Document v102z7YBO8X7xZNENEN56vbrY

DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown, Pennsylvania 18901 Telephone: (215) 348-3868 __________________ Fax: (215) 348-5081 _____________ AR226-3055 Study Title Guinea Pig Dermal Sensitization - Modified Buehler Method with H-21891 Laboratory Project TD 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 Author George E. Moore, B.S. for E.I. du Pont 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: E. I. du Pont de Nemours and Company - H-21891 Guinea-Pig-DermaLSensitization---Modified-Buehler-Method DuPont HLO 504-96 THIS PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS Paig2imparty Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORII]ES - DOYLESTOWN, PENNSYLVANIA 18901 ' DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road Doylestown, Pennsylvania 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT White Eagle Project Number: Sponsor: E. I. du Pont de Nemours and Company Test Article: H-21891 Test: Guinea Pig Dermal Sensitization - Modified Buehler Method Protocol Number: (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. Study Director Sponsor: DuPont Specialty Chemicals E.I. du Pont 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 Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method GENERAL INFORMATION Test Material Substance Tested: DuPont HLO 504-96 Synonyms/Other Codes: CAS Registry Number: Haskell No.: Composition: 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 Pont de Nemours and Company Barley Mill Plaza Wilmington, Delaware 19880-0038 WHITE EAGLE TOXICOLOGY LABORATORffif^ D c S ? ^ O ^ /t ^ ^ s v A h a l Clt8S:0< n t a i n T S C A C B I White Eagle Project Number:! E. I. du Pont 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: l-chloro-2,4-dinitrobenzene Approximately 100% (supplied by Eastman Kodak Co.) DNCB Eastman Kodak Company, Lot #A11R CAS Registry N o.: 97-00-7 (supplied by Eastman Kodak Co.) Stability: 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 WHITE EAGLE TOXICOLOGY L A B O R A T O R ^ '^D C^JE^rO w 5s,t i ^ ^ i s 9 ^ l f i ^ f f r lt a ,n T S C A C B I White Eagle Project Number: E. I. du Pont 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 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. PagdDfompany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: E. I. du Pont 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) Study Director Reviewed/Audited by: Date Quality Assurance Manager ^ -2 0 -q l Date Paj WHITE EAGLE TOXICOLOGY LABORATORI in TSCA CBI White Eagle Project Number: E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 White Eagle Project Number: Sponsor Study Number: Test Article: Study Initiation: H-21891 06/03/96 Inspections Protocol Procedures Raw Data Draft Report Final Report Quality Assurance Statement Pates 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: 2 T/Cook, B S. Quality Assurance Manager Pag>mpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number E. I. du Pont 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 S ystem .................................................................................................... 12 Justification for Route of Administration.............................................................................12 LACUC Review ................................................................................ 12 Animals ..........................................................................................................................................12 Husbandry Conditions..............................................................................................................13 Dose Range-finding .............................................................; ...................................................13 Induction S ta g e ............................................................................................................................ 13 Challenge Stage ................ 15 Statistical A nalysis............................................................ 16 Mortality, Morbidity and Term ination.................................................................................16 Data R etention............................................................................................................................. 16 P erso n n el...................... 16 Test Article and Positive Control Characterization ......................................................... 17 Interpretation of R esu lts........................................................................................................... 17 Table I - Scale for Evaluation of Dermal Irritation ........ 17 WHITE EAGLE TOXICOLOGY L A B O R A T O R m S ^ D C ^ f T S C A C B I White Eagle Project Number: E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 TABLE OF CONTENTS (continued) Page Results ....... .................................................................................................................................18 Conclusion ............................................................................................................................ ....2 0 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 C o n trol......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 Page&SbTpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DCSYLESTOWN, PENNSYLVANIA 1890 DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown, Pennsylvania 18901 ___ ___________________________ Telephone:_(215)_348-3868________________________________ Fax: (215) 348-5081 (Rev. 04/96) White Eagle Testing Facility: Sponsor: Test Artide: Test Article Description: Test Article Received: Vehicle Control Article: Vehicle Control Description: Positive Control Article: Positive Control Description: Positive Control Vehicles: Positive Control Vehicles: Description: Dose-Rangefinding Initiated: In-Life Starting Date: Test: White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown, PA 18901 E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714 H-21891 Tan liquid 05/29/96 Normal Saline Solution (Vedco Inc., St. Joseph, MO 64504 Lot #5080419, Exp. 8/98) Clear liquid l-chloro-2, 4-dinitrobenzene (Eastman Kodak Co., Rochester, N.Y. Lot #A 11R , Exp. 12/26/98) Yellow crystal Ethanol (95%)/Acetone (99.7% pure) Clear liquids 06/05/96 In-life 06/18/96 . Completion Date: 07/19/96 Guinea Pig Dermal Sensitization - Modified Buehler Method* Ritz, H.L. and Buehler, E.V., Planning, Conduct, and Interpretation of Guinea Pig Sensitization Patch Tests, current Concepts in Cutaneous Toxicity, Drill & Lazar editors, Academic Press 1980. WHITE EAGLE TOXICOLOGY LABORATORIE! in TSCA CBI White Eagle Project Number E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method DuPont HLO 504-96 Objective of Test: To assess the contact dermal sensitization potential of the test article. Testing 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 Toxicity, 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 FTFRA 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. TACTIC 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. A nim als: 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. . Page, WHITE EAGLE TOXICOLOGY LABORATORI) in TSCA CBI White Eagle Project Number: E. I. du Pont de Nemours and Company - H-21891 DuPont HLO 504-96 Guinea-Pig-Dermal-Sensitization^Modified-Buehler-Method Animals: (continued) Husbandry Conditions: The animals were individually identified by color coding with solventresistant 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 LABORA'^ ^ ^ % ^ E ^ O w { , t f e A s ' R . ? ? ? l l C^ 5 t f n t a i n T S C A C B I White Eagle Project Number: DuPont HLO 504-96 E. I. du Pont de Nemours and Company - H-21891 G uin eaPig-D erm aLSensitization ^M odified3uehler_ 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 dining 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 Elastikon . 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 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 0.1% 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 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 LABORATroOaR^IES^ f f i 9 I M ^ ? ntain TSCA 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 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. Challe.nge_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 clipped right flank). Based on the range finding 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). WHITE EAGLE TOXICOLOGY LABORATORI] iin TSCA CBI White Eagle Project Number: E. I. du Pont 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 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). 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 analysis was performed using CSS: Statistica, 3.1 copyright, StatSoft Inc. 1991. Mortality. Morbidity and Termination: No deaths occurred during the study. All animals were euthanized with an intraperitoneal injection of sodium pentobarbital at the conclusion of the study. 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. 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 Pagedtfnpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project N u m b e r f ^ P P DuPont HLO 504-96 E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method_______________________________ Test Article and Positive Control P T iarartprizatinn: 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 the positive control material to validate the test system for this particular sample. P a g a ^ p a n ^ S a m t i z ^ ^ ^ o ^ n t a i n TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DYL! 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_______________________________ 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. Results: 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 fortyeight hours after the challenge application. The incidence of sensitization was 0/20. WHITE EAGLE TOXICOLOGY L A B O R A T O I^ K ^ X ? n .E ^ r iW W !'lM A l S f t (^ l f l P f e S f l n t a i n T S C A C B I 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 artide group. 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 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 vehide or test artide 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 LABORATTORIES O R E ? nt ai n TSCA 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) 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% 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. Conclusion: 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 Rf Moore, B.E Study Director Reviewed/Audited by: 9 -A O -9U Date Date WHITE EAGLE TOXICOLOGY LABORATrOORMIESK ^C ^E ^O W N ^'^ 's vA N ^fe'0*'3'n TSCA ^B l White Eagle Project N u m b e r :f | ^ H B r E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method T ABT .F. 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 0 0 0 48 Hours 0 0 0 0 Temporary Animal Number 142 143 144 145 Screen # 2 - Right Side 75% concentration of H-21891 in 0.9% saline 24 Hours 0 0 0 0 48 Hours 0 0 0 0 Body Weights (g) Temporary Animal # 1 4 2 Temporary Animal # 1 4 3 Temporary Animal # 1 4 4 Temporary Animal # 1 4 5 393 380 380 366 Page 21 WHITE EAGLE TOXICOLOGY L A B O R A T O Iffii^ W fie E a irS R ifiiE S S i B fiH in a tffiO n ta in TSCA CBI White Eagle Project Number:! E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 2 ('continued') Dose Range-finding Scores DuPont HLO 504-96 Temporary Animal Number 146 147 148 149 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 Screen # 4 - Right Side 25% concentration of H-21891 in 0.9% saline 24 Hours 0 0 0 0 48 Hours 0 0 0 0 Body Weights (g Temporary Animal # 1 4 6 Temporary Animal # 1 4 7 Temporary Animal # 1 4 8 Temporary Animal # 1 4 9 375 382 418 465 WHITE EAGLE TOXICOLOGY LABORATrOrl a ^ ^ a ti;^ i s 9 ^ i R q t o ntain TSCA CBI White Eagle Project Number: | DuPont HLO 504-96 E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Behler Method TABLE 3 Guinea Pig Dermal'Irritation/Sensitization Scores Test Article - H-21891 Induction Study Animal Number Application 1 24 Hrs 48 Hrs Application 2 24 Hrs 48 Hrs Application 2 24 Hrs 48 Hrs 1 00 00 00 2 00 0+ +0 3 00 +0 00 4 00 00 00 5 00 +0 00 6 00 00 00 7 00 00 0+ 8 00 +0 00 9 00 +0 00 10 00 00 ++ 11 00 00 00 12 00 00 00 13 00 ++ .+ + 14 00 00 00 15 00 00 00 16 . 00 +0 00 17 0 0-. 00 00 18 00 00 + + 19 00 0 `0 .0 0 20 00 + 0 00 Note: All induction applications were performed with 0.5 mL of the test article, as supplied. Pai WHITE EAGLE TOXICOLOGY LABORATOR mtain TSCA CBI White Eagle Project Number: I DuPont HLO 504-96 E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 3(continued! Guinea Pig Dermal Irritation/Sensitization Scores Test Article - H-21891 Challenge Study Animal Number 24 Hrs 48 Hrs l 00 2 00 3 +0 4 00 5 0+ 6 00 7 00 8 +0 9 00 1 0 0 11 0 0 12 0 0 13 0 0 14 0 0 15 0 + 16 0 0 17 - 0 + 18 0 0 19 0 ' 0 20 0 0 Note: The challenge application was performed with 0.5 mL of the test article, as supplied. WHITE EAGLE TOXICOLOGY L A B O I U T O W E ^ ^ ^ ^ ^ ^ s f t ^ S l O f e f t n t a i n TSCA 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 TABLE 4 Guinea Pig Dermal Irritation/Sensitization Scores Vehicle Control Studv Animal Number Hrs: Induction - 0.9% Saline 1 24 48 2 24 48 3 24 48 21 00 00 00 22 00 00 00 23 00 00 00 1 24 00 0 0' 0 0 Challenge H-2189 24 48 00 00 00 00 Challenge 0.9% Saline 24 48 00 00 00 00 25 00 00 00 00 00 26 00 00 00 00 00 27 00 00 00 00 00 28 00 00 00 00 00 29 00 00 00 00 00 30 00 00 00 00 00 Note: The challenge application of the test article sites was performed with 0.5 mL of the test article, as supplied. WHITE EAGLE TOXICOLOGY LABORATOl in TSCA CBI White Eagle Project Number: I E. I. du Pont de Nemours and Company - H-21891 DuPont HLO 504-96 Guinea Pig Dermal Sensitization - Modified Buehler Method . TABLE 5 Guinea Pig Dermal Irritation/Sensitization Scotps Study Animal Number 31 32 33 34 35 Positive Control flDNCBl and Naive Positive Control Induction Application 1 24_hirs 48 Hrs Application 2 24JTrs 48 Hrs Application 3 . 24 Hrs 48 Hrs 0+ 1+ 11 .0 0 +0 +1 ++ 11 11 00 11 11 00 11 11 Challenge 24 Hrs 48 Hrs 11 +0 02 +1 0+ Positive Control Naive 36 no induction applications 37 no induction applications 38 no induction applications 39 no induction applications 40 ' no induction applications Note: All sites stained yellow; however, erythema could be scored. Challenge 24JrJrg 48 Hrs 00 00 00 00 00 Induction: DNCB Preparation = 0.1% DNCB w/v in 50% ethanol (95% d e a r 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 Page,26 WHITE EAGLE TOXICOLOGY LABORATOi tin TSCA CBI White Eagle Project Number: E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABT.F, 6 Daily Observations 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: All animals in all groups appeared to be normal. Day 7: All animals in all groups appeared to be normal. 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: All animals in all groups appeared to be normal. Day 15: All animals in all groups appeared to be normal. Day 16: All animals in all groups appeared to be normal. Day 17: - All animals in all groups appeared to be normal. Day 18: All animals in all groups appeared to be normal. Day 19: All animals in all groups appeared to be normal. Day 20: All animals in all groups appeared to be normal. Day 21: All animals in all groups appeared to be normal. Day 22: All animals in all groups appeared to be normal. Day 23: All animals in all groups appeared to be normal. Day 24: All animals in all groups appeared to be normal. Day 25: All animals in all groups appeared to be normal. Day 26: All animals in all groups appeared to be normal. Day 27: All animals in all groups appeared to be normal. Day 28: All animals in all groups appeared to be normal. Day 29: All animals in all groups appeared to be normal. Day 30: All animals in all groups appeared to be normal. Day 31: All animals in all groups appeared to be normal. Day 32: All animals in all groups appeared to be normal. DuPont HLO 504-96 ... WHITE EAGLE TOXICOLOGY LABORATORIES - rntain TSCA CBI White Eagle Project Number: E. I. du Pont de Nemours and Company - H-21891 DuPont HLO 504-96 Guinea Pig Dermal Sensitization - Modified Buehler Method TABEE 7 Test Article - H-21891 Individual Body Weights (g) Study Animal Number Initiait 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 18 470 19 512 20 541 Mean Std.Dev. 507 25 Vehicle Control - Saline Day 7 586 492 558 529 577 504 540 574 518 567 535 567 499 531 554 562 539 505 560 582 544 29 Pay 14 Pay 21 Pay 28 641 694 756 531 589 626 588 638 658 562 611 672 611 657 699 540 579 626 571 623 665 609 656 707 561 603 629 619 669 716 597 616 681 625 686 743 550 601 623 571 639 690 620 678 714 625 687 747 591 645 709 537 581 634 595 647 683 627 700 752 589 640 687 34 38 45 Final Dav 32 Change (-1 tt> 32) 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 + 225 697 + 189 45 29 Study Animal Number 21 22 23 24 25 26 27 28 29 30 Initiait 501 533 464 557 489 484 521 464 537 506 Baal Ray.Z Day 14 Pay 21 Day 28 Dav 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 M to 32V +175 +209 +266 +231 +237 + 190 +232 + 163 + 178 +282 Mean Std.Dev. 506 31 558 602 657 715 722 31 38 44 52 51 +216 40 t -- Body weights taken on Day -1 (the day before the first induction application of test article or vehicle). WHITE EAGLE TOXICOLOGY LABORATORIES ntain TSCA CBI White Eagle Project N u m b e r ^ ^ ^ ^ ^ f E. I. du Pont de Nemours and Company - H-21891 Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 7(continued) Individual Body Weights (gl Positive Control ODinitrochlorobenzenel Study Animal Number Initiait Final Day 7 Day 14 Day 21 Day 28 Day 32 31 499 32 542 33 528 34 491 35 457 547 598 636 697 703 547 631 694 734 753 581 642 696 768 769 553 603 661 738 743 525 615 685 745 756 Mean 503 Std.Dev. 33 551 618 674 736 745 20 19 26 26 25 DuPont HLO 504-96 Change (-l.tp.32) +204 +211 +241 +252 +299 +241 38 Naive Positive Control ('Dinitrochlorobenzenel Study Animal Number Initiait Final Day 7 Dav 14 Day 21 Dav 28 Day 32 36 514 37 486 38 522 39 537 40 477 567 635 703 764 770 508 585 637 703 699 565 622 684 745 761 618 687 734 792 792 535 577 634 677 684 Mean 507 Std.Dev. 25 559 621 678 736 741 41 44 43 46 47 t = Body weights taken on Day -1 Change M to 321 + 256 +213 +239 +255 +207 + 234 23 WHITE EAGLE TOXICOLOGY LABORATOl iin TSCA CBI White Eagle Project Nrnberg E. I. du Pont 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 Page WHITE EAGLE TOXTCOLOGY LABORATORI! intain TSCA CBI White Eagle Project Number DuPont HLO 504-96 PROTOCOL (Rev.-04/ 96) Guinea Pig Dermal Sensitization - Modified Buchler Method Testing Facility: White Eagle Toxicology Laboratories 2003 Lower State Road Doyles town, PA 18901 2-1$ 9 / Sponsor E.I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Newark, DE 19714 Test Article: -- H -----------C t k o l ' f r x / . r A _________;_____________ White Eagle Project Number Test Article Description: Test Article Receipt Date: Proposed In-Life Starting Date: Proposed In-Life Completion Date: &>&/!{/f t 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 bv the Sponsor as of s / 1~> /^L . 3 2. The Sponsor has labeled the Test Article container(s) with the name, expiration date, batch number, and where applicable storage conditions H i E S N O 3. The Test Article has been determined by the Sponsor to be stable under specific test and storage conditions as of *5 / n / . . If applicable, reserve samples of Test Article/Vehicle MIXTURES are to be RETURNED TO SPONSOR FOR ANALYSIS and STABILITY TESTING Y E S Q 'N O EPA CONFIDENTIALITY STATEMENT S'A. Sponsor will include their own EPA Confidentiality Statement in the Final Report B. White Eagle will include standard EPA Confidentiality Statement in the Final Report If "B", select one of the following: No Data Confidentiality Claims 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: 2 Study Director PROTOCOL APPROVAL Sponsor: -3 -76 Date Sponsor Representative ln h f1 Date WHITE EAGLETOXICOLOGY LABORATORIES - ntaio TSCA CBI White Eagle Project Number: DuPont HLO 504-96 White Eagle Toxicology Laboratories 2003 Low er State Road D o.ylcstow n., Pennsylvania, 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 Protocol Number: Protocol N am e; Guinea Pig Dermal Sensitization - Modified Buehler Method This protocol contains information that is proprietary and confidential to White Eaele *o which W h L ^ ^ r ,CntS WUhOUf P" 0r aPPr Val f WhHC Eaglc Toxico"Sy Laboratories WHITE EAGLE TOXICOLOGY LABORATORIES 1W TS0A CBI White Eagle Project Numbe DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 003 Lower Stale Road Doylestown, Pennsylvania, 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 Protocol Mfimlmp [lev. 04/96) Project Numhpr: (to be completed by While Eagle Toxicology Laboratories) Sponsor: White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown, PA 18901 E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, DE 19714 ' l e s t Article: {to be specified) le s t Article Description: {to be specified) Positive Control Article: Positive Control Description: l-chIoro-2, 4-dinitrobenzcne (Eastman Kodak Co., Rochester, N.Y.) Yellow crystal Posi live' Con lrn| Vehicle.;: Positive Control Vehicle Description;- Ethyl Alcohol (95%) Acetone (99.7%) Clear liquids ICSt.Arliclc Receiver!- {to be specified) Proposed Tn-T.ifc Starting Date: {to be specified) Proposed Tn-T.ifn Completion n.-itp- {to be specified) Test: Objective of To;!- Guinea Pig Dermal Sensitization - Modified Buehler Method* To assess ihe contact dermal sensitization potential of the test article. lesting 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 bubpart E - Specific Organ/Tissue Toxicity, September 1985, 40 CFR Part 798; the OECD Guidelines for testing of Chemicals, No. 406 Skin Sensitization, adopted July 17, 1992; EEC 92/69 Annex V Part .6 (992) 93/21 (1993); and the MAFF Japan Testing Guidelines for loxicology Studies, Dermal Sensitization Study, 59 NohSan No. 4200, 1985. ` Ritz I'LL, and Duchlcr, U.V Planning, Conduct, and Interpretation of Guinea Pir Sensitization Pitch Tests, current Concepts in Cutaneous Toxicity, Drill & Lazai editors, Academic Press 1980 WHITE EAGLE TOXICOLOGY LABORATORIES - Dl ntain TSCA CBI White Eagle Project Number: DuPont HLO 504-96 Protocol N u m b e r : 04/96) Guinea Pig Dermal Sensitization - Modified Buchlcr Method Page 2 of 9 Good r.lhnratnry Statement This study is conducted in com pliance w ith the U S EPA PiRRA C' Laboratory Practice Standards (40 CPR 160) U S EPA ? S T "2>; CCD' ^ lustifiralinp fffr lest System: q u S 'S ' S ^ " "E l c o E 6 of Ch" " b lype of Study and L s i l ^ h - . r M,AFF JaPan S id e lin e s for this studies. 7 3 '0ng hls ory of use in dermal sensitization justification for Rontn of Adminislrnlion: Animals: The specific route of adminisini.Vm tA ,, , guidelines for this study. rma ) is required in the testing n T L ' f i ^ k " 1' S , " ' 1? ! . " 6 3 " ' 5 5 0 p * " . < 1 Lnbs, Jamcsburg, NT 08831, U.S.D.A 5 m i ^ t m - A d S A rc "/ " `` '"O Inc., Boyertown, PA 19512 r i c n a t ,,__ ,, Ace Animals Camm Research Lab Animals, W . m NI W b r i ^ T i-' 4' 0 '0" " 005. The source of animals will 11 T ' J . 7 ? ' U-S D-A - License #22animats (except for screens} are d.0<: u TM e n l .e d ,n l* e study records. The cuopmonputceormcpclneteinoniedof the stud^y. ,The'6 anniimmaaltss'ar'enilriaanl`dno'mTMizcecdklyusainndg w rx , r i ni: t e " to e , m p s " d " taM.1 body ( A N W r i t a i k i i S , "a!v 2 " I d ' ra' " d" * by A," lySlS f performed using CSS- Statistics 3 1 m I, n" mlx:r generalion arc challenge n a i v e t e " imTs' V S ? ' S" ' f " i" ? ^ initiation of the study, bu, am weighed prim ,,, C h a lle n g e 6 d ^ ' s m '/ r n t i r S lire G u fd f t m ' , " " r<la" " wf,h (National ,,,s,i,,, ,e s Health J u b S tio n N o" ^ AniTM 'S animals during e study/ bservallons performed daily on all JA CU C Ppvii-iy displaying the p ro ie^ num bL dMe sex, and responsible technician's fnitials * T l0r C ding With waler- ,den.`ificd wifh a " 8 card ' an,maI numbers' SrouP' InslitU(ionalAnma!hC n rran d Use CommUlee ^ A C U C ) ^ i d ^ with acceptable standards for animal welfare and h u S c a r e mP" eS W1..TE EACLE TOXICOLOGY LAUORATORIES - DOYI.ESTOWN, PENNSYLVANIA WHITE EAGLE TOXICOLOGY L A B O I L A T O I ^ K fff1*-3 ' nT S C A CBI White Eagle Project Number: DuPont HLO 504-96 Protocol N u m b e r ^ m | ^ ( R e v . 04/96) Guinea Pig Dermal Sensitization - Modified Buehler Method Husbandry Conditions: Temperature: 18C - 26C (64.4F - 78.8F) Page 3 of 9 Relative Humidity, 50 20% , Light: 12-hour light/dark cycle Diet: Purina Certified Guinea Pig Diet #5 0 2 6 and tap water are 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. Selection of Vehicle: Dose Rangefinding: Caging: Stainless steel with 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 test 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) are 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 a powdered test article up to 50% are prepared as a percentage of test 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 the irritation screen. The Sponsor will then 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 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. If the induction site is moved, an additional area (sufficient for patching) is closely clipped at the new site. 1 wi-irru eacuiioxicolocyiauoratories doylestown. Pennsylvania WHITE EAGLE TOXICOLOGY LABORATORIES - in TSCA CBI White Eagle Project Number: DuPont HLO 504-96 Protocol-Number: ev.-04/96) Page 4 of 9 Guinea Pig Dermal Sensitization - Modified Buehler Method Method of Study Performance: l e s t Article Group (continued) Induction Stage: (continued) : Based 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 the test 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 with the lest material. The 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 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 the new site. A 0.5 mL portion of the vehicle (used for the test article group) is applied to a 25 mm Mill Top Chamber1" 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 lest site, plastic material is wrapped snugly around the trunk of the animal and covered with elastic tape. If the test article does not require a vehicle (liquid d sed 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 an 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 may bo moved for the next induction application, with its relative location fully documented. * WHITE EAGI.E TOXICOLOCY LABORATORIES - DOYLESTOWN, PENNSYLVANIA . Pagi3$npany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: DuPont HLO 504-96 Protocol Number: Rev. 04/96) Guinea Pig Dermal Sensitization - Modified Buehler Method Page 5 of 9 Performance: Positive Control A group of five (5) guinea pigs is closely clipped 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. If the induction site is moved, an additional area (sufficient for patching) is closely clipped at the new site. r A 0.5 mL portion of 0.1% l-chIoro-2,4-dinitrobenzcne (DNCB) w/v in 50% ethanol: 0.9% saline 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" 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 the lest site is wiped with the appropriate solvent. The lest 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 DNCB. The 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. Challenge Singe: Test Material Group After the third induction application, the animals are 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 range finding 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 rechallenge application of all animals may be 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. Dates of application and observations are recorded. Approximately 24 and 48 hours after the challenge (and rechallengc if necessary) application, the sites are examined for dermal irritation and/or signs of elicited sensitization, according to the scale included in Table I. w i ir n : e a c i .e t o x i c o l o c y l a b o r a t o r i e s - d o y l e s t o w n . Pe n n s y l v a n i a WHITE EAGLE TOXICOLOGY LABORASTORiIES ! TS CA CBI White Eagle Project Nuiiber: DuPont HLO So Protocol Number:! P(Rev. 04/96) Guinea Pig Dermal Sensitization - Modified Buchler Method Page 6 of 9 .Challenge Singe: Vehicle Control 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 the right flank lising 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 application and observations are recorded. Approximately 24 and 48 hours after the challenge application, the sites ore examined for dermal irritation and/or signs of elicited sensitization according to the scale included in Table I. Positive Control Group . Rcchallcnge: 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% DNCB in acetone, using the procedures described previously, is applied to a challenge site (on the right flank). A group of five.naive animals is ailso 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 sensitization according to the scale included in Table I. The Sponsor is notified of animals that are considered sensitized. Verbal instructions for rechallenge are 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 rcchallenged. The 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. . IcsLA itidc ` 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. WHITE EAGLE TOXICOLOGY LAIIORATORIES - DOYLESTOWN. PENNSYLVANIA PagecSSnpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: DuPont HLO 504-96 Protocol Number: ev. 04/96) Guinea Pig Dermal Sensitization - Modified Buohler 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 White 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 the event of a significant finding (P<0.05) for the data as a whole. Statistical analyses are performed using CSS: Statislica, 3.1 copyright, StatSoft Inc. 1991. . 1 All animals found dead during the observation period or euthanized in extremis are necropsied to establish, if possible, the cause of death or illness. AH test animals are euthanized by CO inhalation or IP pentobarbital at the completion of the study. \ 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. rs Records Maintained: Records to be maintained include: - irritation scores for screen animals - irritation scores for redness and swelling (induction and challenge) - daily clinical observations . f - 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 Finn! 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,; WHITK EAGLE TOXICOLOGY UVUORATOK1ES - DOYLESTOWN, PENNSYLVANIA . : PagC$n, WHITE EAGLE TOXICOLOGY LABORATORIES ~ TSCA CBI White Eagle Project Number: DuPont HLO 504-96 Protocol Number: 04/96) Guinea Pig Dermal Sensitization - Modified Buehler Method Page 8 of 9 Interpretation of Results: All non-eschar and non-necrotic test 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/wilhout swelling Note: 0.5 scores will be 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 BrouP determined for both 24 and 48 hours. Grades + 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 > . one (1)) to the positive control material to validate the test system. 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 be used to decide whether material will be classified as a sensitizer or not. WHITE UACI.ETOXICOI.OCV l-AIIORATORIES - DOYl.liSTOWN, PENNSYLVANIA . . Pa; WHITE EAGLE-TOXICOLOGY LABORATOl igptainTSCA CBI White Eagle Project Number: DuPont HLO 504-96 Protocol Number: ev. 04/96) Guinea Pig Dermal Sensitization - Modified Buehler Method Signatures: Submitted by White Eagle Toxicology Laboratories: Page 9 of 9 George E/M oore, B.S Study Director Approved by: C ^A ,w Sponsor Representative Date Date WHITE EAGLE TOXICOLOGY I-ADORATORIES - D0YI.ES1OWN. PENNSYLVANIA WHITE EAGLE TOXICOLOGY LABORATORIES itainTSCA CBI White Eagle Project Number:1 E. I. du Pont 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 L A B O R A T O I ^ ^ ^ W 3 S l ^ W ;l s f i.9 S l P f e r,t a i n TSCA CBI DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower State Road, Doylestown, Pennsylvania 18901 Telephone: (215) 348-3868 ----------------------------------------- FaxT(215)~348;5081-----------------------------------:------- White Eagle Project Number: Protocol Number: Sponsor: Test Article: Test: GLP Compliance: DEVIATIONS FROM PROTOCOL #1, #2, #3, #4, #5 r(Rev. 04/96) E. I. du Pont 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. These deviations are not thought to have affected the integrity of this study. Submitted by: George EMoore, B.S. Study Director Date Reviewed by: Date Approved by: l l IS \ \ b Date Page dim pany Sanitized. Does not contain TSCA CBI White Eagle Project Number: E. I. du Pont 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%) Pa; WHITE EAGLE TOXICOLOGY LABORATOR intain TSCA CBI White Eagle Project Number: 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-hexylcinnamaldehyde, tech, 85%) T.ahnratory 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 Pont de Nemours and Company Haskell Laboratry 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. WHITE EAGLE TOXICOLOGY LABORATORIES TSCA CBI White Eagle Project Number] DuPont HLO 504-96 DuPont HLO 306-96 THIS PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS Page 2 WHITE EA GLE TO XICOLOGY LABORATORIES DOYLESTOW N. PEN NSYLVANIA 18901 PiLCferoany Saniti2e d . _ , D ^ ^ ^ ^ t a i n TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - Dl TOWN, PENNSYLV White Eagle Project Number:! DuPont HLO 504-96 WHITE EAGLE TOXICOLOGY LABORATRLf t i f 96 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 E. I. du Pont de Nemours and Company a-hexyldnnamaldehyde, tech, 85% Guinea PigDermalSensitization - 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 F1FRA 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 O.Olg. 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. A H ' George Er Moore, B.5. Study Director 6 --T C Date Sponsor: ~ DuPont Agricultural Products Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, DE 19714 ' Page 3 WHITE EAGLE TOXICOLOGY LABORATORIES - DO :ain TSCA CBI White Eagle Project Number: DuPont HLO 504-96 VVhileEagleProject.Number: DuPont.HLO_30fc96 E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization Modified Buehler Method DuPom HLO 306-96 Substance Tested: Svnonvms/Oiher Codes: CA S Registry Number Haskell No.: Purity: Major. Imputit i : Stability: Physical Form: Sponsor Structure: GENERAL INFORMATION Test Material Octanai, 2-(phenylmethylene)- HCA Hexyl cinnamaldhyde a-Hexylcinnamaldehyde 2-Hexylcinnamaldehyde Hexyl cinnamic aldehyde a-Hexylcinnam ic aldehyde a-Hexylcinnamyl aldehyde a-N-hexyl-beta-phenylacrolein Octanai, 2-(phenyImethylene) Lot Number 10021 HF 101-86-0 21780 85% (Supplied by Aldrich Chemical Co.; Milwaukee, WI 5 3 2 3 3 ); Expiration date: 5/99 None known to be o f toxicological significance at this time. The test substance appears to be stable under the conditions o f the study; no evidence o f instability was observed. Y ellow liquid DuPont Agricultural Products E . L du Pont de Nemours and Company Barley M ill Plaza Wilmington, Delaware 19880-0038 Pta-CH=C--ICH21S-H Study Initiated - Completed:. 4/19/96 - 6/26/96 In-Life Phase Initiated - Completed: 4/30/96-5/31/96 -4 - Page 4 WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 190I = Pag<4fiipany 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-hexylrinnamaldehyde, tech, 85% Guinea Pig 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 artide animals were induced and challenged by ocduded application of HCA to dipped intact guinea pig skin. The same procedures were carried out on contemporaneous control groups except that the test artide was replaced by vehide. 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 artide 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 th induction phas 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 vehide 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 approximatdy 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 vehide 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 approximatdy 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 approximatdy 24 and 48 hours after the challenge application of either the vehide (acetone) or test artide (1.0% HCA in acetone) sites. WHITE EAGLE TOXICOLOGY LABORA! :an TSCA CBI 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 Guinea Pig Dermal Sensitization - Modified Buehler Method with Positive Control (a-hexyldnnamaldehyde, tech, 85%) Summary fmntinnpd) It was concluded therefore, that, under the study conditions utilized, repeated administration of 2.0% and 1.0% a-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 Offidal Journal of the European Communities (EEC Directive 93/21), a-hexyldnnamldehyde, tech, 85% is a skin sensitizer. Study Director Reviewed/Audited by. Quality Assurance Manager Page 6 WHITE EAGLE TOXICOLOGY LABORATORIES . DOYLESTOWN. PENNSYLVANIA 1(901 Pag50ipany 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method White Eagle Project Number: Sponsor Study Number: Test Article: Study Initiation: a-hexyldnnamaldehyde, tech, 85% (HCA) 04/19/96 Inspections Protocol Procedures Raw Data Draft Report Final Report Quality Assurance Statement Dates 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 Page 7 ___ W HITE EAGLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA IS901 ' . ' Pa] WHITE EAGLE TOXICOLOGY LABORATOl intain TSCA CBI White Eagle Project Number: DuPont HLO 504-96 _W h ite E a g le P ro je c tN u m b e n ^ m p ____________________________ DuPont HLO_30S;96 TABLE OF CONTENTS . 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 T e s t.......................................... 10 Testing Guidelines ................................................................................................................11 Good Laboratory Statement................................................................................................ 11 Justification for Test System................................................................................................ 11 Justification for Route of Administration..........................................................................11 Animals.................................................................................................................................... 11 IACTJC Review ...................................................................... Husbandry Conditions........................................................................................................ 12 Test Article Vehicle ...............................................................................................................12 Dose Rangefinding...................................................................................... 12 Induction S tag e....................................................................................................................... 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 ................................................................................... Conclusion............................................................................................................................... 18 Signatures................................................................................ Page 8 WHITE EACLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA IB90I 12 16 19 WHITE EAGLE TOXICOLOGY LABORATTORoIESt^ ^ O ^ ^ ^ s R .W A 0^ ^ ^ TSCA CBI White Eagle Project Number: DuPont HLO 504-96 White Eagle Project N u m b e r ^ ^ ^ ^ DuPont HLO 306-96 TABLE OF CONTENTS (continued) Page Table 2 - Challenge Screens ............................................................................................. 20 Table 3 - Guinea Pig Dermal Irritation/Sensitization Scores Test Article 2.0% H C A ......................22 Table 4 - Guinea Pig Dermal Irritation/Sensitization Scores Vehicle Control 2.0% HCA ........... 23 Table 5 - Guinea Pig Dermal Irritation/Sensitization Scores Test Article 1.0% H C A ......................24 Table 6 - Guinea Pig Dermal Irritation/Sensitization Scores Vehicle Control 1.0% HCA ................... 25 Table 7 - Guinea Pig Dermal Irritation/Sensitization Scores Test Article 0.5% H C A ............... ....2 6 Table 8 - Guinea Pig Dermal Irritation/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 W H ITE EA GLE TO XICOLOGY LABORATORIES - DOYLESTOW N. PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATORIES TSCA CBI 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 Sponsor Test Article: Test Artide Description: Test Artide Rereived: Vehide Control Articles: Vehicle Control Descriptions: White Eagle Toxicology Laboratories 2003 Lower State Road Doylestown, PA 18901 E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714 a-hexylrinnamaldehyde, tech, 85% (HCA), Lot # 10021 HF, Exp. date 5/99, Supplied by White Eagle Labs. Clear liquid 05/01/94 Ethyl Alcohol (95%) (Clearspring Distilling Co., Gearmont & 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) Gear liquids Initiated: In-Life Study Initiated: Test: Objective of Test: 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, ct-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 Toxicity, Drill & Lazar editors. Academic Press 1980. Page 10 WHITE EA GLE TOXICOLOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA I 901 Pa] WHITE EAGLE TOXICOLOGY LABORATORIES - mtain TSCA CBI White Eagle Project Number: DuPont HLO 504-96 White Eagle Project Number I DuPont HLO 306-96 E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Testing Guidelines: 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. Good Laboratory Statement: This study was conducted in compliance with the U.S. EPA F1FRA 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). Justification for Test System: 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 Justification for type of study and has a long history of use in dermal sensitization studies. Route of Administration- 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 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 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 P a y -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. Ginical 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 W HITE EA GLE TO XICO LO G Y LABORATORIES - DO YLESTOW N . PEN N SYLVA N IA 18901 Pas WHITE EAGLE TOXICOLOGY LABORATORI! TSCA CBI 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method 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. Husbandry Conditions: Temperature: 18C - 26C (64F - 78F) 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. Test Article Vehicle: Dose Eangefinding Caging: Stainless steel with elevated wire mesh flooring, 4-5 guinea pigs/cage. 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. . 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 faint 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 20% , 1.0% and 0.5% in 95% ethanol induction application animals respectively. Page 12 W H ITE EAGLE TO XIC O LO C Y LABORATORIES - DOYLESTOWN. PENNSYLVANIA 1B901 White Eagle Project Number: DuPont HLO 504-96 -White-Eagle-Eroject-Number:l -DuPont-HLO-306-96- E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tedi, 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 dosely 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 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 sue 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 included 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. Yehide Control Group Three groups of ten (10) guinea pigs were dosely dipped 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 repeated as necessary. A 0.5 mL portion of the vehide (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 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 7 days. Page 13 W HITE EAGLE TO XICO LOGY LABORATORIES DOYLESTOWN. PENNSYLVANIA 18901 w w t t f F An r F m y rrO L O G Y LABORATORIES TSCA CB1 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-hexylrinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Challenge Stagp: Test Material Onnp 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 flank). 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 Statistical i^na^ysis: Mortality. Morbidity and Termination: 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 !~ L ,, k (caudaI)- Each of three groups also received either 1.50%, 0.75% or 0.25% HCA in acetone on the right flank. 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). Individual body weight percentage gain was statistically evaluated by Analysis of Variance (ANOVA), with the Neuman Keuls test applied in tire event of a significant finding (P<0.05) for the data as a whole. Statistical analyses were performed using CSS: Statistica, 3.1 copyright StatSoft Inc. 1991. & Animals # 1 5 and #45 (1.0 % HCA test article group) were found dead during the induction phase of the study p a y s 14 and 16, respectively). All other animals were euthanized at the completion of the study with IP injection of pentobarbital. 1 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. rage 14 w r e r e EAGLE TOXICOLOGY LABORATORIES - D O Y U 5T O W N . PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATORIES TSCA CBI 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - 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. - Interpretation pf Results: All non-eschar and non-necrotic test sites were scored on a scale of 0 to 3 as indicated in Table 1. - TABLE I 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 0.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 elicited a positive reaction (Scores > . 1) to HCA to validate the test system. - Page 15 W HITE EA G LE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATORIES - DOY TSCA CBI 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-hexyicinnamaldehyde, 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% HCA 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. Page 16 W HITE EAGLE TO W COLO CY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 . -W HITE EAGLE TOXICOLOGY LABORA1 Y^aLnE^1 o 8?finta in TSCA CBI 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method R esu lts: (continued) Test Article Group - Induction and Challenge (continued) o^ancA 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 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% H CA 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 vehicle (acetone) or test article (1.5% HCA in acetone). LP% 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 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 vehicle control group of animals and any of the other five groups of animals (Analysis of Variance). Page 17 WHTTE EA GLE TOXICOLOGY LABORATORIES DOYLESTOtVN. PENNSYLVANIA 11901 ^TO W N ^* PENNS& v M M nteinTSCACB' White Eagle Project Number: DuPont HLO 504-96 White Eag l_ e _ P ro je c t.N u in b e r ^ ^ p y ___________________________ DuPont HLO-306-96 E. I. du Pont de Nemours and Company - a-hexylannamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Results: (continued) Vehicle Control Group - Induction and Challenge (continued) L Q % 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 4S 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). One 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. C o n d u s ion : The test ardde, a-hexylannamaldehyde, 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 EAGLE TO XIC O LO G Y LA BO RA TO RIES-D O YLESTO W N . PENNSYLVANIA 18901 Pag(62npany Sanitized. Does not contain TSCACBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: DuPont HLO 504-96 W h ite _ E a g Ie P r o je c tN u m b e r:^ P d ------------------------------------------- DuPont HLO-306-96 E. I. du Pont de Nemours and ompany - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method Sign a tur e s : Submitted by White Eagle Toxicology Laboratories: Study Director Reviewed/Audited by: Date Quality Assurance Manager L -zL -<m Date W HITS EACLE TOXICOLOGY LABORA IES - DOYLESTOWN. PENNSYLVANIA 1B01 PageOiibipany 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 - o-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method IABLE2 Challenge Screens Animal Number 61 62 63 64 Screen #1 - T c;jrfP 1.50% concentration in Acetone 24 Hours + 0 0 0 r. + 48 Hours t 0 0 0 0 Animal 61 62 63 64 Screen # 2 - Righf Sitlf 0.75% concentration in Acetone 21HOUIS o 0 0 0 48 Hours o 0 0 0 Body W eights fe) Animal #61 Animal #62 Animal #63 Animal #64 415 393 - 409 401 t - Site stained light pale yellow; however, erythema could be scored 20 WHITE EAGLE TOXICOLOGY LABORATOIRES D O Y L ST O W N , PENNSYLVANIA I 901 Pa* WHITE EAGLE TOXICOLOGY LABORATORIES in TSCA CBI White Eagle Project Number: DuPont HLO 504-96 White Eagle Project Number: I DuPont HLO 306-96 E. I. du Pont de Nemours and Company - a-hexylrinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 2 (continued) Challenge Screens Animal Number 65 66 67 68 Screen # 3 - Left Side 0.50% concentration in Acetone 24 Hours 0 0 0 0 48 Hours 0 0 0 0 Animal Number 65 66 67 68 Screen # 4 - Right Side 0.25% concentration in Acetone 24 Hours 0 0 0 0; 48 Hours 0 0 0 0 Body Weights (gl Animal #65 Animal # 6 6 Animal #67 Animal # 6 8 . 377 400 380 373 Page 21 W HITE EAGLE TO XICOLOGY LABORATORIES - DOYLESTOWN. PENNSYLVANIA 18901 ' . > P, WHITE EAGLE TOXICOLOGY LABORATORIES - Dl TSCA CBI White Eagle Project Number: DuPont HLO 504-96 White Eagle Project Number: j -DuPontH LO 306-96- E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABI.E 3 2,0% HCA Animal Numhw 17 18 27 31 32 35 41 47 55 57 Guinea Pie Dermal Irritatin/Spnsifization Srnrps Reading - Hours Aftpr Annliralinn Mirnko, 1 24 48 2 24 48 2 24 48 24 Hrs* 48 Hrs 00 +0 00 00 00 00 00 +0 00 00 11 11 00 00 +0 +0 10 2+ 10 00 SS SS ++ ++ 33 SS 33 SS 22 11 32 2+ 11 00 1+ 33 12 21 +0 +1 * Readings are performed approximately 24 hours after the challenge application S = Eschar Note: All 6-hour exposures. Induction applications - 2.0% HCA in 95% ethanol Challenge application -1.5% HCA in acetone W H ITE EAGLE TOXICOLOGY L A B O R A T OIE^S^- DOYLESTOWN. PENNSYLVANIA 18901 ' Pag<C66npany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number DuPont HLO 504-96 % White Eagle Project Numbers _DuPont_HLO_30fc96_ E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABI.E 4 2.0% HCA - Vehicle Control Guinea Pip Dermal Irritatinn/Sensitization Scores Animai Number 3 10 16 22 23 28 30 34 ' 37 56 Heading-.Hours-After Application Number Challenge 1' 2 3 Acetone 24 43 24 48 24 48 24 Hrs 48 Hrs 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 +0 0+ 0 0 00 00 00 00 00 00 00 00 00 00 00 00 Challenge L 5 % HCA 24 Hrs 48 Hrs +0 00 00 00 00 00 00 0' 0 00 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 Page 23 W HITE EA G LE TO XICOLOGY LABORATORIES DOYLESTOWN, PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY L A B O R A T O n t a in TSCA CBI I White Eagle Project Number: DuPont HLO 504-96 "White Eagle ProjectNumberJ DuPont HLO 306-96 E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 5 1.0% HCA Guinea Pig Dermal Irritation/Sensitization Scores A n im a l Number 4 5 7 11 12 15 42 45 48 59 Beading- Hours After Application Number 122 24 48 24 48 24' 48 Challenge 24JHrs* 48 Hrs 00 +0 11 +0 00 00 00 +0 00 00 00 +0 00 00 00 +0 00 +0 ++ + +' 00 00 FD 00 +0 00 00 00 00 FD 00 00 ++ 2+ 00 00 00 20 * 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 nafe.-' Page 24 W HITE EAGLE TO XICO LOG Y LABORATORIES - DOYLESTOWN. PENNSYLVANIA 18901 r o ^ %WHITE EAGLE TOXICOLOGY LABORATORIES a S f f l s s ' t e r lai" TSCA cbi 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE fi L0% HCA - Vehicle Cnntml . Animal Number 1 9 20 36 43 46 49 50 51 54 Guinea Pig Dermal Initation/Sensiiizatinn Reading * Hours After Annliratinn Number rhallen. 1 24 48 2 24 48 8 24 48 Acetone 24-Hrs* 48. His 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 +0 00 00 00 00 00 00 00 00 00 00 00 00 00 0o 00 00 00 00 Challenge 0.75% HCA 24 H g 48 Hr-; 00 00 00 +0 00 0 0. 00 0' 0 00 00 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 W HITE EAGLE TOXICOLOGY L A D O R A T OI^ESS^-DDOYLESTOWN. PENNSYLVANIA IW01 m ' Page; WHITE EAGLE TOXICOLOGY LABORATOR in TSCA CBI White Eagle Project Number:! DuPont HLO 504-96 -White-Eagle-Project-Numbenl m -DuPont-HLO-306-96- E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 7 0.5% HCA Animal Number 2 13 19 33 38 39 40 44 . 53 58 Reading - Hours After Application Number 1 24 4S 2 24 43 3 24 43 00 00 00 00 00 ++ 00 00 ++ 00 00 11 00 00 00 00 00 00 00 00 00 00 00 ++ 00 00 ++ 00 00 00 24 Hrs* 0 0 0 0 0 0 0 + 0 0 48 Hrs 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 - 0.5% HCA in 95% ethanol Challenge application - 0.25% HCA in acetone % Page 26 WHITE EAGLE TO XICO LOG Y LABORATORIES - D O Y LESIO W N . PENNSYLVANIA 1B901 - " : Pase^O WHITE EAGLE TOXICOLOGY LABORATOl TSCA CBI White Eagle Project Number:! DuPont HLO 504-96 JVVhitjMgle_ProjeLNumber: ---------------------------------------- DuPont-HLO-306-96 E. I. du Pont de Nemours and Company - a-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 8 Q.5% HCA - Vehicle Control Animal Number 6 8 14 21 24 25 26 29 52 60 Guinea Pig Dermal Imtation/SensihVation Scores Reading - Hours After Application Number Challen 1 24 48 2 24 48 8 24 48 Acetone 24 Hrs* 48 Hrs 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 o r0 00 00 00 00 00 00 00 oa 00 00 00 00 00 00 00 00 00 0o 00 0o 00 00 00 00 Challenge 0.25% HCA 24 Hrs 48 Hrs 00 00 00 00 00 0 0. 00 0' 0 00 00 ' Readinss are performed approximately 24 hours after the challenge application Note: All 6-hour exposures. Induction applications - 95% ethanol Challenge application - 0.25% HCA in acetone Challenge application - Acetone - Page 27 WHITE EAGLE TOXICOLOGY LABORAT' O B E S - DOYLESTOW N, PENNSYLVANIA 1(901 WHITE EAGLE TOXICOLOGY LABORATORIES' - DC ntain TSCA CBI 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method TABLE 9 Daily Observations 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: All animals in all groups appeared to be normal. Day 7: All animals in all groups appeared to be normal. Day 8: All animals in all groups appeared to be normal. Day 9: All animals in all groups appeared to be normal. Day 10: AH animals in all groups appeared to be normal. Day 11: AH 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 # 1 5 (1.0% HCA test article group) was found dead. No gross abnormalities were noted at necropsy* All other animals in aU groups appeared to be normal. Day 15: All animals in all groups appeared to be normal. Day 16: Animal # 4 5 (1.0% HCA test article group) was found dead. Other than green liquid in the gastrointestinal tract, no gross abnormalities were noted at necropsy. AH other animals in aU groups appeared to be normal. Day 17: AH animals in aU groups appeared to be normal. Day 18: A1I animals in all groups appeared to be normal. Day 19: AU animals in aU groups appeared to be normal. Day 20: AU animals in all groups appeared to be normal. Day 21: All animals in all groups appeared to be normal. Day 22: AU animals in aU groups appeared to be normal. Day 23: AH animals in aU groups appeared to be normal. Day 24: All animals in all groups appeared to be normal. j-ay 25; All animals in all groups appeared to be normal. Day 26: AU animals in aU groups appeared to be normal. Day 27: AU animals in aU groups appeared to be normal. Day 28: AU animals in aU groups appeared to be normal. Day 29: AH animals in aU groups appeared to be normal. Day 30: AU animals in aU groups appeared to be normal. Day 31: AU animals in aU groups appeared to be normal. Day 32: AU animals in aU groups appeared to be normal. . Page 28 w h i t e e a g l e t o x i c o l o g y l a b o r a TO R IES DOYLESTOWN, PEN NSYLVANIA 18901 '1 . ' . PageCSbipany Sanitized. D oes 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization Modified Buehler Method TABLE IQ , Test Article - 2.0% HCA Individual Body Weights fg) Animal Number Initiait Final Day 7 Day 14 Day 21 Day 29 Day 32 Change (-l.tO.32) 17 456 18 435 27 480 31 460 32 437 35 470 41 432 47 474 55 420 57 483 514 564 621 702 689 494 550 606 689 689 564 628 610 679 677 510 575 635 709 699 499 552 689 758 763 553 622 670 750 771 505 571 626 715 717 571 645 707 777 790 476 522 569 624 637 548 618 703 763 789 +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 - Ethanol/Acetone Animal Number Initiait Final Day 7 Day 14 Day 21 Day 29 Day 32 Change f-1 to 321 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 LABORATORIES - DOYLESTOWN. PENNSYLVANIA IB M ' PageGSnpany 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization^ Modified Buehler Method Test Article-1.0% HPA TABLE 11 Individual Body Weights M Animal Number 4 5 7 11 12 15 42 45 48 59 Mean Std.Dev. Initiait 459 479 442 406 434 469 433 424 517 475 454 32 Day-Z 549 538 494 475 502 537498 500 598 570 526 39 yehide Control - Ethanol/Acetone Final Day 14 Day 21 Day 29 Day 32 613 685 741 729 629 697 785 811 538 568 641 638 517 580 639 626 559 619 694 716 552 596 650 651 563 648 712 763 766 647 711 792 807 585 646 713. 718 50 61 65 74 Chance f-1 to 321 +270 +332 + 196 +220 +287 + 218 +249. +332 + 262 51 Animal Number Initiait Final Day 7 Day 14 DayJU. Day 29 Day 32 Change f-l to 321 1 475 9 455 20 426 36 479 43 407 46 465 49 469 50 433 51 480 54 453 53.1 607 662 716 735 501 556 641 724 728 482 540 599 581 582 559 639 712 776 809 462 507 564 615 618 547 614 590 697 721 518 590 603 666 667 468 519 554 627 627 537 614 677 721 731 563 632 636 718 704 +260 +273 + 156 +330 +211 +256 + 198 +194 +251 +251 Meaji 454 Std.Dev. 25 517 582 624 684 692 37 48 51 60 68 +238 49 .t BodyWights taken on Day -1 (the day before the first induction application of test article or * = Animals #1 5 and #45 were found dead on Days 14 and 16 respectively Page 30 WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY L A B O R A T O ^ S ^ C ^ ^ T O ^ , t i^ ^ iS '5 R 9 R n il01^5fj?n ta in T S C A C B I 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 TABLE 12 T est Article - 0.5% H P A Individual Body Weights fgl Animal. Number 2 13 19 33 38 39 40 44 53 58 Initiait 458 385 442 464 451 462 413 481 507 * 474 DajLZ 521 440 527 529 519 521 462 560 580 560 Mean 454 Std.Dev. 35 522 43 Vehicle Control - Ethanol/Arelonp Day 14 Bay_21 Day 29 577 652 723 482 542 599 569 656 714 603 685 752 595 662 737 586 654 709 500 564 623 63 684 739 632 708 754 618 693 763 579 650 711 51 55 56 Final Day 32 731 603 733 788 759 686 639 739 792 770 724 63 Change (-J 10 32) + 273 +218 +291 +324 +308 + 224 + 226 +258 +285 + 296 +270 37 Animal Number Initiait 6 455 8 411 14 472 21 431 24 396 25 421 26 499 29 451 52 516 60 411 Mean - 446 Std.Dev. 40 Day 7 533 479 560 517 490 505 591 519 508 484 519 35 Final Day 14 Pay 21 Day 29 Day 32 608 670 754 773 549 614 685 701 627 701 766 776 590 653 705 716 568 618 705 704 573 628 691 688 680 759 825 845 554 581 631 588 559 610 651 644 547 613 686 687 586 645 710 712 42 53 57 72 Change U to 32) +318 +290 +304 +285 +308 +267 +346 + 137 +128 +276 +266 74 Body weights taken on Day -1 (the day before the first induction application of test article or vehicle). rag e 31 W HITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 ' " .. : Pa. . WHITE EAGLE TOXICOLOGY LABORATOl ntain TSCA CBI White Eagle Project Number: DuPont HLO 504-96 ----White-Eagle-Project-Number: DuPont.HLQ.30M6. E. I. du Pont de Nemours and Company - a-hexyldnnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method APPENDIX A COPY OF PROTOCOL COVERSHEET AND PROTOCOL Page 32 W HITE EAGLE TO XICO LOG Y LABORATORIES DOYLESTOW N. PENNSYLVANIA 18901 ' . Page, . WHITE EAGLE TOXICOLOGY LABORATOR onta in TSC CBI White Eagle Project Number: DuPont HLO 504-96 FILE No. 515 0& si9 '96 09:23 ID:HASKELL LAB TOXICOLOGY/ 302 366 5207 -DuPont HL(&H PROTOCOL Guinea Pig DcrmST iCBung faculty; W hile Eagle Toxicology Laboratories 2003 Lower Slate Koad Doylestown, PA 18901 * Test Article: --H __ (Rev. 04/96) Modified Uuchlcr Method oponson C.I. du Pont do Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine jNMeewwaarrk*. UDSE 19714 d L S eJ?j Jh L ~J ) g Va While Eagle Project Number. Test Article Description: Test Article Receipt Dale: Proposed In-Life Starting Date: Proposed In-Life Completion Dale: i-Z S -IL J r--* a -lC r To be Completed by White Eagle EP A C O N FID EN TIA LITY STA TEM EN T X a - Sponsor wiU indude their own EPA Confidentiality Statement in the Final Report a D. White Eagle will include standard EPA Confidentiality Stalement in the Final Report If "D", select one of the following: a N o Data C onfidentiality C laim s a Data C onfidentiality (Haims This study docs not unnecessarily duplicate previous experiments nor is there an acceptable in-vjtro method that can substitute for the use of onimala. ; _ Ruff1 X t f , PROTOCOL AMENDMENTS a - -cvmc/ . i Z / z A , ) <6 S / n \ n Z u cjL --tta U a t. - JX iSL .f l ^ e S ty Z tf -a x jL - 9fi ? //? T esting Facility: . I PROTOCOL APPROVAL Sponsor: it il= 2 U Date alive D ate i ' Page 33 : W H tT t EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA IB90I WHITE EAGLE TOXICOLOGY LABORATroOa^RIE^S ? ^ W i s ^ m ^ ? ntain TSCA CBI White Eagle Project Number: DuPont HLO 504-96 DuPont-HLO-3Ub-yb" White Eagle Toxicology Laboratories 2003 Low er Stale Road D oylcslow n,Pennsylvania, 18901 Telephone: (215) 348-38G8 Fax: (215) 348-5081 Protocol N um ber: Protocol N am e: Guinea Pig Dermal Sensitization - Modified Buchlcr Method y u s protocol contains inform ation that is proprietary and confid en tial to W h ile Eagle Toxicology Laboratories. U se o f this inform ation is restricted to the p arlies to w hich W hite Eagle Toxicology Laboratories h as issued it in the course o f bu sin ess. A ny duplication or distribution o f the contents w ithout prior approval of W hite Eagle Toxicology Laboratories is strictly prohibited. = ! Page 34 WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN. PENNSYLVANIA 18901 '' - v Pa WHITE EAGLE TOXICOLOGY LABQRATO: intain TSCA CBI White Eagle Project Number:' DuPont HLO 504-96 ------DuEontHLO-309T WHITE EAGLE TOXICOLOGY LABORATORIES 2003 Lower Stale Road, Doylcstown, Pennsylvania 18901 Telephone: (215) 348-3868 Pax: (215) 348-5081 JR cv . 04/96) - T e s tin g F a c ility: Sponsor T est A rtide: XcaLArlidc D escription: T est A rticle R eceiv e d : V ehicle Control Articles: Vehicle Conimi D escriptions: Proposed In-Life Startin g Dali*: P roposed In-I-ifp C om pletion D ale: T est: (to be completed by W hite Eagle Toxicology Laboratories) W hile Eagle Toxicology Laboratories . 2003 Lower Stale Road D oylcstow n, Pennsylvania 18901 E. I. du Pont dc Nemours and Company ` Haskell Laboratory for Toxicology and Industrial M edicine Etkton Raad, P.O . Box 50 New ark, DE 19714 Positive Control (a-hcxytdnnam aldchydc, tech, 85% ) Clear liquid Supplied by W hile Eagle Toxicology Laboratories Ethyl Alcohol (95%) Acetone (99.7% ) G e a r liquids (to be specified} {to be specified) Guinea Pig Dermal Sensitization - Modified Duchlcr Method* Testing G uidelines: To validate the test system using a know n sensitizer, a-hcxylcinnam aldchydc (HCA). T h e m ethods em ployed in the testing o f th is lest article a rc sim ilar to those described in the U .S. EPA Pesticide A ssessm ent G uidelines, Subdivision F, H azard Evaluation: Human Sc D om estic Anim als, Scries 81-6. 1982; EPA Health Effects Testing G uidelines Subpart E - Specific Organ/Tissuc Toxicity, Septem ber 1985, 40 CPR Port 798; the OECD Guidelines for Testing o f Chem icals, N o. 406, Skin Sensitization, adopted Ju ly 17, 1992; E E C 92/69 A nnex V , Part It.6 (1992), 93/21 (1993); and the M A FF Jap an Testin g G u id elines for Toxicology Studies, Dermal Sensitization Study, 59 N ohSan No. 4200, 1985. 'K 'tz ,. H .L. and Biichlcr, E.V ., Planning, Conduct, and Interpretation of Guinea Pie Sensitization Patch Tests, current Concepts in Cutaneous Toxicity, Drill & Lazar editors Academ ic Press 1980. w itlT E EACLE TOXICOLOGY LABORATORIES - DOYLESTOWN. PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATORIES mtain TSCA CBI White Eagle Project Number! DuPont HLO 504-96 Prolocol N um bc (Rev. 04/96) Guinea rig D erm al Sensitization - Modified Duchlcr M ethod D u P on tH t0-3S5-95Page 2 of 7 Slalo m en t: luslifiration fnr le s t System : Justification ioUtaulC-Qf A dm in istra tio n : T his study is conducted in com pliance with lhe U .S. EPA FIFRA Good Laboratory P ractice S tan d ard s (4 0 .C F R IGO), U .S . EP A T S C A G ood Laboratory Practice Standards (40 CFR 702). O EC D Principles o f Good Laboratory Practice (c(81)30(Final)( Annex 2), and M AFF Jap an Good Laboratory Practice Standards (59 N ohSan N o. 3850). T he laboratory albino guinea pig is the system o f choice sin ce it is required in the EPA , EE C , O EC D , and M A FF Jap an guidelines for thistype o f study and has a long history o f use in derm al scnsilizalion studies. Tire specific route o f adm inistration (derm al) is required in the testing guidelines for this study. " Anim als: . The adult m ale Hartley guinea pig, w eighing 300 to 550 gram s, is used for this study. Tire anim als arc obtained from D avidson Mill Orccding Lobs, Jam csburg. NJ. 08831 U .S.D .A . License #22-A -003, Ace Anim als In c ., U oycrlow n, 1*A 19512 U .S .D .A . L ic en se # 2 3 - 0 -0 0 9 , Elm Hill Breeding Labs, Chelm sford, MA 01824 U .S.D .A . License #14-0-006 or Camm Research Lab Anim als, W ayne, NJ 07470 U .S.D .A . License # 2 2 005. Tire source o f anim als w ill be docum ented in the study record s' T he anim als (except for screens) arc w eighed prior to initiation, w eekly and upon -completion o f the study. Tire anim als a re random ized using computer generated random num bers into groups and the initial body w eights P a y -1) arc statistically evaluated by A nalysis o f Variance (ANOVA). Statistical analyses and random num ber generation arc performed using C SS: Statislica, 3.1 copyright, StatSoft Inc. 1991. Re challenge naive vehicle controls, if utilized, arc n o t w eighed p rior to initiation o f the study, but a rc weighed prior to re-challenge. T he anim als are housed and m aintained in accordance with standards set forth in the Guide for the C are and U se o f Laboratory A nim als (National Institutes o f H ealth Publication N o. 86-23). Tire guinea pigs a rc acclim ated to the laboratory fo r at least 7 days prior to th e initial induction application. Clinical observations arc perform ed daily on all anim als during the study. Tire anim als are individually identified by color coding w ith w alcr- nnd/or solvent-resistant m arkers. Each cage is identified w ith a cage card displaying the study num ber, dale initialed, anim al num bcr(s), group, sex, and responsible technician's initials. . IA C U C Review : Tire Prolocol for this study has been review ed by W hile Eagle's Institutional Anim al Care an d Use Com m ittee (IA CU C) and com plies with acceptable standards for anim al w elfare and hum ane care. w urro EACuit o x ico lo g y labo rato r ies - do ylestow n. Pe n n s y lv a n ia i Page 36 : WRITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 P a gf(8SVnpany'Sani1 WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, 9 C0 ? n t a i n T S C A C B I White Eagle Project Number: DuPont HLO 504-96 DuPont-HLO306-96-- - Protocol N um ber: 04/96) Page 3 of 7 Guinea Pig D erm al Sensitization - Modified Bu chlcr M ethod Husbandry. C o n d itio n s: T em p era tu re: 18"C - 26C (64.4*F - 7 8 .8 T ) - Relative Hum idity: 50 20 % t Light: 12-hour light/dark cycle D ie t: Purina Certified G uinea Pig D iet # 5 0 2 6 and tap w ater are provided ad libitum . Dascd on our current know ledge, no contam inants arc known to be in this diet or w ater w hich m ight be expected to interfere w ith the objectives of th e study. Caging: Stainless steel w ith elevated w ire m esh flooring, 1-5 guinea pigs/cagc. Test A rticle V chirle: Dose R an g e fin d in g: Ethyl 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. : H istorically, 5 .0 % and 2 .5 % 1-lCA in 95% eth an ol h ave p ro d u ced n o irritation to faint irritation after the first induction application and necrosis after the second or third induction application, based on this steep derm al response, conccniralions of 2.0% , 1.0% and 0.5% w ill be used for the induction phase of this study. Tw o groups of 4 unexposed anim als will be w eighed and exposed to different challenge concen tration s o f H CA in aceton e by the p atching technique d escribed in the induction stage. C oncentrations o f 1.50% , 0.75% , 0.50% an d 0.25% in acetone will b e utilized to determ ine non-irritating levels to b e used- as challenge concentrations. Performance: Test Article Group Induction Stage: T hree groups o f ten (10) guinea pigs arc closely clipped over the induction site on their left flank (approxim ately 1.5 X 1.5 incites) during the day prior to the day o f initiation and repeated as necessary. If the induction site is m oved, a n additional area (sufficient for p atch ing ) is closely clipped at the new site. Based upon historical data, dose levels o f 2.0% , 1.0% and 0.5% w /v of HCA in 95% ethanol, will be used for the induction stage. A 0.5 m L portion o f th e appropriate concentration o f H C A in 95% eth an o l is app lied to a 2 5 m m H ill T o p C h am b er1" w h ich is placed o n to th e test site and covered with a piece o f dental dam (approxim ately 2 in ch es X 2 inches). A fter positioning the cham ber and the occlusive dental dam onto the prepared skin test site, plastic m aterial is w rapped snugly around the trunk of the anim al and overwrapped w ith clastic tape. WHITE BACLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA : : Page 37 : W fjIT E EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN. PENNSYLVANIA 1SSOI _ ___aanv banitizei WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PE ntain TSCA CBI White Eagle Project Number: DuPont HLO 504-96 DuPont-HLO-309iT Protocol N u m b e r] (Rev. 04/96) G uinea P ig D erm al Sensitization - Modified Buchlcr M ethod Page 4 of 7 (co n tin u e d ) A pproxim ately six hours later, the d iam ber is rem oved and the lest site is wiped with an appropriate solvent. T he treated site is scored for irritation, using the scale included in Table I, a t approxim ately 24 and 48 . hours from the initial application. T he procedure is perform ed once a w eek for three w eeks, for a total o f three, six-hour applications w ith the appropriate concentration of MCA. T he interval between induction exposures m ay vary from 6 to 8 days. Should pronounced irritation be observed at the induction site, the site m ay be moved for the next induction application, with its relative location fully docum ented. T h ree groups o f ten (10) guinea pigs arc closely clipped over the induction site towards the head on their left (lank (approxim ately 1.5 X 1 .5 inches) during th e day prior to the day o f initiation and rep ealed as necessary. If the induction site is m oved, an additional area, (sufficient for patching) is closely clipped at the new site. A 0 .5 m L po rtion of th e vehicle (95% eth an ol) is app lied to a 2 5 m m I-Iill T o p C h am b er1" w hich is placed on to th e lest s ite and covered w ith a piece o f dental dam (approximately 2 inches X 2 inches):' After positioning the cham ber and the occlusive dental dam onto the prepared skin lest site, plastic material is wrapped snugly around the trunk o f the anim al and overwrapped with clastic tape. Approxim ately six hours later, the cham ber is rem oved and th e test site is wiped with an appropriate solvent. The treated site is scored for irritation according to the scale included in Table I at approxim ately 24 and 48 hours from the initial application. Tire procedure is perform ed once a w eek for three weeks, for a total o f three, six-hour applications o f 95% ethanol. T h e interval between induction exposures may vary from 6 to 8 days. Should pronounced irritation be observed at the induction site, the site m ay be moved for the next induction application, w ith its relative location fully docum ented. . Challenge Singe: T est M aterial Group A fter the third induction application, the anim als are rested for approxim ately 14 to 16 days. At the term ination o f the rest p eriod, a d iallcn g c application in acetone, using the procedures, described previously, is applied to a challenge site (on the right flan k). T h e challenge application remains on for approxim ately 6 hours. Upon removal o f the challenge application, the contact area is w iped w ith an appropriate solvent. In the event o f questionable findings, a rcchallcng c application o f all anim als is perform ed on a second naive site n o sooner than six days after prim ary challenge, along w ith a group o f 5 naive anim als taken from th e colony pool, serving as vehicle controls. D ales o f application and observations arc recorded. A pproxim ately 2 4 and 48 hours after the challenge (and rcchallcng c if necessary) application, lire sites are . exam ined for derm al irritation and/or signs o f elicited sensitization, according to the scale included in T ab le I. . m irrCCACLETOXICOUXnr LAnORATORICS- DOYUSTOWN, PENNSYLVANIA ; Page 38 ' WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA IS901 . Pag WHITE EAGLE TOXICOLOGY LABORATOR l o i n t a i n TSCA CBI White Eagle Project Number: DuPont HLO 504-96 DuPont-HLO:306=9 Prolocol N u m b c r r ^ P ^ ^ (r cv. 04/96) Guinea Pig Derm al Sensilizalion - Modified Duehlcr Method Challenge-Stage: Vehicle Control H m ip Page 5 of 7 A fter the third induction application, the anim als are rested for ap p ro xim ately 14 to 16 days. A t thp term ination o f the rest p e rio d , th e vehicle control group is challenged with aceton e on the left flank (caudal) and the appropriate concentration of HCA in acclon c on the right flank using the procedures described previously. T h e challenge application rem ains on for approximately 6 hours. U pon rem oval of the challenge application, the contact areas arc wiped w ith an appropriate solvent. i aPpl>ralion and observations a rc recorded . A pproxim ately 24 and 48 hours after the challenge application, the sites arc exam ined for derm al irritation and/or signs of elicited sensitization according to the scale included in Table I. G Kcchallenge: Data Retention- If the study is not positive, the Sponsor is notified and the test anim als a rc rcch a llcn g cd 6 to 8 days after th e p rim ary ch allen g e . A " r . W f 31.3 G enerated, all reco rd s accu m u lated d u rin g the p erfo rm a n ce of this study and a copy o f the final report w ill be retained a t W hite tiagte Toxicology Laboratories for a period o f ten (10) years, after which tim e the data will be shipped to the Sp onsor or, discarded up on w ritten request from the Sponsor. r Positive Conim i . ChararlrriT-.-ilipn- D etails regarding the identity, strength, stability, p u rity , and com position of the positive control ore provided by the M anufacturer and are on file at W hite Eagle Toxicology Laboratories, D oylcslow n, Pennsylvania. T lic positive control has been exam ined and lias been fou nd lo be soluble in ethanol and acclon c. Statistical A n aly sis: M ortality. M orbidity and . Term ination: Protocol Changes: A ny m ixtures Sponsor. or dilutions will not be analyzed by J W hite Eagle or the Statistical M ethods: Individual body w eight pcrccnlare ra in is statistically evaluated by Analysis o f V ariance (AN O VA), w ith the Neurruin K culs test applied in the event o f a significant finding (P < 0 .0 5 ) for the data as a whole. Statistical analyses are perform ed using CSS; Statistica, 3.1 copyright, StatSoft Inc. 1991. b All anim als found dead during the observation period or euthanized in c sltc m is are nccropsicd to establish, if possible, the cause o f death or fitness. All lest anim als arc euthanized by C O , inhalation or IP pentobarbital at the com pletion o f the study. 1 All changes in o r revisions o f the approved prolocol and the reasons will be docum ented, signed by the study director, dated and m aintained with the prolocol. wurm CACCE TOXICOLOGY LABORATORIES. DOYUSTOWN. PENNSYLVANIA ; : Page 39 ' WHITE EAGLE TOXITOLOCY.LABORATORIES - DO YUSTO W N . PENNSYLVANIA I890I : PagefSSnpany Sanitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES' - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: DuPont HLO 504-96 D uPont H LO 306-96-- Protocol N u m b er ((R ev. 04/96) Page 6 of 7 G uinea Pig D erm al Sensitization - Modified Duchlcr M ethod Records M aintained: Records to be m aintained include: - irritation scores for screen anim als - irritation scores for redness and sw elling (induction and challenge) - daily clinical observations - initial, w eekly, and final body w eights (including group m eans, standard deviation, and d ian gc between initial and final) - MCA preparation ' calculations and statistical analyses random ization scheme Final Report: A final report is written w hich includes, but is not lim ited to, the item s d ie d in U .S . E P A FIFRA G ood Laboratory P ractice S tan d ard s (40- C FR Port 160), Subpart J, Records and Reports. U .S . EPA TSCA Good Laboratory Practice Standards (40 C FR 792), Subpart ] Records and Reports, OECD Prindplcs of G ood Laboratory Practice, c(81)30 A nnex 2 (Final). Section IL Part 9.2, C on ten t o f the Final Report. and M A FF Jap an Good Laboratory Practice Stand ards, 59 N ohSan N o. 3850, Chapter VIII, Report and Records. - Interpretation of R esu lts: All non-eschar and non-nccrolic test sites arc scored on a scale o f 0 to 3 ns indicated in the following Table I. W hen edem a is present with scores o f + , 1, or 2 for erythem a, it will be docum ented. TADLEI S c ilc-for Evaluation of D erm al Irritation 0 *=> n o reaction 0.5 = very faint redness (usually nonconflucnt) 1 = faint redness (usually confluent) 2 = m oderate redness 3 = severe redness wilh/w ilhout sw elling N ote: 0.5 scores will be recorded in the raw data as a + Scores o f 1 or greater in the lest group arc required to be indicative o f sensitization. If scores o f one (1) or greater a rc seen on the v ch id c control anim als, then the reactions of the test artid e group anim als that exceed the m ost severe vehicle control reactions arc considered to be positive scores. INCIDENCE is reported as th e num ber of positive anim als in ca d i group divided by the total n u m b er o f anim als tested in that group. SEVERITY is reported as the sum o f the lest grades divided by the total num ber of anim als tested in a given group determ ined for both 24 and 48 hours. Grades + arc equal to 0.5 for calculation o f severity indices. All average grades arc to be rounded o ff to the nearest tenth o f a unit. A minimum of-tw o out o f ten positive control anim als m ust be sh ow n to have d ia le d a positive rca d io n (Scores one (1)) to the positive control material to validate the lest system . WHTIt EAGU1TOXICOLOCY LABORATORIES . DOYLESTOWN, PENNSYLVANIA : Page 40 ' : WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN. PENNSYLVANIA 18V01 ' PageCM npany S anitized. Does not contain TSCA CBI WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 White Eagle Project Number: DuPont HLO 504-96 DuPont HLO 306-96' % Protocol N u m ber l(R ev . 04/96) Page 7 of 7 G uinea Pig Derm al Sensitization Modified Duchlcr Method S ig n atu res: Subm itted by W hile Eagle Toxicology Laboratories: G eorge E./M oorc, U.S. Study D irector Approved by: jvOl*\ Sponsor Representative D ate . .......................................... WHITE CAGLETOMCOLOCY LAnOIlATCmiES - DOYUSrOWN. PENNSYLVANIA . - I ......... . : i Page 41 WHITE EAGLE TOXICOLOGY LABORATORIES DOYLESIOWN. PENNSYLVANIA 18701 WHITE EAGLE TOXICOLOGY LABORATr OoRIESf ^ D ^ ^ l ^ S ^ ^ t e n t a i n T S C A C B I 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-hexylcinnamaldehyde, tech, 85% Guinea Pig Dermal Sensitization - Modified Buehler Method APPENDIX B DEVIATIONS FROM PROTOCOL Page 42 WHITE EAGLE TOXICOLOGY LABORATORIES - DOYLESTOWN, PENNSYLVANIA 18901 WHITE EAGLE TOXICOLOGY LABORATORIES intaih TSCA CBI White Eagle Project Number: DuPont HLO 504-96 DuPont H LO 306-96 WHITEEAGLE-TOXICOL0GY-LABORA-TORIES 2003 Low er State Road, D oylestow n, Pennsylvania 18901 Telephone: (215) 348-3868 Fax: (215) 348-5081 W hite Eagle P roject N um ber: Protocol N um ber: Sponsor T est A rticle: T e st: CLP Com pliance: DEVIATION S FROM PROTOCOL # 1 , # 2 , # 3 (Rev. 04/96) E. I. du Pont de Nem ours and Company a-hexylcinnam aldehyde, tech, 85% G uinea Pig Derm al Sensitization M odified Buehler M ethod 40CFR160 40CFR792 OECD M AFF Japan 1) T he R elative H um idity during the In-Life p h ase o f this stu d y w as 24% 88% instead o f 30% -70% as stated in the protocol. 2) Due to a H oliday, anim al body w eights w ere taken on D ay 29 (w eek 5) instead of Day 28 (w eek 4). T he follow ing is an am endm ent to protocol w hich w as n ot approved in w riting until the com pletion o f the study; therefore, it is p resen ted a s a deviation. 3) Protocol C over Sheet and Protocol Change From : a-hexylcinnam aldehyde, tech, 85% Change T o: a-hexylcinnam aldehyde, tech, 85% These deviations are not thought to have affected the integrity of this study. Subm itted by: Reviewed by: Approved by: G eorge E. fvioore, B .S. Study D irector D ian eT . Cook, B.S. Q uality A ssurance M anager Sponsor Representative Page 43 " D ate C. - 2 C - e D ate Date WHITE EAGLE TOXICOLOGY LABORATORIES mtain TSCA CBI