Document g2Q43z2QOgJyVdK45x2ZdjZE3

AR226-3086 DuPont H L Q -f 998-00729 Study title A cute D erm al Irritatio n /C o rro sio n Study w ith H -22481 in R ab b its FINAL REPORT H askell Laboratory P roject ID : H L O -1998-00729 P& dbm iine Laboratory Project ID : W IL-189160 O rganization for Econom ic C ooperation and Development (O EC D ), P art 404 (1992) European Econom ic Com m unity (EEC) 92/69 Annex V - M ethod B 4 (1992) 1 A uthor Tom G . K ern, B .S. Study C om pleted On Janua !5 , 1998 Perform ing Laboratory W IL Research L aboratories, Inc. 1407 G eorge Road A shland, O hio 4480S-9281 for E. I. du Pont de Nem ours and Company H askell Laboratory fo r Toxicology and Industrial M edicine Elkton Road, P . . B ox 50 Newark, D elaw are 19714 Page I o f 38 I r WIL RESEARCH LABORATORIES, INC. A SubsidiaryofGreat Lakes Chemical Corporation, Ashland, OH 44805*9281 (419)289-8700 ............ - ....... ........ .............................. Company Sanitized. Does not contain TSCA CBI Project No.: W IL-!89160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits SPECIFIC COUNTRY REQUIREM ENTS (This page was intentionally left blank; client will insert appropriate statement.) 2- - Com pany SanitirpH n nP, kpa i Project N o.: W IL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO -1998-00729 Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits G O O D LABORATORY PRACTICE CO M PLIANCE STATEM ENT This study w as conducted in compliance with EPA FIFRA (40 CFR 160) mid EPA TSCA (40 CFR 792) G ood Laboratory Practice Standards, and OECD Principles o f Good Laboratory Practice [C (81)30(Final), Annex 2] with die following exception: Although H-22481 was characterized in a DuPont Laboratory, the test substance w as not characterized according to Good Laboratory Practice. This deviation did not affect the validity o f die study. S ubm itter: Sponsor: E. I. du Pont de Nemours and Company E. I. du Pont de Nemours and Company V Sponsoring Com pany R ep resen tativ e:____ Registration M anager -3Onmnanu Ranltborl n. TCf>AAn Project No.: W IL -189160 Sponsor: E. I. du Pont de Nemours and Company DuPont H L O -1998-00729 Acute Dermal Irritation/Corrosion Study with H-22481 in Rabbits GENERAL INFORM ATION Haskell Number: 22481 Stability: Sponsor: Study Initiated-Completed: In Lile Phase Initiated-Completed : In the absence of visible evidence to die contrary, the test substance was assumai to be stable under die conditions of administration. DuPont Specialty Chemicals E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 June 19, 1997 - January 15, 1998 June 20, 1997 - June 28, 1997 AH raw data and the final report will be stored in the archives of WIL Research Laboratories, Inc., Ashland, Ohio, in the archives o f 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). -4fiQaPaW .SaBit.m d...Onac ratal;.rrantaiwTSGA 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 TABLE OF CONTENTS Specific Country Requirements Page Good Laboratory Practice Compliance Statement General Information Summaiy 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 2. Incidences of Dermal Response 3. Individual Body Weights (Grams) Page 19 20 21 INDEX O F APPENDICES A. Scoring Criteria for Dermal Reactions B. Protocol Page 22 24 -5- , ' Com pany Sanitized. Does not contain T S C A CBI Project No.: W1L-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 SUMMARY: Acute Dermal Irritation/Corrosion Study with H-22481 in Rahhitc 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 o f the test snhstanTM 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 m accordance with the method of Draize (Appendix A) and corrosion evaluations were marfp immediately following unwrap of the three-minute and one-hour exposure sites. The four- hour exposure sites were observed for dermal irritation at 30-60 m im .^ 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 cnhgjrfpd 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 raim ia^d according to EEC Commission Directive 93/21 relative to the general classification and labeling requirements for dangerous suDstances published in the Official Journal of the European Communities (EEC Directive 93/21), H-22481 is not classified as an irritant -6fiaoBaattSainlti-raif! StiShfiSi Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company PERSONNEL AND REPORT SUBMISSION: Kev 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. Report Prepared BV: DuPont HLO-1998-00729 Biologist II - Acute Toxicology Director o f Metabolism and Analytical Chemistry Assistant Director of Pathology and Veterinary Medicine Biologist H/Report Writer II - Acute Toxicology Reviewed Bv: R. Kiplinger, B.S. Manager of General Toxicology Approved and Submitted Bv: Study Monitor: G. Study Director Toxicology Associate Date s / 'z h f Date -7- Com panv Sanitized n n P, nnt l 'a g i a t a .TSCA..f!Rl Project No.: W IL-189160 Sponsor: E. 1. du Pont de Nemours and Company QUALITY ASSURANCE UNIT STATEMENT: DuPont HLO-1998-00729 Date(s) of 6/20/97 7/22.26/97 7/23.26/97 Phase Inspected Date(s) Findings Reponed to Study Director Evaluation of Dermal Reactions Study Records (1-1) Draft Report 6/20/97 7/28/97 7/28/97 Date(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 amendment(s), if any. Quality Assurance findings, derived from the inspections) during the conduct of the study and from the inspections o f 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. Manager of Quality Assurance Date -8- Cnm nanv S a n it i^ n nog tcpa o p, OBJECTIVE AND INTRODUCTION: The objective o f the study was to determine the irritative and corrosive potentials of the test substance following a single exposure to the skin o f 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]. 1 i i MfcffiaifliitiifBfit MHMad&fcfitt Project No.: W IL -189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 MATERIALS AND METHODSStudv Title: WIL Studv N o.: Experimental Start Date: Experimental Termination Date: Test Facility: Studv Sponsor: Studv 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. Kern, B.S. Senior Group Supervisor - Acute Toxicology Tracy A. Filliben Toxicology Associate Test Svstem: Species: Strain: Justification for Species Selection: Source: Number on Studv: Bodv Weieht Ranee: 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- Com pany Sanitized. Does not contain T S C A CBI Project No.: WIL-189160 Spoasor: E. I. du Pont de Nemours and Company B Test System (continued): DuPont HLO-1998-00729 Age at Start o f Study: Young adult Method o f Identification: Eartag Housing: 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. Quarantine: The animals were acclimated to laboratory conditions for a minimum of seven days prior to initiation of dosing. Food and Water: / 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 libitum. 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. Environmental Conditions: 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 o f 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- Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont 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 of deionized 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 Dermiform 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 LeveK'sVGroupfsVTreatment 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 o f 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-minate 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- B Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company BA IA ANALYSIS AND INTERPRETATION OP r e s t ii t u . DuPont HLO-1998-00729 gSeeneerraall ccla^sssiifSicantionn^adndTlaibte1?lin"g8re*quEirEemC eCntosmfomrisdsainognDTMir.ecstuivbest9an3c/2e1s. relative to the * Interpretation according to Annex VI Skin Corrosion o r Irritation C riteria: 1. Corrosive bfM ce will be considered to be corrosive if it produces full thiekness STftet sfuSbstat n^ce^wtilnl bie tcllfaSsUsifriield.aatskcaoSrtr0o0s6ive and adsusirginngeddtehtee ssyt mfobroslk"inCi"rraitnadtioUnse. Risk phrases shall be assigned in accordance with 35 "CAUSES SEVERE BURNS* If, when applied to healthy intact animal skin, full thickness 0f tissue occurs as a result o f up to three-m inute exposure o r if this result can be predicted. R34 "CAUSES BURNS" If, when applied to healthy intact animal skin, fall thickness destruction o f skin tissue occurs, as a result o f up to four hour exposure o r if this can be predicted. 2. Irritant The tost substance will be classified as "IRRITANT" and w ill require the "Xi" symbol and the indication o f danger "IRRITANT" in accordance with the followinc criteria: 6 R38 "IRRITATING TO SKIN" If, w hen applied to healthy intact animal skin for up to four hours, cignifirarif inflam m ation is caused and is present 24 hours o r m ore after the e n d o f the exposure period. Inflammation is significant if the mean values o f the scores for ^ eSChar fOIf ation o r edema forauttran corresponds to one o r m ore o f the following mean values calculated for each animal separately and has been observed m two or m ore animals: w -14- Project No.: WIL-189160 Sponsor: E. I. du Pont de Am ours and Company DuPont HLO-1998-0O729 DATA ANALYSIS AND INTERPRETATION OF RESULTS (continued): D erm al Lesion Erythem a and eschar form ation 1 Edema M ean V alue 2 .0 o r more 2 .0 o r more A ll scores at each o f die reading tim es (24, 48, and hours) fo r an effect should be used in calculating the respective m ean values. inflam m ation o f the skin is also significant if it persists in a t least tw o animal a t the end o f the observation tim e. Particular effects such as hyperplasia, scaling, discoloration, fissures, scabs, and alopecia should be taken into account. -, -15- Com pany Sanitized. D oes not contain T S C A CBI Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 RESULTS: Mortality: N o deaths w ere observed during the study. D erm al Observations (Tables 1 and 21: F o r the screen anim al, there was no evidence o f corrosion follow ing any o f the three different exposure tim es (three-m inute, one-hour, and four-hour exposures). The three- m inute exposure site had erythema (score o f 1). There was no derm al irritationobserved for the one-hour exposure. Erythem a (score o f 1) was noted on two four-hour exposure sites a t the 30-60 mim observation. O ne rabbit had erythema (score o f i) and edem a (score o f 1) through 48 hours Brown staining was noted on all three four-hour exposures throughout the study. A ll fa* responses com pletely subsided by 72 hours o r earlier. There w ere no other derm al findings. C linical Observations fTable 3* N o clinical signs o f toxicity were observed during die study. Body W eights (Table 3 ): * N o rem arkable changes o r differences were observed In body weights during the study. I PH -'.'I -16Pnmnam# -Qi viam Liiaam aat^aatm a a s M ift Project N o.: W IL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont H LO -1998-00729 C O N C L U S IO N S : U nder the conditions o f testing and according to the guidance provided in EEC D irective 93/21, H-22481 is not classified as an irritant. D ate I i i 4 i 1 1 :i 4 I i -17- C o m p a n y S a n itiz e d . D o e s n o t c o n ta in T S n A p.r i Project No.: W IH 89160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 ?5 { Acute Derm al Irritation/C orrosion Study w ith H-22481 in Rabbits TABLES 1-3 i - 18- Com pany Sanitized. D oes not contain T S C A CBI i Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company TABLE 1 DuPont HLQ-1998-00729 ACUTE DERMAL IRRITATION/CORROSION STUDY WITH H-22481 IN RABBITS INDIVIDUAL DERMAL SCORES E rythem a Animal Sex .Site 1 hr 24 hr 48 h r J t t l L 23575 M A is Is Is Os 23585 M A Os Os Os Os 23602 F A Is Os Os Os Anim al 23575 23585 23602 M M F Edema Site J J l 24 h r J S J l 72 h r A11 10 A0 0 0 0 A0 0 0 0 hr = Hour M = Male s = A pplication site stained brown A -- Four-hour sites F = Female Animal Number 23575 23585 23602 Erytheipa 0.67 0.00 0.00 -Edema 0.67 0.00 0.00 19 L Company Sanitized. Does not contain TSCA CBI Project N o.: WIL-18916G Sponsor: E. I. du Pont de Nemours and Company ) TABLE 2 DuPont HLO-1998-00729 ACUTE DERMAL RRITATION/CORROSON STUDY W ITH H-22481 IN RABBITS INCIDENCES O F DERMAL RESPONSE (SCORES)1 Score 0 1 1 hr 1/3 2/3 Eiythem ah 24 hr 2/3 2/3 1/3 1/3 UM 3/3 0/3 Score 0 1 m 2/3 1/3 2QCIu3 U M SM 2/3 2/3 1/3 1/3 UM 3/3 0/3 y "According to EPA test guidelines. bFour-hour sites evaluated. hr = Hour i : 20 Project No.: WIL-189160 Sponsor: E. . du Pont de Nemours ami Company /) TABLE 3 DuPont HLO-1998-00729 ACUTE DERM AL IRRITATION/CORROSION STUDY W ITH H-22481 IN RABBITS INDIVIDUAL BODY W EIGHTS (GRAMS) ..G ro u p 0.5 g/site Animal 23575 23585 23602 M M F Initiation CDav 01 3025 3325 3318 Term ination 3117 3368 3338 Sb M ale Female r CLINICAL OBSERVATIONS No clinical findings were noted for any anim als during die study. 21 GomganySanitzed^Doe^To^ontair^SCACBI Project No.: WI!^189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-00729 Acute Derm al Irritation/C orrosion Study Willi H-22481 in Rflhfaitt APPENDIX A Scoring C riteria fo r Derm al Reactions y - 22- Company Sanitized. Does not contain TSCA CBI Project No.: WIL-189160 Sponsor: E. I. du Pont de Nemours and Company DuPont HLO-1998-OQ729 APPENDIX A ACUTE DERMAL IRRITATION/CORR0SION STUDY W ITH H-22481 IN RABRTTS SCQBJNG CRITERIA FOR DERMAL REACTIONS V alu e Evaluation o f Dermal Reartinnc* Erythem a and Eschar Formation 0 N o erythem a 1 V ery slight erythem a (barely perceptible, edges o f area not w ell defined) 2 M ild erythem a (pale red in color and edges definable) 3 M oderate to severe erythem a (definite red in color and area well defined) 4 Severe erythem a (beet o r crim son red) to slight eschar form ation (injuries in depth) Edema Formation 0 No edema 1 Very slight edema (barely perceptible, edges o f area not well defined) 2 M ild edema (edges o f area well defined by definite raising) 3 M oderate edema (raised approximately 1 mm) 4 Severe edema (raised m ore than 1 mm and extending beyond area o f exposure) *D raize, J.H ., 1959. The Appraisal o f die Safety o f Chem icals in Foods, D rugs and Cosmetics D erm al Toxicity, pp. 46-59. Assoc, o f Food and Drug O fficials o f the U .S ., Topeka, Kansas. 23 C o mjgany S a n itiz e d . D o e s n o t c o n ta in T S C A C B I .24- DuPont H U M 998-00729 Study Number W1L-, ./j ? /4 fo cm p MASTER PROTOCOL COVER SHEET Sponsor: E i. du Poolde Nemou cad Company SpwuofFfiffjgct (Sponsor) B. ftpMCTLMaiifiaBWB; 1) O. PERSONNEL INVOLVED IN THE STUDY A. Sponsor Representative Tttle: ToxicologyAssociate (Sponsor) 2) m . STUDY SCHEDULE DATA C. ftOPMOd Audited Drsft Renew D ie: 3) IV. TESTMATERIALDATA a . MentifisaiMij A aA ? A * /* ? - .s /t/f? (WIL) (WIL) (WIL) (Sponsor) (Sponsor) (Sponsor) WIL RESEARCH LABORATORIES, INC. A Subsidiary of Great Lakes Chemical Corporation, Ashland, OH 44805-9281 (419)289-8700 25 Com pany Sanitized. Does not contain T S C A CBI 'M L -J & .____ (WIL) Master Protocol Cover Sheet Page Two I DuPont HLCM998-00729 E. Physical Description: F. Storage Conditions: .kffmn WfrT-ig S f f M Amine temperature (WIL) (Sponsor) (Sponsor) (Sponsor) 1) Sponsor Representative is specified. 2) Proposed scheduling infioraiation is added to the protocol. 3) Test material dam are added *otin protocol. Approved By: EX du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industria! Medicine EUctonRoad P.O. Box50 Newwk.DE 19714 Prepared By: WE. Research Laboratories.!:. Ashland. OH 44865*9281 Representative fr /i3 k 7 Date 7 *ramG. K bn^LE Study Director /rtA Date 26 Company S a n ita ri nnp. n n tr.n ta in X S C A ./at DuPont HLO-1998-00729 PROTOCOL Acute Dermal Inritation/Corrosion (OECD/EEC Guidelines) Master Protocol N oi J H B \ For E. I. du Pom de Nemours mid Company Haskell 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 WILRESEARCHLABORATORIES, INC. ASubsidiaryofGreatlakes Chemical Corporation,Ashland. OH44805-9281 (419)289-8700 27 -Qamoanv Sanitized. Does not contain TRPA n m ACUTE DERMAL KRI7ATION/CORROSION STUDY IN ALBINO RABBITS DuPont H LO -1998-00729 Master Protocol No; I- OBJECTIVE OF STU D Y To determine the initative and corrosive potential o f the test material following a exposure to the skin o f albino rabbits. B r w ^ ^ f b ' c designed and the study will be conducted in general compliance with tte Organization for Economic Cooperation and Development (OECD) Guidelines for Testing ofC henucals. Section 404 and European Economic Communities (EEC) Official Journal o f the European Communities, 92/69, B4 (1992). The study will be conducted in compliance with the U.S. EPA FIERA and TSCA Good Laboratory Practice Standards (40 CFR Parts 160 and 792, respectively) and the OECD Principles o f Good Laboratory Practice [C (8l) 30 (Final) A nn 2]. n . PERSiJC)13UHVQLVED IN THE STUDY To be specified by Master Protocol Cover Sheet B- W1L Study Director Tom G. Kern, B.S. Group Supervisor - Acute Toxicology c . W SLPeputy Director Jonathan M . Hurley, B.S. Senior Biologist - Acute Toxicology D. 1- Tames L. Scbardein, M .S., A.T.S. D irector o f Research 2. Christopher P. Chengeiis, Ph.D ., D .A .B.T. Senior Toxicologist 3. Jozef J.W .M . Mertens, Ph.D ., D .A .B.T. Staff Toxicologist 4. Gary R. Kiplinger, B.S. M anager o f General Toxicology Page 2 of 12 28 Com pany Sanitized. D oes not contain T S C A CBI Acute Dermal Imtation/Conrosion Study in Albino Rabbits DuPont HLO-1998-00729 n- NVnr.VFD INthe firrmy 5. Ronald E. Wilson, B.S. Director of Informational Systems 6. Daniel W. Sved, Ph.D. Director of Metabolism and Analytical Chemistry 7. Sally A. Keets, A.S. Manager of Vivarium , 8. Deborah L. Little M anager of Quality Assurance 9. Kerin Clevidence, B.S. Group Supervisor o f Gross Pathology and-Developmeutal Toxicology Laboratory 10. Charlene M. Lindsey, M .A. M anager of Technical Report Writing 11. Carney B. Jackson. D .V .M ., D .A .C .V .P., D.A.C.V.P.1 Assistant Director -f Pathology and Veterinary 12. Jenny R. Randall Biologist II - Acute Toxicology m study SCHFnmyp^TA A- i t o e d Experimental Starr Date. To be added by Master Protocol Cover Sheet B` mQ?ed ^ n mcnfal Termination Date: To be added by M aster Protocol Cover Sheet C s ffiged AuditCd Draft RePrt D"f' : To be added by Master Protocol Cover Sheet IE S T SUBSTANCE 1M T ai A. dgm ifim w ii: B- Lm Number.- c. Purity: D. Stability: To be added by M aster Protocol Cover Sheet To be added by M aster Protocol Cover Sheet To be added by M aster Protocol Cover To be added by M aster Protocol Cover -Sheet Page 3 of 12 29 Com pany Sanitized. Does not contain T S C A CBI Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 TV- TEST SUBSTANCE DATA (continued) E. Basical Description: F. Storage Conditions: G. Personnel Safety Data: V. TEST SYSTEM i A. Species: B. Strain: 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 New Zealand White C. Source: Covance Research Products, Inc. Denver, PA or Kalamazoo, MI D. Mimfacr OR Study: Three animals (except as noted in section VM. C. of this protocol). E. S: Males and/or females F. Body Weight Range: 2.0 kg or greater at initiation of dosing. r G. Approximate Age: Young adult H. Identification System: Each animal will be uniquely identified by a plastic eartag displaying die animal number. Individual cage cards will be affixed to each cage and will display the animal number, group and study number. I- Justification for Selection: This species and strain is generally recognized as appropriate for primary skin irritation studies. The experimental design uses the procedures and standards required by the current and proposed international guidelines. Page 4 of 12 30 Company Ssmijgg:d. Does nnt rnntain TSfA P.RI tri 'wifc'lfrHi t&SSiiiil Acute Dermai Irxitation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 V1- SPECIFIC MAINTENANCE scH E nrn.B A. Animal Housing wUI 1x5housed individual,y " suspended wire-mesh cages in an environment!! controlled room. Animals will be housed in clean cases e^m m datove Research LaboratonesW,UI!n*c. are fullyataicecarsetdi^ted^ bev athec Ahssnwcieaetikn.t. T t ae ^f *tCSS**t wt ^u * Accreditation of Laboratory Animal Care I n t e ^ t i o L ^ A ^ A ? ' Assessment and B- Environmental Conditions St" 4* .SiS T iT Z i c. Drinking W ater Reverse osmosis treated municipal water will be available ad Khh*m. pat m-mr.Yi.uL automatic watering system are changed regularly according to' S t a S ^ S r S f P r ^ M ^ W a t e r applying the laboratory is analyzed for contaminants a c t m K ^ Standard Operating Procedures. No contaminants known to be in die water are m interfere with the validity of the study. me wiser are expected to V D. Basal P M Feeds, Inc. Certified Rabbit LabDiet 5322 will be offered at approximately iso during the smdy. Periodic analyses of the cenified feed for the presence pesucides are performed and provided by the manufacturer to ensure that noire are m-m-m in concentrations that would be expected to affect the outcome of dre study. vn- ex perim en tal design A- Animal Receipt and Ac.~lr iation Each animal will be inspected by a qualified technician upon receipt mriirrrf tn v , m good health and suitable as test animals will be acclimated to laboratory c o t i d i S fo r^ ,iaum o;f seven days. AH rabbits will be weighed initially and permanently identified win. SSt * rabbit Page 5 of 12 31 Com pany Sanitized. Does not contain TS C A CBI Acme Dermal Irritation/Corrosion Study in Albino Rabbits VE. EXPERIM EN T B Si (continued) DuPont HLO-1998-00729 aSmScor^ros.io*n.dTThe dteramalSrou!te listae p"o"s1sible"rotuat'e"o*f e*xp*osuPre in humf *anst.onal Marion C. Bod> Weights Tlte body weight o f each animal will be determined on study day 0 and at ro-mm.i :- . R ^ ^ u m n g on study beyond day 7 will be weighed at m L e f t e J ^ ^ y POTAGE LEVEI.^ A|up-n>FY The same dose level will be used for all three animals (0.5 ml or 0.5 g per test site). B. Treatment Gmnnc Win be utilized; each animal will sen mils own conn. All rat ste te n t e S S ' c . Initiation Prayt I f it is anticipated that the test substance may be severely irritating and/or corrosive a study will be dosed to evaluate the irrita tiv e /c Z siv e E E o f S T S a , j " . T1f ee s,tes on single animal will receive individual exposures o f the te. a t S * for `bf.presen" ofsevere irritation/corrosion in m eS ^ oltow u a the unwrap. If no senous skin reaction is present on the th ie e - m in u te S tte 2 exposure site will be dosed and observed for the presence o f severe w rim W r^ f * ^ "nm ediaidy following die unwrap. I f no = : siK " i,: * ' * - < - -- iPM o f tbe Sponsor. If conosion is taerved r i t e S fS d T S immediately be terminatwi if nejrhp.r a m m c i . . rr_ , y xposure, the study will ohwrv^rf a*.. ^ nettner a corrosive effect nor a severe irritant effect is Page 6 of 12 32 Com pany Sanitized. D oes not contain TS C A CBI Acute D ental Irritation/Conosion Study in Albino Rabbits DuPont H LO -1998-00729 Vni- DOSAGE L E V E T S A fjpT py^i LRfiOIMEK (continued) Will constitute approximately 2 5 o f the total abnormalities or injuries will be excluded. The test sites will be to S u e d la te S to tf? * ^ 1 ^ o f the back and delineated with four dots dr w ith ------ t0.fly TMriIme approximately 2.5 centimeters apart ( - * ! ! , " * * * "* Approximately 0.5 ml o f the undiluted test substance or 0 5 g o f solid o r . . applied evenly to each test site covering an area at aroraxim aLv t o t o s (If pnpaicd) will be p e rfe L d / S `'" Ii l ? I1.be (addi&onal coa). Solid materials wU! be a i r t a , S ^ S i S ? - * - * * Spoll?or vehicle (amount recorded) to form a thick paste The nf documented in-die study records. P amount o f vehicle used will be Each test site will be immediately covered with a two-ply, approximately one-i* ^ s e c u re d in place with s S lrntating Vt!a^pef .PeCf onllaItrhs wgmilZl Cbebaapnpdliaegdintgo eafh "a nsmeciuirfeadr wa i.thnns_ev_e_nj^w anpings o*f nnoonn_- periods to prevent ingestion of the test material and/or wratminas F or the n * * " - applied is assumed Only one test substance will be applied to each rabbit. ^ ** K . OBSERVATIONS / ^ e r Mch exposure, the bandages will be removed and the residual test substance eentlv wined fs~ 7n; As tesm "s f imsoist?eningmmoia!t"er"iaflswmi,ahywb"e usre<das(to be documsecnpteod Sin,"stuidvsreco2rds) ins?!- t remove residual test substance upon approval by the Study Director. * * 0nier test sites will be examminineUd"aSnadfttehre16d5e1gsruebesloafnceervthmemovaalanodn <fohurTM-horuerrenxSplonsure sites onl.v1' tuhe ^ a iz e Techmque (Appendix A). Additional examinations will be performed at iS i n . , . , i , 24. 48 and 72 hours after patch removal If irritation is n apProJUm*tely Z 1. T l " "T , after ^ ^xajn`natio spd teconims of ~ Page 7 of 12 33 if -Sanitized. Does .JTotcojitemTSCAjCB^ Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 IX. OBSERVATION^ (continual) Individual animals may be terminated if no irritation is present at the 72- hour or any observation. If irritation is present at the end o f seven days, observation will continue daily until it subsides o r is obviously irreversible up to 14 days (additional cost). TTie study need not ** aBt o t o "le s * - a o r L bv f l. X. GROSS PA TH O r/X iY The animals will be euthanized by an intravenous inj e r ^ of termination and discarded. Collection o f skin samples from the four-hour exposure sites mav be collected and preserved in 10* neutral-buffered formalin for h isto n a th o iS T x ^ L S * th , ,, f f lc t e n n p m o f 0 Sponsor (sd d tto n l cosO A on major organ systems o f the thoracic and visceral cavities will be found dead o r euthanized in extremis. m y M- BAIA ANALYSIS AND INTERPRETATIONOF REfinffi ^^^ ^^^ toc^oncspoiidiD g tc each sniiiul fbrcipiteiu sodcdsms will beRcutisd at eacn observation period. Mean group -cores will be for each dermal (erythema and edema) by summing the scores obtained from all animai at the 24- 48-and 72-hour observations (excluding the 3 and 60 minute exposures). The th r^ 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 Commission Directive 93/21 relative to the general gnr<- - -- for dangerous substances to EEC Interpretation according to Annex VI Skin Corrosion or Irritation Criteria: 1. Corrosive The test substance will be considered be corrosive if it produces foil destruction o f the tin tissue on at least one animal during the test for skin b r itu ta The substance will be classified as corrosive and assigned the symbol "C" and the indication o f danger "corrosive." Risk phrases shall be assigned in accordance with the R35 "CAUSES SEVERE BURNS" . If, when applied to healthy intact animal skin, foil thickness of ^ ttKue occurs as a result o f up to three minute exposures or if this result can be Page 8 of 12 34 Com pany Sanitized. Does not contain T S C A C R l Acute Dermal Irritation/Corrosion Study ln Albino Rabbits DuPont HLO-1998-00729 M T A ANALYSf&ANP INTERPRETATION OF P r a t r ^ ^ R34 "CAUSES BURNS" ir. when applied to Healthy intact animal skin, full thickness destruction o f skin tissue occurs as a result o f up to four hours exposure or if this can be predicted. 2. Irritant Tbe test substance will be classified as "IRRITANT" and will require the "Xi* svmhrn and the indication o f danger "IRRITANT" in accordance with SfiStow m g^ R38 "IRRITATING TO SKIN" If. when applied to healthy intact animal skin for up to four hours inflammation *s caused and is present 24 hours o r more alter the end o f the exposure period.* Inflammation is significant if die mean values o f die scores ft ehher me following mean va*lu-es: 01 corresponds to one o T S T S i D erm al Lesion I Erythema and eschar formation 0 Edema M ean Score 2.0 o r more 2.0 o r more All scores at each o f the reading times (24, 48, and 72 hours) for an effect will be used in calculating the respective mean values. Inflammation o f the skin is also significant if it persists in at least two at the end o f the observation time. Particular effects such as hyperplasia -hTM discoloration, fissures, scabs, and alopecia should be taken into account XII. REPORT f0ll?W`"S: 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 5 * 2 " n i f CSt ^ s ta n c e based on dermal irritation properties. One copy o f die audited draft report will be provided approximately six weeks following completion o f the smdv Tam copies o f the report will be provided upon finalization. Page 9 of 12 35 Company Sanitized. Does not contain TSCA CBI ! J Acute Dermal Inritation/Corrosion Study in Albino Rabbits Xm . RECORns DuPont HLO-1998-00729 laboratories, Inc. M V WORK PR Q D T rrr 1 CoUected " * maiMaifled by W IL Research Sponsor will have title to all documentation records, raw a*** eiMM L XV- QUALITY H e study will be audited by the WIL Quality Assunnce Unit m i. m - Quality Assurance Unit prior to subm ission^ the s S L S T T ^ te f ldited by * # WIL accurately describes the conduct and the findings tf to e s m d ^ m K * " ^ 0081 iq x ,It XV1- PROTOCOL M OD lFTr^-noM Tliis Master Protocol shall be used for multiple smrfj- s a be prepared for each study which will use this design T te Cover Sheet shall constitute the protocol for an i n S d n i ^ o o r e ig e t i t M a t a t , C o , Sheet -- , r^- ^ ** M aster ***>1 iS S E -S S i S f how" ? : ; * % -. Page 10 of 12 36 Com pany Sanitized. D oes not contain T S C A CBI S) ,) ' T Acute Dermal Irritation/Corrosion Study in Albino Rabbits DuPont HLO-1998-00729 X m ANIMAL WETJFARE ACT COMPLIANCE BK " * Sponsor should m ate pa"rtic`Vula'r "no1te* o"f*die*fol*lo*w*ing": W e lfta A i . The Sponsor signature on this protocol documents for the Study Director the S n o iw * *** sm<*y *escr* ed * is protocol does not unnecessarily duplicate previous uiscomrort, distress or pain to animals. All mrfw<ig ar. : .m. j . f in written laboratory Standard Operating Jbrcedurea. ^ a3cr* e^ m ^ protocol or 3' r C*K0I"C P8" OT<**" Urat csnoot be relieved w in be painlessly m banized as deemed appropriate by the veterinary staff and Study D irector The aScptoionnsoinr wasilltibmeealydvaismedanbnyetrhaesSptousdsyibDlei.rector o f all citauunsLa nncceess wwmhcfnc choruoldu lledaSd to thSis 4. Methods o f euthanasia used during this study are in regulation. X v m - PROTOCOL A P P P n v ^ E. I. du Pont de Nemours and Company Haskell Laboratories for Toxicology and Industrial Medicine Elkton Road P.O . Box 50 Newark, DE 19714 -------- f tA'Ilt- %L[\ la.Lf Carol Finlayj B A. (j Sponsor Representative - UI11 Date WIL Research Laboratories. Inc. Ashland, OH 44805-9281 Tom G. Kern, BT Study Director A /A - 7 Dare s u ^ Q .O r js .^ . a c y Fliben Sponsor Representative _____ ___________________ Dare Page 11 of 12 37 Com pany Sanitized. D oes not contain T S C A CBI Acute Dennal Irritation/Corrosion Study ia Albino Rabbits DuPont HLO-1998-00729 -V te. 0 1 2 3 4 APPENDIX A s s m m . W lia SIA^QR DERMAL REA m oM C* Eggtotion o f Pennal Reactions* ---- S y tiiq n a arnl Eschar Formation No erythema V y slight erythema (barely perceptible, edges or area not well defined) M ild erythema (pale red in color and edges definable) M oderate to severe erythema (definite red in color and area well defined) severe erythema (beet or crimson red) to slight eschar formation (injuries in depth) Edema Formating 0 No 1 2 V y slight edema (barely perceptible, edges o f area not well defined) M ild edema (edges o f area well defined by dtifiwfo D 3 M oderate edema (raised approximately 1 mm) 4 Severe edema (raised more than 1 mm and extending beyond area o f exposure) 1959 `n *e Appraisal o f the Safety of Chemicals in Foods D ru mid r w --i~ Dermal Toxicity, pp 46-59. Assoc, o f Food and Drug Officials o f the U .S ., S p S a , Page 12 of 12 38 1 Com pany Sanitized. Does not contain T S C A CBI