Document mDQKNNEEXqaRjLjq96vGrL0O

AR226-3086 TRADE SECRET DuPont HLO-1998-00729 Study Title Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits FINAL REPORT HaskeH Laboratory Project ID: HLO-1998-00729 P&rfonninp Laboratory Project ID: WIL-1891fiO Data Requirement Organization for Economic Cooperation and Development (OECD), Part 404 (1992) European Economic Cooununitv (EEC) 92/69 Annex V - Method B4 (1992) Author Tom G. Kern, B.S. Study Completed On Janua '5,1998 Perfonning Laboratory WIL Research Laboratories, lac. 1407 George Road Ashtend, Ohio 44805-9281 for E. I. du Pont de Nemours and Company Hastell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newaric, Delaware 19714 Page 1 of 38 WIL RESEARCH LABORATORIES,(NC. A Subsidiaryof Great Lakes Chemical Corporation,Ashland. OH 44805-9281 (419) 289-8700 Company Sanitized. Does not contain TSCA CBI Projecl No.: WIL-189160 Spun-sor: E. 1. du Pom de Nemours and Company DuPont HLO-1998-00729 Acute Dermal Irriiation/Corrosion Study with H-22481 in Rabbits SPECIFIC COUNTRY REQUIREMENTS (This page was intentionally left blank; client will insert appropriate statement.) -2- Comoanv Saniti^d nno. ^ ^^. -rcnA ^0. J Project No.: WIL-189160 Sponsor: E. I. du Font de Nemours and Company DuPont HLO-1998-00729 Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with EPA HPRA (40 CFR 160) and EPA TSCA (40 CFR 792) Good Laboratory Practice Standards, and OECD Principles of Good Laboratory Practice [C(81)30(Final), Annex 2] with die following exception: Although H-22481 was chaiacterized in a DuPont Laboratory, the test substance was not characterized according to Good Laboratory Practice. This deviation did not affect the validity of die study. Study Director: Submitter: E. I. du Font de Nemours and Company Sponsor: E. I. du Font de Nemours and Company fomA Kem, B.S. ' Supervisor - Acute Toxicology WIL Research Laboratories. Inc. Date //'^/fAW Sponsoring Company Representative^ Registration Manager -3- Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 Substance Tested: Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits GENERAL INFORMATION Svnonvms/Codes: Haskell Number: Composition: 22481 Known Impurities: CAS Registry Number: Stability: Sponsor: Study Initiated-Completed: In Life Phase Initiated-Completed: In the absence of visible evidence to me contrary, the test substance was assumed to be stable under the conditions of administration. DuPont Specialty Chemicals E. I. du Font de Nemours and Company Wilmington, Delaware 19898 June 19, 1997 - January 15, 1998 June 20, 1997 - June 28, 1997 All raw data and the final report will be stored in the archives of WIL Research Labc/atories, Inc., Ashland, Ohio, in the archives of Haskell Laboratory for Toxicology and Industrial Medicine, E. I. du Pont de Nemours and Company, Newark, Delaware, or at Iron Mountain, 200 Todds Lane, Wilmington, Delaware (formerly DuPont Records Management Center, Wilmington, Delaware). .4. iiiifiiQanainMii.SaBtfc.gidii.iinnaB nrtiTi-mntain T5iCA CDI Projeci No.: WIL-189160 Sponsor: E. !. du Pont de Nemours and Company DuPont HLO-1998-00729 Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits TABLE OF CONTENTS Specific Country Requirements Page Good Laboratory Practice Compliance Statement General Information Summary Personnel and Report Submission Quality Assurance Unit Statement Objective and Introduction Materials and Methods Observations Data Analysis and Interpretation of Results Results Conclusions Page 2 3 4 6 7 8 9 10 13 14 16 17 INDEX OF TABLES 1. Individual Dermal Scores 19 2. Incidences of Dermal Response 20 3. Individual Body Weights (Grams) 21 INDEX OF APPENDICES A- Scoring Criteria for Dermal Reactions 22 B. Protocol 24 -5Company Sanitized. Does not contain TSCA CBI Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits SUMMARY- The primary dermal irritation and corrosion potentials of H-22481 were evaluated in this study with New Zealand White rabbits. The study consisted of single 0.5-gram doses of the test substance that were applied to the clipped, intact skin of three albino rabbits under semi-occlusive dressings. Initially, a single screen animal was dosed to evaluate the irritative/corrosive potential of the test substance. This single rabbit received three sequential applications of the test substance for three minutes, one hour, and four hours. No severe dermal irritation or corrosion was observed following any exposure to the screen animal. Based on these results, two additional rabbits received single four-hour exposures of the test substance. Dermal observations were made in accordance with the method of Draize (Appendix A) and corrosion evaluations were made immediately following unwrap of the three-minute and one-hour exposure sites. The fourhour exposure sites were observed for dermal irritation at 30-60 minutes and 24. 48. and 72 hours after patch removal. For the screen animal, there was no evidence of corrosion following the three-minute, onehour, and four-hour exposures. Very slight erythema was observed on the three-minute exposure site. There was no dermal irritation for the one-hour site following unwrap. The test substance induced erythema (score of 1) on two of the four-hour exposure sites. One animal had edema (score of 1). Brown staining was noted on all three rabbits. There were no other dermal findings. All irritation was reversible and completely subsided by 72 hours or earlier. Mean values were calculated for each animal seperately by using numerical scores obtained from the quantitative evaluation of each dermal response (erythema and edema) observed in the rabbits at the 24-, 48-, and 72-hour observations (excluding the three-minute and onehour exposures). These values are as follows: Animal Number 23575 23585 23602 Erythema 0.67 0.00 0.00 Edema 0.67 0.00 0.00 Based on the overall average of the mean values for erythema and edema calculated according to EEC Commission Directive 93/21 relative to the general classification and labeling requirements for dangerous suostances published in the Official Journal of the European Communities (EEC Directive 93/21), H-22481 is not classified as an irritant. -6- n^^.. t^:^ n^ ^ "nntnin TTflLCRL Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 PERSONNEL AND REPORT SUBMISSION: Key Personnel: Jenny R. Randall Daniel W. Sved, Ph.D. Carney B. Jackson, D.V.M., D.A.C.V.P., D.A.C.V.P.M. Biologist II - Acute Toxicology Director of Metabolism and Analytical Chemistry Assistant Director of Pathology and Veterinary Medicine Report Prepared BV: Q ^ i UA^ //Anita F. Wilmot, B.S. Biologist n/Report Writer II - Acute Toxicology '1/yhs Date Reviewed Bv: dfc;^v (JonathaMn . Hurley (B^. SeniorjBiologist - Acute Toxicology qjfJR^Kiplinger/B.S. Manager of General Toxicology Approved and Submitted Bv: ';F6mG. Kem. B.S. Study Director Study Monitor: r^' r /^ ' i'. ," ^. ^^it/)d^t i^ ^ u.a.7^7j Tracy A. Filliben ^Toxicology Associate ' h^ha Date 1/^1^ Date ///sAy Date s l ^ Date -7- Comnanv S^nif.^d nno. ^ ^ ^ TQ^ A nn. Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company QUALITY ASSURANCE UNIT STATEMENT: DuPont HLO-1998-00729 Date(s) of Inspection^ 6/20/97 7/22.26/97 7/23.26/97 Phase Date(s) Findings Reported to Study Director Evaluation of Dermal Reactions Study Records (1-1) Draft Report 6/20/97 7/28/97 7/28/97 Daie(s) Findings Reported to Management 7/29/97 8/29/97 8/29/97 This study was conducted and inspected in accordance with the Good Laboratory Practice Regulations, die Standard Operating Procedures of WIL Research Laboratories, Inc., and the protocol and protocol amendments), if any. Quality Assurance findings, derived from the inspections) during the conduct of the study and from the inspections of the raw data and draft report, are documented and have been reported to the Study Director. A status report is submitted to management monthly. The raw data, the retention sample(s), if applicable, and the final report will be stored in the Archives at WIL Research Laboratories, Inc.. or another location specified by the Sponsor. ^^.^A^^Xtt^L Deborah L. Little Manager of Quality Assurance ^/9^ Daie -8- nnmnanv Saniti^ n^ ^ ^t^ Ten A ^pl Project No.: WIL-189160 Sponsor: E. I. du Font de Nemours and Company DuPont HLO-1998-00729 OBJECTIVE AND INTRODUCTION: The objective of the study was to determine the irritative and corrosive potentials of the test substance following a single exposure to the skin of albino rabbits. The protocol was designed and the study was conducted in general compliance with the Organization for Economic Cooperation and Development (OECD) Guidelines for Testing of Chemicals, Section 404 and European Economic Communities (EEC) guidelines in the Official Journal of the European Communities, 92/69, B4 (1992). The study was conducted in compliance with the U.S. EPA FIFRA and TSCA Good Laboratory Practice Standards (40 CFR Pan 160 and 792, respectively) and the OECD Principles of Good Laboratory Practice [C(81) 30 (Final) Annex 2]. -9- Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 MATERIALS AND METHODS: Study Title: WIL Study No.: Experimental Start Date: Experimental Termination Date: Test Facility: Study Sponsor: Study Director: Sponsor Representative: Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits WIL-189160 June 20, 1997 June 28, 1997 WIL Research Laboratories, Inc. Ashland, OH 44805-9281 E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, DE 19714 Tom G. Kem, B.S. Senior Group Supervisor - Acute Toxicology Tracy A. Filliben Toxicology Associate Test System: Species: Strain: Justification for Species Selection: Source: Number on Study: Body Weight Range: Albino rabbit New Zealand White The New Zealand White rabbit is generally recognized as appropriate for primary skin irritation studies. Considerable historical control data have been accumulated with this species for this type of study. Covance Research Products, Inc. Denver, PA Two males and one female 3025 to 3325 grams at initiation of dosing -10- Company Sanitized. Does not contain TSCA CBI Project No.: WIL-189160 Sponsor: E. I. du Font de Nemours and Company DuPont HLO-1998-00729 Test System (continued): Age at Start of Study: Method of Identification: Housing: Quarantine: Young adult Eartag Individual suspended wire-mesh cages. The animals were maintained by the animal husbandry staff of WIL Research Laboratories, Inc., in accordance with Standard Operating Procedures. * The animals were acclimated to laboratory conditions for a minimum of seven days prior to initiation of dosing. Food and Water: Environmental Conditions: PMI Feeds, Inc. Certified Rabbit LabDiet 5322 at approximately 150 grams per day. Analysis of feed was performed and provided by the manufacturer. Reverse-osmosis treated municipal water was provided ad libitwn. Water was analyzed in accordance with Standard Operating Procedures. Contaminants were not present in animal feed or water at levels expected to interfere with the objective of this study. Results of analyses are available upon Sponsor request. Animal room with controlled temperature (66.067.1F), humidity (50.0-52.1%). and light (12 hours light/12 hours dark). Test Substance Preparation: Individual 0.5-gram doses of the test substance were weighed into plastic weigh boats that were covered, labeled, and transported to the animal room for dosing. Sufficient deionized water was dispensed for moistening the test substance. Route and Rationale of Test Substance Administration: The route of test substance administration was direct application to clipped, intact skin. This route of administration is standard for assessment of local dermal irritative/corrosive potential. -11- Companv Sanitized. Does not nnnf.m TCPA r^. Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPunt HLO-1998-00729 Method of Test Substance Administration: On the day prior to dosing, the hair was removed from the backs and flanks of the rabbits using an Oster small animal clipper. The clipped area on each animal constituted approximately 25% of the total body surface. The 0.5-g test sites, delineated with waterinsoluble ink, were located lateral to the midline of the back on each rabbit. Approximately 0.1 milliliters ofdeionized water were added to each dose. Each dose was applied to an area of skin approximately 6 cm2 under a secured two-ply, one-inch square gauze patch that was overwrapped with gauze bandaging and secured with Dennifonn tape. The wrappings were used to retard evaporation and keep the test substance in close contact with the skin. Collars were applied to each animal for the one- and four-hour exposure periods to prevent ingestion of the test substance and/or wrappings. Only one test substance was applied to each rabbit. Dosage Level(s)/Group(sVTreatment Regimen: The dosage level was 0.5 g/site. There was one group of three rabbits. Initially, a single animal was selected and treated with the test substance in order to minimize unnecessary use of additional rabbits in case the test substance was corrosive or severely irritating. One screen animal received three separate semi-occluded applications of the test substance for the three-minute, one-hour, and four-hour exposure periods. Following the three-minjte and one-hour exposures, the patches were removed and the sites were immediately evaluated for corrosion and dermal irritation (see Appendix A for scoring criteria). No other observations were conducted on the three-minute and one-hour exposure sites. The four-hour exposure site was evaluated for dermal irritation at 30-60 minutes after patch removal and further observed in accordance with the protocol. As no corrosion or severe dermal irritation was observed on these sites, two additional animals subsequently received a single, four-hour, semi-occluded exposure. At the end of each exposure, the collars (if applicable) and bandages of each rabbit were removed and the sites were wiped with disposable paper towels moistened with tepid tap water. Brown staining was noted on the application sites throughout the study. On day 5, two days after the scheduled termination, the four-hour exposure sites of the two remaining anima's on study were washed with a 1 % solution of Johnson baby shampoo at the Sponsor's request. Upon washing, all evidence of brown staining was removed. This procedure demonstrated that the skin of the application sites was not discolored. The initial single animal screen was euthanized prior to the Sponsor's request and therefore not washed. -12- Companv Sanitized. Dn^ nnt nnnf.in TSP.A nm Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company OBSERVATIONS: Mortality: The rabbits were observed twice daily (morning and afternoon) for mortal^ of the study. ^ Dermal Observations: The three-minute and one-hour dose sites were observed once immedia removal for the presence or absence of corrosion. Dermal irritation w| graded in accordance with the method of Draizs (Appendix A). The four-hour application sites wbre observed for erythema, eote findings approximately 30-60 minutes and 24,48, and 72 hours after] irritation was graded in accordance with the method of Draize (/ areas of untreated skin were used for comparison. Clinical Observations: The rabbits were observed for clinical signs of toxicity at each dermal i the three-minute and one-hour exposures). Body Weights: Body weights were obtained and recorded on study day 0 termination). Termination: Upon termination, the rabbits were euthanized by intravenous pentobarbital solution and discarded. -13Company Sanitised, Project No.: WIL-189160 Sponsor: E. 1. du Pont de Nemours and Company DuPont HLO-1998-00729 DATA ANALYSIS AND INTERPRETATION OF RESULTS: A. EEC Classification The numerical values correspondtag to each aninial for erythema and edema were reconied at each observation period. Mean values for each dennal response (erythema and edema) were calculated for each animal seperately from numerical values obtained at the 24-, 48-, and 72-hour observations (excluding me three-minute and one-hour exposures). The results were interpreted according to EEC Commission Directive 93/21 relative to we general classification and labeling requirements for dangerous substances. Interpretation according to Annex VI Skin Corrosion or Irritation Criteria: 1. Corrosive The test substance will be considered to be corrosive if it produces full thickness destruction of me skin tissue on at least one animal during we test for skin irritation. The substance will be classified as corrosive and assigned me symbol "C" and me indication of danger "corrosive." Risk phrasesshall be assigned m accordance with me following criteria: 35 "CAUSES SEVERE BURNS" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to three-minute exposure or if this result can be predicted. R34 "CAUSES BURNS" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs, as a result of up to four hour exposure or if this can be predicted. 2. Irritant The test substance will be classified as "IRRITANT" and will require the "Xi" symbol and the indication of danger "IRRITANT" in accordance with me following criteria: R38 "IRRITATING TO SKIN" If, when applied to healthy intact animal skin for up to four hours, significant inflammation is caused and is present 24 hours or more after me end of me exposure period. Inflammation is significant if me mean values of the scores for either erythema and eschar formation or edema formation corresponds to one or more of the following mean values calculated for each animal separately and has been observed in two or more animals: -14- Project No.: WIL-189160 Sponsor: E. I. du Font de ^'incurs and Company DuPoat HLO-1998-00729 rATION OF RESULTS (continued): Dermal Lesion Erythema and eschar formation Edema Mean Value 2.0 or more 2.0 or more All scores at each of the leading times (24, 48, and ?2 hows) for an effect should be used in calculating Hie respective mean values. Inflammation of me skin is also significant if it persists in at least two animals at the end of the observation time. Particular effects such as hvperplasia, scaling, discoloration, fissures, scabs, and alopecia should be taken into account. -15Company Sanitized. Does not contain TSCA CBI Project No.: WBL-189160 Sponsor: E. I. du Font de Nemours and Company DuPont HLO-1998-00729 RESULTS: Mortality: No deaths were observed during the study. Dermal Observations (Tables 1 and 2Y. For the screen animal, there was no evidence of corrosion following any of the three different exposure times (three-minute, one-hour, and four-hour exposures). The threeminute exposure site bad erythema (score of 1). There was so dennal irritation observed for die one-hour exposure. Erythema (score of 1) was noted on two four-hour exposure sites at the 30-60 minute observation. One rabbit had eiythema (score of 1) and edema (score of 1) through 48 hours. Brown staining was noted on all three four-hour exposures throughout the study. All dennal responses completely subsided by 72 hours or earlier. There were no other dennal findings. Clinical Observations fTable 3): No clinical signs of toxicily were observed during the study. Body Weights fTable 3): i> No remarkable changes or differences were observed in body weights during (he study. -16- Ppmr^.g^a.-^i n.^.^..^..^Tg^ A-r^j Project No.: WIL-189160 Sponsor: E. I. du Font de Nemours and Company DuPont HLO-1998-00729 CONCLUSIONS: Under the conditions of testing and according to the guidance provided in EEC Directive 93/21, H-22481 is not classified as an irritant. //^^f Date -17Company Sanitized. Does not i Project No.: WII^l89160 Sponsor: E. I. du Font de Nemours and Company DuPont HLO-1998-00729 Acute Dermal Irritation/Corrosion Stodv with H-22481 in Rabbits TABLES 1-3 -18CgmDajMSanitizgd^Doesno^onteir^SCACBI Project No.: WIL.189160 Sponsor: E. I. du Font de Nemours and Company TABLE 1 DuPont HLO-1998-00729 ACUTE DERMAL IRRITATION/CORROSION STUDY WITH H-22481 IN RABBITS INDIVIDUAL DERMAL SCORES Animal Sex 23575 M 23585 M 23602 F Erythema Site Ihr '24 hr 48 hr A Is Is Is A Os Os Os A Is Os Os 72 hr Os Os Os Je. Site 23575 M A 23585 M A 23602 F A hr = Hour M = Male s = Application site stained brown A == Four-hour sites Edema Ihr 24 hr 48 hr 72Jir 1 1 1 0 0 0 0 0 000 0 F = Female Animal Number 23575 23585 23602 Ervmema 0.67 0.00 0.00 0.67 0.00 0.00 19 Company Sanitized. Does not contain TSCA CBI Project No.: WIL-189160 Sponsor: E. I. du Font de Nemours and Company TABLE 2 DuPont HLO-1998-00729 ACUTE DERMAL IRRITATION/CORROSION STUDY WITH H-22481 IN RABBITS INCIDENCES OF DERMAL RESPONSE (SCORES)* Score Ihr 0 1/3 1 2/3 Erythema'1 24 hr 48 far 2/3 2/3 1/3 1/3 72 hr 3/3 0/3 Score Ihr 0 2/3 1 1/3 Edema1' 24 hr 48 hr 2/3 2/3 1/3 1/3 "According to EPA test guidelines. ''Four-hour sites evaluated. hr = Hour 72 hr 3/3 0/3 20 Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company TABLE 3 DuPont HLO-1998-00729 ACUTE DERMAL IRRITATION/CORROSION STUDY WITH H-22481 IN RABBITS INDIVIDUAL BODY WEIGHTS (GRAMS) Group Animal 0.5g/site 23575 M 23585 M 23602 F Initiation (PgyO) 3025 3325 3318 Termination f72hour^ 3117 3368 3338 M ss Mate F = Female CLWICAL OBSERVATIONS No clinical findings were noted for any animals during the study. 21 Company Sanitized. Does not contain TSCA CBI Project No. :WIl^l89160 Sponsor: E. I. du Font de Nemours and Company DuPont HLO-1998-00729 Acute Dennal Irritation/Corrosion s^y yift H-22W n RBWyte APPENDIX A Swws CnitCTiafor Pwmal RMttioBg -22Company Sanitized. Does not contain TSCA CBI Project No.: WIL-189160 Sponsor: E. I. du Font de Nemours and Company APPENDIX A DuPont HLO-1998-00729 ACUTE DERMAL IRRITATION/CORROSION STUDY WITH H-22481 IN RABBITS SCORING CRITERIA FOR DERMAL REACTIONS Value Kvahiatinn nf Dermal R^nn.* Erythema and Eschar Formation 0 No erythema 1 Very slight erythema (barely perceptible, edges of area not well defined) 2 Mild erythema (pale red in color and edges definable) 3 Moderate to severe erythema (definite red in color and area well defined) 4 Severe erythema (beet or crimson red) to slight eschar formation (injuries in depth) Edema Formation No edema Very slight edema (barely perceptible, edges of area not well defined) Mild edema (edges of area well defined by definite raising) Moderate edema (raised approximately 1 mm) Severe edema (raised more than 1 mm and extending beyond area of exposure) *Draize, J.H., 1959. The Appraisal of me Safety of Chemicals in Foods, Drugs and Cosmetics. Dermal Toxicity. pp. 46-59. Assoc. of Food and Drug Officials of me U.S., Topeka, Kansas. 23 Company Sanitized. Do&s not contain TSCACBI PcNo WL8160 - {Se Ammolngs i El i 5 ::: Tv hEEhS d e Sa g0 3 wll 4 Ren Er 4 Solid i SFEagH A in hel E Ee i ia HE gr od 3 : Gobn Re Hows SORE phe eH . |i BBbEil | a ee il Ee I| B| 8 ` hi | |! * "i i || ------i-- t -- AG y DuPontHLO-1998-00729 StudyNumber: WL. /^9/o fWH,i MASTER PROTOCOL COVER SHEET Spon*aK EJ.duPoatdeNetnouBadCofflpuiy Mcaer Protocol Nof|-J--_---- SpomorPFojeetNo-M------ A. Title ofSmdv: AcineDenniIImtxacm/QmosuaStucfyra Albino RcbbiB (SPOOKS) B. 1) n. PERSONNEL PWOLVEPIM THE gmPY Name: TracyA.Filiiben Title: TaxicoioeyAuocme 2) m. STOPY SCHEDULE DATA A. ftBBMM) Bmrnmenuil SUM BMBB. C. 3) IV. TSrMATEMM.PATA A. B. D. Sl^ite Subte (Sponger) (SpoBMr) ^7 WB.) ^/^7 (WIL) WE.) (Sponsor) (Sponsor) (Spontor) (Spoozor) WIL RESEARCH LABORATORIES, INC. A Subsiduuy of Great Ldces Chemical Corporation. Ashltnsi. OH 44805-9281 (419)289-8700 25 Company Sanitized. Does not contain TSCA CBI wn..^giS2__ CWL) Master Protocol Cover Sheet Page Two DuPont HLCM998-00729 E. Phvaeri Pe-riptinn- T^rk ^^ ^^^ ^<-^ F. SxMget-Mid.rions- Ainbieilt teBlpensure G. BaMmaei gfffrff ffflff' Nonnxl Eabcntoty proeedices. EBEflamteBOTB'MteHfia H. CASJiumbBt: (WIL) (Sponsor) (Spoaior) (Spoosar) C. RMMB for PMiacol MaJHUMtion1) Sponsor Repeseaaxive is specified. 2} PropOBedicfaedulnig uifociiuiion u >ddmd to the protocol. 3) Tea autrnddtta BB added KfttejaotoeoL Approved By: EJ-duPoatdeNeiiuMBS and Company HffccD Laboratsxy for Toxicolofy nd lodusttui Medicine BIJOooRoad P.O.Box 50 Newaric.DE 19714 ^//sh^ Ctate fWfuuASy WILReKfiichLabantories.Iac. AAIud.OH 448GS4281 ^ ^ : ^inG. IBBBfcBA / StudyDireeior ^9A^ Dae 26 Company SanitteRd nno. ^^^^.^TQ^A ^o. DuPont HLO-1998-00729 PROTOCOL Acute Derma! Irritation/Corrosion Study in Albino Babbits (OECD/EEC Guidelines) Master Protocol No' For E. I. du POM de Nemours and Company Haskel! Laboratories for Toxicology and Industrial Medicine Elkton Road P.O. Box 50 Newark. DE 19714 By WIL Research Laboratories, Inc. Ashland. OH 44805-9281 June 4, 1997 WIL RESEARCH LABORATORIEISN,C. A Subsidiaryof Great Lakes Chemical CorporationA, shland. OH 44805-9281 (419)289-8700 27 Cc ACUTE DERMAL IRRITATION/CORROSION STUmZ&ALBIKO.gABBEES Dupont HLO-1998-00729 Master Protocol w No I. OBJECTIVE OF STUDY To determine the iiritative and corrosive potential of the test material following a single exposure to the skin of albino rabbits. This protocol has been designedand the study will be conducted in general compliance with the Organization for Economic Cooperation and Development (OECD) Guidelines for Testing OofffCichiaelmJicoaulrsn,alSoecfttihoen E4u0r4opaenadnECuorompmeaunnitEiecso,no9m2/i6c9C, oBm4m(1u9n9it2ie).s (EEC) guidelines in the The study will be conducted in compliance with die U.S. EPA FIFRA and TSCA Good Laboratory Practice Standards (40 CFR Parts 160 and 792, respectively) and me OECD Principles of Good Laboratory Practice [C(8l) 30 (Final) Annex 2]. n. PERSONNEL INVOLVED IN THE STUDY A. Sppnffor Representative To be specified by Master Protocol Cover Sheet B. WjL Sttidy Director Tom G. Kern, B.S. Group Supervisor - Acute Toxicology C. W1L Deputy Director Jonathan M. Hurley, B.S. Senior Biologist - Acute Toxicology D. Study Responsibilities 1. Tames L. Schariein. M.S.. A.T.S. Director of Research 2. Christopher P. Chengelis, Ph.D.. D.A.B.T. Senior Toxicologisi 3. JozefJ.W.M.Mertens, Ph.D.. D.A.B.T. Staff Toxicologist 4. Gaay R. Kiplinger, B.S. Manager of General Toxicology Page 2 of 12 28 ______Company Sanitized. Does not contain TSCA CBI Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 n. PERSONNEL INVOLVED IN THE STUDY (continued) 5. Donald E. Wiison. B.S. Director of Informational Systems 6. Daniel W. Sved, Ph.D. Director of Metabolism and Analytical Chemiscy 7. Sally A. Keets, A.S. Manager of Vivarium 8. Deborah L. Little Manager of Quality Assurance 9. Kerin Clevidence, B.S. Group Supervisor of Gross Pathology and-Developmeatal Toxicology Laboratory 10. Charlene M. Lindsey, M.A. Manager of Technical Report Writing 11. Caraey B. Jackson. D.V.M.. D.A.C.V.P.. D.A.C.V.P.M. Assistant Director. -' Pathology and Veterinary Medicine 12. Jenny R. Randall Biologist n - Acute Toxicology m. STUDY SCHEDULE DATA A. Proposed Experimental Start Date: To be added by Master Protocol Cover Sheet B. Proposed Experimental Termination Date: To be added by Master Protocol Cover Sbeci C. Proposed Audited Draft Report Date: To be added by Master Protocol Cover Sheet W. TEST SUBSTANCE DATA A. Identification: To be added by Master Protocol Cover Sheet B. Lot Number; To he added by Master Protocol Cover Sheet C. Purity: To be added by Master Protocol Cover Sheet . D. Stability: To be added by Master Protocol Cover Sheet Page 3 of 12 29 Company Sanitized. Does not contain TSCA CBI Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 IV. TEST SUBSTANCE DATA (cantirmed) E. Physical Descrintinn: F. Storage Conditions: G. Personnel Safety Data: V. TEST SYSTEM i A. Species: To be documented by WIL Research Laboratories, Inc. and added by Master Protocol Cover Sheet To be added by Master Protocol Cover Sheet To be added by Master Protocol Cover Sheet Albino rabbit B. Strain: New Zealand White C. Source: D. Number on Study: Covance Research Products. Inc. Denver, PA or Kalamazoo, MI Three animals (except as noted in section VTB. C. of mis protocol). E. Sex: F. Body Weight RanpeG. Approximate AptH. Identification System- 1 Justification for Selection: Males and/or females 2.0 kg or greater at initiation of dosing. Young adult Each animal will be uniquely identified by a plastic eartag displaying die animal number. Individual cage caidf will be affixed 10 each cage and will display the animal number, group and study number. This species and strain is generally recognized as appropriate for primary skin irritation studies. The experimental design uses me procedures and standards required by the current and proposed international guidelines. Page 4 of 12 30 Company Sanitized. Does notconto.-n TRUA P.RI Acute Dermal Imitation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 VI. SPECIFIC MAINTENANCE SCHEDULE A. Acimai Housing The animals will be housed individuallyin suspended wire-mesh cages m an environmentally controlled room. suitable material Animals will be housed that will be changed in clean caps elevated above age-baud or why at least three tones each week. The facilities at WIL Research Laboratories. Inc. are ftiliy accredited by the Association for Assessment Accreditation of Laboratory Animal Care International (AAALAC International). and i B. Environmental Conditions Controls will be set to maintain temperature at 67 4F and relative humidity approximately 30-70%. illumination Fluorescent lighting controlled by light timers win at provide for a 12-hour light/dark phoioprnod. be recorded oner daily. Temperature and relative humidity will C. Drinking Water Reverse osmosis treated municipal water will be available ad Sbitum. Filters servicing the automatic watering system are changed regularly according to Standard Operating Procedures. Water Standard Operating supplying the laboratory is analyzed for coataminanis accoiding to interfere with the Procedures. validity of the No contaminants study. known to be in the wafer are expected to D. Basal Diet Vn. PMI Feeds, during Inc.0 Certified Rabbit LabDiet 5322 will be offered at approximately 150g/day the study. Periodic analyses of the cenified feed for the presence of heavy metals and pesticides in are performed and provided by the manufacturer to ensure that none are present concentrations that would be expected to affect the outcome of thf study. EXPERIMENTAL DESIGN A. Animal Receipt and Ac"lr lation Each animal will be inspected by a qualified technician upon receipt. in good health Rabbits judged to be and suitable as test animals will be zcclimaled to laboratory conditions for a a^n'""e^ainiiamg. ofDseuvreinngdatyhse. aAclcllirmababtiiotsn wpiellriboed,weighed initially and permanently identified with changes in general appearance and each rabbit will be behavior. Prior to initiation observed twice of dosing, daily for judged to be suitable test subjects will be identified. those animals Page 5 of 12 31 Company Sanitized. Does not contain TSCA CBI Acute DHIBIJ Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 Vn. EXPERIMENTAL DESIGN fcontmued) B. Route and Rationale of Test Material A< The route of administration win be dennal to order to lest die potential for dermal irritation aad corrosion. The dennal route is a possible rouie of exposure in humans. C. Bod. ^Veiyhts Thr body Rabbits weight of each animal will be determined on study day 0 and at termination. Mffainrng on study beyond day 7 will be weighed at least once a week if die study is extended. Vm. DOSAGE LEVELS AND TREATMENT REGIMEN A. TiTBfflnffltlevels The same dose level will be used for aU three animals (0.5 ml or 0.5 g per test site). B. Treatment groups Following the acclimation period, three rabbits will be arbitrarily selected from available stock based upon health and a body weight of 2.0 kg or greater. No separate control group will be utilized; each animal will serve as its own control. All test sites will be left intact. C. Initiation Procedure If it is anticipated that the test substance may be severely irritating acd/or corrosive, a single animal screen study will be dosed to evaluate the irritative/corrosive potential of me test substance. Three sites on this single animal will receive individual exposures of (he test bsuebdsctasnecdeafnodr othbrseeervmedinufotersd, ieonperehsoeuncr,e aonfdsfeovuerrehoirurirtsa.tioTnh/ceotrhroreseio-mniinmutme eedxipaotesulyrefoslliotewwinilgl the unwrap. If no serious skin re&ction is present on me three-minute she, the eimxpmoesduiraeteslyhefowlloilwl ibneg dtohseeudnawnrdapo. bsIefrvneod seforiroutsheskpirnesence of severe irritation/coonrreo-hsoiounr site, the four-hour exposure she wil; be dosed and reaction is present on the observed approximately one-hour after tes a-ubstance removal. 30-60 minutes If severe irritation (erythema, cden'4 and/or eschar) is observed after any exposure period on the single animal screen, no farmer tfesiiag will be conducted without die specific written request of die Sponsor. immediately If corrosion is observed after any exposure, me study will be tenninaied. If neither a corrosive effect nor a severe irritant effect is aonbismearvlse,d eaafctherremceeivfionugr-ahosuinrgelexpfoosuur-rhe,outhr eexsptousduyrew. ill be completed using two additional Page 6 of 12 32 Company Sanitized. Does not contain TSCA CB1 Acute Dennal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 Vm. DOSAGE LEVELS AND TREATMENT REGIMEN fcontm,.n D. Method of Administration Approximately 24 hours prior to dermal applications, the back and flanks of each rabbit will be clipped ftee of hair with an Oster* small .tnimal clipper. The clipped area on each animal will constitute approximately 25% of the total body surface srea. Animals with dermal abnormalities or injuries will be excluded. The lest sites will be located lateral to die midline of the back and delineated with four dots made with water-insoluble ink spaced approximately 2.5 centimeters apart (~6 cm2) arranged in a square. i Approximately 0.5 ml of the undiluted test substance or 0.5 g of solid or semi-solid will be applied evenly to each test site covering an area at approximately 6 cm2. Analysis of dosing solutions (if prepared) will be performed at the specific written request of the Sponsor (additional cost). Solid materials will be slightly moistened with water or other suitable vehicle (amount recorded) to form a thick paste. The amount of vehicle used will be documented in-the study records. Each lest s;?c will be immediately covered with a two-ply, approximately one-inch square gauze patch. The patches are secured in place with surgical porous tape. The trunk will then be wrapped with gauze bandaging that is secured with several wrappings of noa- irritatiag tape. Collars will be applied to each animal for me one- and four-hour exposure periods to prevent ingesuon of the test material and/or wrappings. For me purpose of mis study, all test substance applied is assumed to be available far absorption by me lest system. Only one test substance will be applied to each rabbit. K. QBSBgVAT|21!!S After each exposure, the bandages will be removed and the residual test substance gently wiped off with disposable towels moistened with water (as thoroughly as possible without irritating me skin). Alternate moistening materials may be used (to be documented in study records) in order to remove residual lest substance upon approval by the Study Director. Approximately-30-60 minutes after lest substance removal on four-hour exposure sites only, the test sites will be examined and the degree of erythema and edema recorded according to me Draize Technique (Appendix A). Additional examinations will be performed at approximately 24, 48 and 72 hours after patch removal If irritation is present at 72 hours, scoring will continue daily up to seven days after dosing. Examination and recording of erythema and edema will only be conducted immediately upon bandage removal on the three-minute and one-hour exposure sites. The animals will also be observed for clinical signs at each dennal evaluation (excluding the 3 and 60-minute exposures). The animals will be observed twice daily for mortality and signs of illness, injury, or abnormal behavior. The areas of application will be clipped free of hair following the 72-hour evaluation, if necessary and as needed during the study to facilitate accurate dermal observations. Page 7 of 12 33 _________________Company Sanitized. Ppe^ not contain TSg^ggJ^ Acute Dennal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 IX. OpSERVATTQNg(continued) Individual animals may be terminated if BO irritation is present it the 72- hour or any subsequent observation. If irritation is present at the end of seven days, observation wil! continue daily until it subsides or is obviously irreversible up to 14 days (additional cost). The study need not Snpoormnsaollry(aedxdcieteiodna1l4cdoasyt)s. after application unless specifically requested and authorized by the X. GROSS PATHOLOGY The animals will be euthanized by an intravenous injection of euthanasia solution upon bteermcoinllaetcitoend aanndd dpirsecsaerrdveedd. inCo1l0le%ctnioenutorafl-sbkuinffesraemdpfleosrmfraolimn ftoher bfoisutro-hpoaumroetxopgoicsualreexasmiteinsamtioany at me specific written request of me Sponsor (additional cost). A gross necropsy examination on major organ systems of the moracic and visceral cavities will be conducted found dead or euthanized in extremis. on all animals XI. DATA ANALYSIS AND INTBRPRETA-nON OF RESULTS A. EEC Classification The numerical values corresponding tc each animal for erytoem find edema will be reconded at each observation period. Mean group 'cores will be calculated fair each dermal response (erythema and edema) by summing the scores obtained from all animals at me 24-, 48- and 72-hour observations (excluding the 3 and 60 minute exposures). The three means corresponding to the three observation periods will be averaged and overall averages will be obtained for each dermal lesion. The results will be interpreted according to EEC Commission Directive 93/21 relative to the general classification and labeling requirements for dangerous substances. Interpretation according to Annex VI Skin Corrosion or Irritation Criteria: 1. Corrosive The test substance will be considered so be corrosive if it produces full thickness destruction of me skin tissue on at least one animal during me test for skin irritation. The substance will be classified as corrosive and assigned die symbol "C" and me finodlliocwatiinogn corfitdearinag: er "corrosive." Risk phrases shall be assignedin accordance with me R35 "CAUSES SEVERE BURNS" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to three minute exposures or if this result can be predicted. Page 8 of 12 34 company Sanitized. Does not contain TSCA CBI Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 XI. DATA ANALYSIS AND INTERPRETAT[QN OF RESULTS fcontmiin R34 "CAUSES BURNS" If, when applied to healthy intact animal skin. full thickness destruction of skin tissue occurs as a result of up to four hours exposure or if this can be predicted. 2. Irritant The test substance will be classified as "IRRITANT" and will require the "Xi" symbol and die indication of danger ''IRRITANT' in accordance with die following criteria: R38 IRMTATING TO SKIN" If, whea applied to healthy intact animal skin for up to four hours, significant inflammations caused and is present 24 hours or more after me end of the exposure period.* Inflammation is significant if die mean values of die scores for either erydiema and eschar formation or edema foxnudna corresponds to one or more of die following mean values: Dermal Lesion Erythema and eschar formation Edema Mean Score 2.0 or more 2.0 or more All scores at each of the reading times (24, 48. and 72 hours) for an effect will be used in calculating the respective mean values. Inflammation of the skin is also significant if it persists in at least two animals at me end of die observation time. Particular effects such as hyperplasia, scaling, discoloration, fissures, scabs, and alopecia should be taken into account. Xn. REPORT The final report will include the following: summary, objective, test substance identification and receipt, methods, individual dermal observations, mortality, individual body weights, a table summarizing mean values for each rabbit, gross necropsy findings (if applicable), and classification of die test substance based on dermal irritation properties. One copy of me audited cdorapfiet sreopfotrht ewriellpboert pwroilvl idbeedpraopvpirdoexdimuaptoenlyfisnixaliwzaeetikosn.following completion of die study. Two Page 9 of 12 35 Company Sanitized. Does not contain TSCA CBI Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 Xm. RECORDS All original WIL raw data records (as defined by the applicable GLPs and WIL SOPs) generated by Research Laboratories, Inc. will be collected aad maintained by WIL Research Laboratories, Inc. XIV. WORS: PRQPVCT Spon^jr will have product generated title to during all dociimeniaiion records, raw data, slides, specimeBS, or other wort: the performance of die study. data, magnetically encoded AH woric product imehiding raw paper " records and specimens will be retained at no charge for a period of Isnixc.mTonhtehrsefaoftlloo.wWin3gLisRseusaenacrechoflatbhoe rfaintoarlierespworitllin die Archives at WIL Research Laboratories, for storage. All work product will be stored in return aH work product to Haskell compliance with regulatory Laboratory requirements. xv. QVWTfASSVWVG " The study will compliance be audited by the WIL Quality Assurance Unit white in progress > Standard with Good Operating LaboratoryPractice regulations and adoerence to n protocol to assure and to WIL Procedures. Quality Assurance Unit prior The raw data and draft report will be audited by the WIL to submission to accurately describes the conduct assd the findings the Sponsor to assure that die final report of die study. XVI. PROTOCOL MODIFICATION This Master Protocol shall be used for multiple studies. A Master Protocol be prepared for each study which will use this design. Cover Sheet shall The Master Protocol and Cover Sheet shall constitute the protocol for an individual study. Director signature on the Master Protocol In addition, die date of Study initiation. Master Protocol Cover Sheet shall be considered the date of study Modification of die protocol may be accomplished during die course of this investigation. However, no changes will be made in the study design without die verbal or written permission of the Sponsor"" In the event that ifec Sponsor verbally protocol, such changes will be made by appropriate requests or approves a change in the amendment. All alterations of me protocol and documentation in the form of a protocol the Study Director sad me Sponsor reasons Representative. for the modification(s)will be signed by Page 10 of 12 36 Company Sanitized. Does not contain TSCA CBI Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 XVII. ANIMAL WELFARE ACT COMPLIANCE This Study will comply with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR). The Sponsor should make particular note of die following: 1. The Sponsor signature on this protocol documents for toe Study Director the Sponsor's assurance that die study described in this protocol does not unnecessarily duplicate previous experiments. 2. Whenever possible, procedures used in this, study have been *igM^ to avoid or minimiyo discomfort, distress or pain to animals. All methods are described in dlis study protocol or in written laboratory Standard Operating Procedures. 3. Animals that experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanized as deemed appropriate by die veterinary staff and Study Director. The Sponsor will be advised by die Study Director of all circumstances which could lead to dus action in as timely a manner as possible. 4. Methods ofeudianasia used during dus study are in confonnaace with tins above-referenced regulation. XVm. PROTOCOL APPROVAL . I. du Font de Nemours and Company HaskcII Laboratories for Toxicology and Industtial Medicine ElktonRoad P.O. Box 50 Newark. DE 19714 i Mtf(. ,^h U' Carol FinlayfB.A. Sponsor Representative ^M-7 Date WIL Research Laboratories, Inc. Ashland, OH 44805-9281 ^Tom G. Kern. B.S. Study Director ^ A - 7 Date < ^ y / 7 ^ ^ KacyPHliben Sponsor Representative ^ A ^Date Page 11 of 12 37 Company Sanitized. Does not contain TSCA CBI Acute Denna! Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 Vatae 0 ' 1 2 3 4 APPENDIX A SCORING CRITERIA FOR DERMAL REACTIONS* Evaluation of Dennal Reactions* Erythema ami Eschar Formation No erythema Very slight erythema (barely perceptible, edges or ami not well defined) Mild erythema (pale red in color ad edges definable) Moderate to severe erythema (defiflite red in color and area well defined) Severe erythema (beet or crimson red) to slight eschar formation (injuries is depth) Edema Formation-- 0 No edema 1 Very slight edema (barely perceptible, edges of area not well defined) 2 Mild edema (edges of area well defined by definite raising) 3 Moderate edema (raised approximately1 mm) 4 Severe edema (raised more than 1 mm and extending beyond area of exposure) "Draize, J.H., 1959. Dennal Toxiciry, The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. pp 46-59. Assoc. of Food and Drug Officials of toe U.S., Topeka, Kansas. Page 12 of 12 38 Company Sanitized. Does not contain TSCA CBI