date Reviewed by: Approved by; DianeT. Cbok, B.S. Quality Assurance Manager W^^^-(y(^- Sponsor Representative Page 43 C -24-^<- Date ^7/^ Date Psg^ WHITE EAGLE TOXICOLOGY LABORATORIES ^yntain TSCA CBI