Document oeXE48aedZ9j1KGLqQogvNML8

TRADE SECRET AR226-3066 HLO- J997-00026 Study Title USP Biocompatibility Testing o f H-22239 (Control Item) and H-22240 (Test Item) Contracting Laboratory North American Science Associates, Inc. 2261 Tracy Road Northwood, Ohio 43619-1397 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 Susan A. MacKenzie, V.M.D., Ph.D., D.A.B.T. Study Completed on January 17, 1997 Q l aboratory Project ID Haskell Laboratory Report No. HLO-1997-00026 Page 1 of 33 Company Sanitized. Does not contain TSCA CBI l 'SP Biuounpatibility IVsting 0|' H-222.^) (Control Item) am! 11-22240(Test Htm) C-ENKRAI- INFORMATinKl Test Substance fl I Substance Synonvms/Other C hiles- NAmSA I ah bfty Physical FormPurity: H-22239 H-22239 (Control Item) % T-16837-00 Blue fabric Composition- Contaminants- CAS Registry NIn- # lest Substance #7 Substance Tester] Svnonvms/nther Cories- NAmSA (.ah NIn H-22240 H-22240 (Test Item) 96T-16836-00 IV i-ni ;1f . l 'SI* llioi'oiupuiibilily lusting ol 11-22239 (Control Item) und H-222-IO ( l est Item) DuPont Central Research ami Development Haskell Laboratory lor Toxicology and Industrial Medicine HLO-1997-00026 January 17, 1997 DuPont Non-Woven Old Hickory, Tennessee USP Biocompatibility Testing o f H-22239 (Control Item) and H-22240 (Test Item! Attached you will find reports from North American Science Associates, Inc. The results for the H-22239 and H-22240 samples are: TestResults Cytotoxicity USP Intracutaneous Test Substance (Agarose Overlay) SCa_____ CSOb USP Muscle Implantation (7 day) H-22239 H-22240 Non-toxic Non-toxic Pass Pass Pass Pass NS NS a Sodium chloride extract b Cotton Seed oil extract c Not significant 0 ,~ h \ , ___` / l i t e f - Susan A. MacKenzie. V.M.D., Mi.D., D.A.B.T. Research Toxicologist Company Sanitized. Does not contain TSCA CBi : . ^... --'-i Confidential MC030-110 J kl I. du Pont de Nemours & Co. HASKELL LABORATORY El.KTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 A1TN: DR. SUSAN A. MACKENZIE Lab No. 96T 16837 00 p o No LGX-593521 ID No. H-22239 CYTOTOXICITY - AGAROSE OVERLAY, SOLID Test Aritele: H-22239 (control item) Test Article Description: Sheet - 1 cm- piece Exnerimental Procedure: monolayer of L-929 mouse fibroblast cells was grown io confluency and verlaid with Minimum Essential Medium supplemented with serum, ant.biottcs, sutral red and 2 % agarose. The test article, a 0.5 cm x 0.5 cm piece of 25871 E^as aTpositive*control. and a 1.0 cm length piece of UP-I as a negative jntrol were placed on the solidified overlay surface Following ncubation for 4 hours the culture was macroscopically examined for evidence of cell L oSL k of o=" '>'. Any - * " " l " " " . i _____ :_11., nAnfirm ppII lvsis. 1 Score Observations ---------1 1-- Normal cell mnmholoevjn proximity to test sample.----- Toxic (T) Cellular death and degeneration associated with the area beneath the test sample and possibly extended I beyond the perimeter of the test sample. W/here a zone of lysis was observed, the distance from the edge of the sample to the edge of the zone was measured and reported in millimeters (mm). --------------- CYTOTOXICITY - AGAROSE OVERLAY, SOLID Results: Test/Control Articles Test Article Negative Control: UP-I NAmSA Positive Control: 725871H Score N N T Zone of Lvsis immi 0 0 7 Results and conclusions apply only to the test article tested. No further evaluation o f these results is by NAmSA. Any extrapolation of these data to other samples is the responsibility of the sponsor. Conclusion: The above test article was nontoxic for L-929 mouse fibroblast cells under the above described test parameters. Comments: Placed the bluest side of the test article down on agarose. Record Storage: All raw data pertaining to this study and a copy of the final report are to be retained in designated NAmSA archive files. Test Article Received: 12-5-96 Date Prepared: 12-11-96 Date Terminated: 12-12-96 Company Sanitized. Does not contain TSCA CB8 Confidential TU013-800 HLO-1997-0C026 Lab No. 96T 16837 00 P.O. No. LGX-593521 STUDY TITLE: USP INTRACUTANEOUS TOXICITY STUDY IN THE RABBIT (Extracts) TEST ARTICLE: H-22239 (control item) IDENTIFICATION NO.: H-22239 SPONSOR: Ac if* vt6A' -O 0i'* & DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 C HAiiiS I 6- World Lradrr In Truing S e n ic e t fo r ihr Medicai O nice tnduury 2NPP2Ahoo6XrtnthewTroaocdy44.1R19oO96a6H6d6684**923496551459 Page I o f 7 Authorization lor duplication o l M a report, eica p t in h o , te raaarvad pending N A m SA 'a unfflui oppro*al. TUUUXUll TABU: OF CONTENTS SUMMARY ..................................................................................... IN T R O D U C T IO N ............................................................................. MATERIALS .................................................................................. M E T H O D S ........................................................................................ RESULTS ........................................................................................... CONCLUSION ................................................................................ RECORD STO R A G E....................................................................... TABLE ^ 1 USP INTRACUTANEOUS TOXICITY OBSERVATIONS i i i .h- d w - oooim lath No. DOT I0KT7 00 Pane Number 3 4 4 4 6 6 6 7 ii Company Sanitized. Does not contain TSCA CB) NAmSA - 7 / ru J rr in fV tlm jc \ r r m r fo r ih f M e ditai I h t i c r hidoti rv A' Authorization*ndutk-atini tt"s *eP"rt i.*f mil m iioIh is esetved pending NAmSA s wrnte* appiovn* V><Mill ..1 Oh 4ti *** Pane 2 of 7 The lost artiele. 11-22239 (control iieint. II 22239. wa:. extracted in 0.9% sodium chloride USP solution and cottonseed oil. NT. These extracts were evaluated lor intracutaneous toxicity in accordance with the guidelines of the current USP. A 0.2 ml dose of the appropriate test article extract was injected by the intracutaneous route into five separate sites on the right side of the back of each rabbit. Similarly, the corresponding blank vehicle was injected on the left side of the back of each rabbit. Two rabbits were used for each pair of extracts. Observations for erythema and edema were conducted at 24, 48. and 72 hours after intracutaneous injection. Under the conditions of this study, there was no evidence of significant irritation or toxicity from the extracts injected intracutancously into rabbits. Each test article extract met the USP requirements. Study and Supervisory Personnel: Donald P. Barda, LAT Michael A. Safron, AAS (HT) Patricia Mitchell Barbara M. Monroe Darcy Irons Cindy Harper Tina M. Cox, BS, LAT Approved by: M iM ( L'ven J. Hennan5ky, PhannD PhD,/t)ABT I>i ni nnr T n ir ir n ln a v Date Completed Company Sanitized. Does not contain TSCA CB! /las Page 3 o f 7 --H -- World l.etuirr in Trtttng S m u rs for ihr M titnal iPrvire hidu*ir\ Aulhori/ahon lo, rtupteal.. n. m,5 -apon, o.copl m hol * * < * * NA" 'SA s t i. - K v h . i.m i N H thw om l OH ITIiU p i n i i- , A< 4f j j Storage Conditions: Vehicles: Preparation: Condition of Extracts: Sample Disposition: Room temperature 0.9% sodium chloride USP solution (SC) cottonseed oil, NF (CSO) A ratio of 120 cnv':20 ml (surface area of test article to volume f vducle) was used for each preparation. The test article was extracted in SC and CoO at 12PC for 1 hour. The extraction vehicles without test article were similarly prepared to serve as control blanks. Test SC: clear CSO: clear Control clear clear Any remaining sample was discarded. METHODS Test System: Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: Rabbit (Orvctolaeus cuniculus) New Zealand White Myrtle's Rabbitry, Inc. No particular gender was prescribed for this test No particular body weight range was prescribed for this test No particular age was prescribed for this test Minimum 2 days Two per pair of extracts Ear tag or tattoo Justification of Test System: The intracutaneous injection test in rabbits is specified in the current USP and has been used historieally to evaluate biomaterial extracts. Animal Management: Husbandry: Conditions conformed to Standard Operating Procedures which are based on the "Guide for the Care and Use of Laboratory Animals. Food: PROLABfc High Fiber Rabbit Diet was provided daily. Page 4 of 1 Tf.il y H.I.TI1 NllmSA - ') \\ tirili I rttdrt in I r ilin g S r r n r r t Ihr hlrdi.nl Hr, n r Indu,Irr Phniw? IIAAX* i.pl m will*, is iPiOn/lwl lio.iilmg NAniSA s wntioii approval Authorization tor duph-Otiun this tftfioit OH 4TU 4 ^ g Q nlalnTSCACBi itized. P eS n u n i on 111.0-1497-00020 Lab No. 96T 16837 00 Water. (oiil.mimaiils: Housing: liccly available, municipal (Toledo. Oil) water was delivered through an automatic watering system. Reasonably expected contaminants in feed or water supplies did not have the potential to influence the outcome of this tes-. Animals were individually housed in sta;:-. . uentified by a card indicating the lab number, animal number, test code, sex, o j. j uaic l, - Environmental: The room temperature was monitored daily. I he temperature range for the rabbit was 64-74P. The room humidity was monitored daily. The humidity range for the rabbit was 30-70%. The light cycle was controlled using an automatic timer (12 hours light, 12 hours dark). Facility: Personnel : Selection: NAmSA is an AAALAC accredited facility. Associates involved were appropriately qualified and trained. Only healthy, thin-skinned animals free of mechanical irritation or trauma that could interfere with the test were selected. Experimental Procedure: Prior to treatment, each rabbit was clipped free of fur from the back and both sides of the spinal column to yield a sufficient injection area. Two rabbits were prepared per pair of extracts. A 0.2 ml dose of test article extract was injected intracutaneously into five separate sites on the right side of the back of each rabbit; 0.2 ml of the control blank was injected into five separate sites on the corresponding left side of the back. Injections were spaced approximately 2 cm apart. The animals were returned to their respective cages following the procedure. Observations for erythema and edema were conducted at 24, 48. and 72 hours after injection. Reactions were scored on a 0 to 4 basis. Any reaction at the injection sites was also noted. The reactions were evaluated according to the following subjective rating scale: ERYTHEMA (ER) 0 No erythema 1 Very slight (barelv perceptible) 2 Well-defined (pink) 3 Moderate to severe (red) 4 Severe (beet redness) to slight eschar formation (injuries in depth) EDEMA (ED) 0 No edema 1 Very slight (barely perceptible) 2 Slight (edges of area well-defined by definite swelling) 3 Moderate (raised approximately I mm) 4 Severe (raised more than 1 mm, may extend beyond the area of exposure) The cumulative average erythema and edema score for each test extract and corresponding control blank was calculated. For each extract, a difference in average scores (test minus control blank) of 1.0 or less was considered to be acceptable. A difference of 0.6 to 1.0 indicated a slight, but acceptable, reaction. A difference > 1.0 was considered to be unacceptable. Additionally, the average score for each test extract and blank was calculated for each interval. Any adverse reaction noted in the test extract was compared to the corresponding blank. t SC*CB1 .DOW" 0' 00" '" " COTOPanVSantt&ed N ilm S il - 10 - \\nrtd / entier in T e itm g S e m i n far the M ditai t>evtte Industry ? ? f .1 I rary Hoad Norlhw nod OH 43619 P h o n e 4 *k)*6#>h<)45S I AX 41 Aulhno/aUon fw finjWh.ntiiin ol this Hport fltr.opl in wlwle. is eservorJ pending KfAnifiA s wntleh appmvul Page 5 o f 7 111,0-1 lJ`.'7-0002( i r u n (io Lab No. 9(T U)837 00 RLSULTS Results of scores for individual rabbits appear in Table I. ihe findings are summarized below: Eviraci SC cso Average Test <-) Average Control = Difference = Assessment 0.0 0.0 0.0 Passed 1.0 1.0 0.0 Passed | | s considered acceptable. The difference between test and control average The difference in average scores was scores did not exceed 1.0 at any observation interva . I I n n i v i n t h e lest article tested. No further evaluation of these results is made by ,her SampleS " thC reSPOnS'b,lily f ^ SPnSOr' CONCLUSION evidence of significant irritation or toxicity from the extracts Under the conditions of this study, there was no injected intracutaneously into rabbits. Each test article extract met the USP requirements. PFCORD STORAGE and a copy of the final report are to be retained in designated NAmSA All raw data pertaining to this study archive files. Does not contain fSC& cB' Sanitized Compaq VAinSA - 11 World l-rndrr in T r u in g Srrrirrs for Ihr M rdiral Drvirr Industry N otthw oori OH Phoni 4 l\J*6 L>'-*4i)c; VAX 4 Authorization(0 i duplication (it th u epvi. whole. leserved penn.q N/CnSA s while., approval v Page 6 of 7 ER = Erythema ED = Edema _ o.9% sodium chloride USP solution CSO = cottonseed oil. NF Sanitized. Does Company contain TSCACB1 Pace 7 of 7 l milulciilia! ruu-4 Kn7 I l l o - I n97-UUl)2i> No '. I lfH37 (ll> I* O No I.GX 591521 STI mV T IT L E : USP MUSCLE IMPLANTATION STUDY IN THE RABBIT (1 Week) TEST ARTICLE: H-22239 (control item) nwismnrATION NCL: H-22239 cjSSP I5 C c' SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 NAiiiSA 13 W orld le a d e r in TfUinft Servirti for the /dedicai Device Industry NorlHwoo! OH Phonfc 4 1 f AX 4 Authomalionloiduplicai*' ul ih'pon. e.cepl resawed ponding MAmSAs milan approvai Page 1 of II 1114 KU7 SUMMARY INTRODUCTION MATERIALS Vtt THODS RI.SIT.IS ................... CONCLUSION ........... RHCORD STRAGU . . I Mtl I Ol CONTENTS Ill.o- m 7-U U 02u L.il) No `K>T 10837 (H) Page Number 3 4 4 4 6 6 6 Company Sanitized. Does not contain TSCA GS^ VAmSA 14 World I.ruder in T rsliny Servier Ihr M edical l>evicr Industry Tracy Muni! PhOl'fc 41*><t OH 43-iy V> | AX S4 < ***,. n .r a p i m w h o . n -oso-vad pnn d .n g NAmSA's . .a n ap p m v a . A ulhoniB lion lo i d u p w a l ' Ol I" Page 2 ol TUO 14-807 SUMMARY HLO-1 997-00026 Lab No. 96T 16837 00 The tcsi article. H-22239 (control item). H-22239. was implanted in muscle tissue of the rabbit. Themusclc tissue was evaluated for evidence o i irritation or toxicity in accordance with the guidelines of the current USP. Implant samples and USP negative control strips were loaded into needles 31111 implanted and were then euthanatized 1 week later. Muscle tissues were excised and the implant sites were examined macroscopically. Under the conditions of this study, the macroscopic reaction was not significant as compared to the USP negative control implant material. The implanted test article met the USP requirements. Study and Supervisory Personnel: Martha Oswanski. BS, LAT Merle L Heinrke, BS, LAT Jason A. Karsten Tina M. Cox, BS. LAT Approved by: Steven . Hermansky, PharmD, PI . DABT Manager. Toxicology Date Completed /mm KAiiiSA - 15 WurU l.tadrr in Tilling S m i r n far Ihr Mtdirai lim e r Induilry 2261 Tracy Hoad N orlhw ood. OH 4.1619 Phone FAX 4HM669465 dU)-66fi-29S4 Authoriietior tor duplicator ot Ihn report, ecapl m whole, is reserved pending NAmSA's w illen approval Page 3 o f 6 TUO 14-807 INTRODUCTION H LO -1 9 9 7 - 0 0 0 2 6 Lab No. 96T 16837 00 The urn anid c identified " r" current USP. The purpose of the study was to evaluate ne poien received on December 5, 1996. were e.pianred - D ^ r 24. ,4 . MATERIALS The sample provided by the sponsor was identified and handled as follows: Test Article: H-22239 (control item) Identification No: Storage Conditions: Control Article: Preparation: Sample Disposition: H-22239 Room temperature USP plastic 1 mm diameter negative control reference strips purchased by NAmSA from the offices of the US Pharmacopeial Convention and cut into 10 mm sections. A minimum of six sections of the test article 1 mm x 10 mm, were loaded into 16 gauge needles The te s t article w<* cut into 1 mn, x 20 mm lengths and then folded in half to obtain 1 mm x 10 mm sections For each rabbit, a minimum of four USP strtps were loaded into S E s S needles * need for .he ms. anlele. Tes, and eonlrol amclcs were sterilized by steam. Any remaining sample was discarded. METHODS Test System: Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: justification of Test System: Rabbit fOrvctolaeus cuniculus) New Zealand White Myrtle's Rabbitry, Inc. , .. , No particular gender was prescribed for this test 3.2 kg to 3.3 kg at implantation No particular age was prescribed for this test Minimum 2 days Two Ear tag or tattoo easily6graded and compared to a known negative control material. Animal Management: Husbandry: Conditions conformed to Standard Operating Procedures which are based on the -Guide for the Care and Use of Laboratory Animals. p00(j. PROLAB High Fiber Rabbit Diet was provided daily. . . A D oes not contain TSC a Company Sanitize . _______________ __ Page 4 NAmSA - 16 - IVrid Lea d e r in T e llin g S ervice s f o r the M e d ica I D evice Industry 2261 Tracy Road NoMhwood. OH 43G19 Phons 419*666*9455 FAX 419*666*2954 Au.hortt.tKx. lo, d u p lican o of Ih report, ex c.p l In w ho*. reM rved pending NAmSA s written epprovai TU0I4-807 Waler: Comaminanis: H L O -l9 9 7 -0 0 0 2 6 U b No. 96T 16837 00 Freely available, municipal (Toledo. OH) water was delivered through an automatic watering system. Reasonably peered com-- in feed or - influence the outcome of this test. -PP*- - <- -" Housing: Environmental: TR. room temperature was monirored daily. The temperature rouge for the rabbt. was The room bnmidi.y was monitored daily. The humid, range for die rabbi, was 30-70%. The Ugh, cycle was controlled using an automatic tin. (12 hours light. 12 hour, dark). Facility: Personnel: Selection: NAmSA is an AAALAC accredited facility. Associates involved were appropriately qualified and trained. Healthy, previously unused animals were selected. Fnpenmentai Kroccums- The fur was clipped f/om the area over * e tirifli 70% alcohol, each Needles containing test anide strips were similarly implanted. " m ile * " * 2 * ^ ' - - -- -- - u s p -- . ., At 1 week, the rabbits were weighed and euthan^ K i K e S T h tS t'an icle s p r i t e s : z v s s z x s - -- - -- - -- *-based drug The paravertebral muscles were dissected free formation or other evidence of irritation follows: 0 No capsule, no adverse reaction (other than minimal hemorrhage) 1 Up to 0.5 mm capsule or reaction area 2 0.6 to 1.0 mm capsule or reaction area 3 l . 1 to 2.0 mm capsule or reaction area 4 > 2 .0 mm capsule or reaction area Average macroscopic scores for test i m p la n Calculate s were rounded off to the nearest 0.1. minus contfol) was regarded as "not significant, 0.6 to " erae" and ** 3..11 as. "mmaarrkeed." TThhee requirements.of theUSP lTM ts ^ cs (test U to 2.0 as "slight," 2.1 to 3.0 as f h difference between test u" ,, men,s ooff lthheetteesstt wweerree nnoot, mmee., aifndthceodnitfrfoelrence between implanted. w o <or m0re) imp,anl * * * * * ' * " " * an'ma not contain TSC^ c ' ____ . .S n i ',D oeS Page 5 of 6 *** ....__ _ - 17 - World leader in Testing Services to , She Medical Device Industry ,, in,duniicationd thisreport, trecapiinwftoi.,s,eaa nendtngNAmSAs niltensppnwl *" 9 TUO 14-807 H L O -1997-00026 Lab No. 96T 16837 00 RESULTS q ^fcai n e r v a t i o n s : All animals appeared clinically normal throughout the duration o f the study. Body weight data for individual rabbits were considered acceptable. M arnwmpir Observations- The findings for the macroscopic evaluation are shown below. Results and conclusions apply only to the test article tested. No further evaluation o f these results is made by NAmSA. Any extrapolation o f these data to other samples is the responsibility of the sponsor. CONCLUSION Under the conditions of this study, the macroscopic reaction was not significant as compared to the USP negative control implant material. The implanted test article met the USP requirements. RECORD STORAGE All raw ta ta pertaining to this study and a copy o f the final report are to be retained in designated NAmSA archive files. TSCA CB1 liz e d .O o ^ not contain C o m p aq Sam' M in S ff - 18 World Leader in Testing Sorrtti fo r ike M edim i Device Industry F2NPA2ho6oXr1nthoT*ra.tocdy44.1R1WOo9a4Hd66864*4939465514S9 A u tn rtu tto n for duplication of this raport. scopi H ho*. t raaanmd ponding NAm SA raion approval Page 6 of 6 NjI iiiS jI W'uriti l.vaiivr in Tfihffi Svitiit*** f o r t h v M v d iv u t /)i'i m i' in d u s t r y HLO-1997-00026 2261 Tracy Road Northw ood, OH 43619 Phone 419*666*9455 FAX 419-666*2954 Confidential MG030-110 E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 ATTN: DR. SUSAN A. MACKENZIE Lab No. 96T 16836 00 P.O. No. LGX-593521 ID No. H-22240 CYTOTOXICITY - AGAROSE OVERLAY. SOLID Test Article: H-22240 (test item) Test Article Description: Sheet - I cm2 piece Experimental Procedure: * A monolayer of L-929 mouse fibroblast cells was grown to confluency and overlaid with Minimum Essential Medium supplemented with serum, antibiotics, neutral red, and 2 % agarose. The test article, a 0.5 cm x C.5 cm piece of 725871H as a positive control, and a 1.0 cm length piece o f UP-1 as a negative control were placed on the solidified overlay surface. Following incubation for 24 hours, the culture was macroscopically examined for evident of cell decolorization to determine the zone of cell lysis. Any decolorized zone present was examined microscopically to confirm cell lysis. Score Nontoxic (N) Toxic (T) Observations -------- 1 Normal cell morphology in proximity to test sample. 1 Cellular Hwith and degeneration associated with the g area beneath the test sample and possibly extended 1 beyond the perimeter of die test sample. Where a D zone of lysis was observed, the distance from the edge | of the sample to the edge o f the zone was measured g and reported in millimeters (mm). N not contain TSCACBI Company Sanitized. Does - 19 -.vili,;. K, ipwrved Page I of 2 NAtnSA' v.-nttrai .ippmwil XAmSXWoriti Li-atier in Trslm n Srnocrs Ior Ihr MrtUral D rvirr h u lu stry HLO-1 9 9 7 -0 0 0 2 6 2261 Tracy Road N orthw ood, OH 43619 Phone 419666*9455 FAX 419^66*2954 M G 030-I10 Lab No. 96T 16836 00 CYTOTOXICITY - AGAROSE OVERLAY, SOLID Results: Test/Control Articles Test Article Negative Control: UP-1 NAmSA Positive Control: 725871H Score N N T Zone of lysis (mm) 0 Results and conclusions apply only to the test article tested. No further evaluation of these results is made by NAmSA. Any extrapolation of these data to other samples is the responsibility o f the sponsor. Conclusion: Comments: H ie above test article was nontoxic for L-929 mouse fibroblast cells under the above described test parameters. Placed the bluest side of the test article down on agarose. Record Storage: All raw Ham pertaining to this study and a copy of the final report are to be retained in designated NAmSA archive files. Test Article Received: 12-5-96 Date Prepared: 12-11-96 Date Terminated: 12-12-96 Does no t contain TSCACBi Company Sanitized. m m Date Completed lZ--*t-96 Authorization lor duplicatiti!' Jhir, - 20 exropl in whole, s icservod [n.-Mfling NAmSA'V. written nppinval Confidential TU013-800 H.O-1 9 9 7 - 0 0 0 2 6 U b No. 96T 16836 00 P.O. No. LGX-593521 STUDY TITLE; USP INTRACUTANEOUS TOXICITY STUDY IN THE RABBIT (Extracts) TEST ARTICLE: H-22240 (test item) IDENTIFICATION NO.: H-22240 C 0^ ,d. 00s X \t* ' tsc* SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 NA111S - 21 World tr a d ir in Tetlimg Serviert fo r ike Mdical Device Induttry 2261 Tracy Road Northw ood. O H 43619 Ph on e 419*666*9455 FAX 419466*2954 A u to m atio n lo t dupfacctnn of Ums rapo*. ateopf m tehola. is taiarvad pandxtg NAm SA's written approval Page I o f 7 TUO13-800 TABLE OP CONTENTS Lab No SUMMARY ...................... IN T R O D U C T IO N .............. MATERIALS ................... METHODS ......................... RESULTS ............................ CONCLUSION ................. RECORD STORAGE------ TABLE ,, I - USP INTRACUTANEOUS TOXICITY OBSERVATIONS .s m . :r4i;jiJUJlp liliill m **1-.*-;*It : HI! i gfl i l l -ic: :# ju s lS \% 14;! -i'*'!> \]\ i l l ! in i l\ l-li h j y : ; !:-h? a -\'YS\ jji m .; iiv i=*?il' 4in |::j \t if ompany Sanitized. Does not contain tsca CBt - 22 - World U a d n In Toiling S tr r ie n to r Iki M idietl D trlct Indnitry 2261 trte yO ctd Northurood, OH 61 Phon* lt69<iSS FAX 41M 664H A A u tw ttfM W o i dupuuuon d this pen. m an* *>*<. h m ttn n a ta rrin g NAmS*' w rthn shxsw iI. |Piige;2 o f 7 jJ TUO13-800 HLO-1 9 9 7 -0 0 0 2 6 Lab No. 96T 16836 00 SUMMARY The test article, H-22240 (test item). H-22240, was extracted in 0.9% sodium chloride USP solution and cottonseed oil, NF. These extracts were evaluated for intracutaneous toxicity in accordance with the guidelines of the current USP. A 0.2 ml dose of the appropriate test article extract was injected by the intracutaneous route into five separate sites on the right side of the back of each rabbit. Similarly, the corresponding blank vehicle was injected on die left side of the back of each rabbit. Two rabbits were used for each pair of extracts. Observations for erythema and edema were conducted at 24, 48, and 72 hours after intracutaneous injection. Under the conditions of this study, there was no evidence of significant irritation or toxicity from the extracts injected intracutaneously into rabbits. Each test article extract met the USP requirements. Study and Supervisory Personnel: Donald P. Barda, LAT Michael A. Safran, AAS (HT) Patricia Mitchell Barbara M. Monroe Darcy Irans Cindy Harper Tina M. Cox, BS, LAT Approved by: ! iIi Steven J. Henhansky, PharmD, PhD^JABT Manager, Toxicology ( 1 * '.(Uu m Ih * ' 2 3 , i % Date Completed Company Sanitized. Does not contain TSCA CB! MinS - 23 World Leader in Testing Services fo r the Medical Device Industry 2261 Tracy Road FNPAhooXrtnhawood44.119O9**H86664449496551459 A u to m ation tor d u ra tio n of this raport. a ic a p l In to la . m a tvad p a n d ty NAm SA'a wnttan approval. Page 3 o f 7 TUO13-800 HLO-1 9 9 7 - 0 0 0 2 6 Lab No. 96T 16836 00 INTRODUCTION The test article identified below was extracted and the extracts were evaluated for biocompatibility in artmAanr* with the guidelines of the current USP. The purpose of the study was to determine whether lennhahli yxlrarffd from the material would cause local dermal irritant or toxic effects following injection into rabbit Hn The test article was received on December 5, 19%. The animals were injected on December 18, 1996, and the observations were concluded on December 21, 19%. MATERIALS The sample provided by the sponsor was identified and handled as follows: Test Article: H-22240 (test item) Identification No: H-22240 Storage Conditions: Vehicles: Preparation: Room temperature 0.9% sodium chloride USP solution (SC) cottonseed oil, NF (CSO) A ratio of 120 cm2:20 ml (surface area of test article to volume o f vehicle) was used for each preparation. The test article was extracted in SC and CSO at 121 C for 1 hour. The extraction vehicles without test article were similarly prepared to serve as control blanks. Condition of Extracts: Test SC: clear CSO: clear Control clear clear Sample Disposition: Test System: Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: Justification of Test System: Any remaining sample was discarded. METHODS Rabbit (Orvctolagus cuniculusl New Zealand White Myrtle's Babbitry, Inc. No particular gender was prescribed for this test No particular body weight range was prescribed for this test No particular age was prescribed for this test Minimum 2 days Two per pair of extracts Ear tag or tattoo The intracutaneous injection test in rabbits is specified in the current USP and has been used historically to evaluate biomaterial extracts. Animal Management: Husbandry: Food: Conditions conformed to Standard Operating Procedures which are based on the "Guide for the Care and Use of Laboratoty Animals." PROLAB High Fiber Rabbit Diet was provided daily. Company Sanitized. Does not contain TSCA CBi M n iS ft - 24 World leader in Ttiiimg Servirti fo r the M edim i Device tndnitry PFN2Ah2o6oXr1tnhawTroaocdy44.1R199Oo44aH66d66*4-293946551459 AulhoniAUan for dupheaton of ft rspon. xcapf m ta b . is f aarv id pandtog NAm SA's wnttart approval. Page 4 o f 7 T U 0 13-800 HLO-1 9 9 7 - 0 0 0 2 6 Lab No. 96T 16836 00 Water: Contaminants: H oeing: Freely available, municipal (Toledo, OH) water w e delivered through an automatic watering system. Reasonably expected contaminants in feed or water supplies did not have the potential to influence the outcome o f this test. Animals were individually housed in stainless steel suspended cages identified by a card inHirating the lab number, animal number, test code, sex, and date dosed. Environmental: The room temperature was monitored daily. The temperature range for the rabbit was 64-74F. The room humidity was monitored daily. The humidity range for the rabbit was 30-70%. The light cycle was controlled using an automatic timer (12 hours light, 12 hours dark). Facility: Personnel: Selection: NAmSA is an AAALAC accredited facility. Associates involved were appropriately qualified and trained. Only healthy, thin-skinned animals free o f mechanical irritation or trauma that could interfere with the test were selected. Experimental Procedure: Prior to treatment, each rabbit was clipped free of fur from the back and both sides of the spinal column to yield a sufficient injection area. Two rabbits were prepared per pair of extracts. A 0.2 ml dose oftest article^ extract was injected intracutaneously into five separate s ite on the right side o f theback of each rabbit; 0.2 ml of the control was injected into five separate sites on the corresponding left side of the bade. Injections were spaced approximately 2 cm apart. The animals were returned to their respective cage following the procedure. Observations for erythema and edema were conducted at 24, 48, and 72 hours after injection. Reactions were scored on a 0 to 4 basis. Any reaction at the injection s ite w e also noted. The reactions were evaluated according to the following subjective rating scale: ERYTHEMA (ER) 0 No erythema 1 Vety slight (barely perceptible) 2 Well-defined (pink) 3 Moderate to severe (red) 4 Severe (beet redness) to slight eschar formation (injuries in depth) EDEMA (ED) 0 No edema 1 Very slight (barely perceptible) 2 Slight (edges of area well-defined by definite swelling) 3 Moderate (raised approximately 1 mm) 4 Severe (raised more than 1 mm, may extend beyond the area of exposure) The cumulative average erythema and edema score for each t e t extract and corresponding control blank w e Vaiiiatwt For each extract, a difference in average scores ( t e t minus control blank) of 1.0 or less w e considered to be acceptable. A difference of 0.6 to 1.0 indicated a slight, but acceptable, reaction. A difference > 1.0 w e considered to be unacceptable. Additionally, the average score for each t e t extract and blank w e ^im larpd for each interval. Any adverse reaction noted in the test extract w e compared to the corresponding blank. M Does o n ia 'n eo tn p atW iiiSA - 25 World leader in Teiiing Services fo r tke Meditai Office Industry 2NPF2Aho6oXr1tnheTwroaocdy44.1R19oO9*a*6H6d666*4*293946551459 A uto risatio n for dupfacation of this report, eccep in to la, la reserved p e n d io N Am SA't w rtten approve Page 5 o f 7 Results of scores for individual rabbits appear in Table I. The findings are summarized below: 1 Extract 1 SC 1 CSO Average Test (-) Average Control = Difference == Assessment 0.0 0.0 0.0 Passed 1.0 1.0 0.0 Passed The difference in average scores was considered acceptable. The difference between test and control average scores did not exceed 1.0 at any observation interval. Results and conclusions apply only to the test article tested. No further evaluation of these results is made by NAmSA. Any extrapolation o f these data to other samples is the responsibility of the sponsor. CONCLUSION Under the comfit"" of this study, there was no evidence of significant irritation or toxicity from the extracts injected intracutaneously into rabbits. Each test article extract met the USP requirements. RECORD STORAGE All raw tat pertaining to this study and a copy of the final report are to be retained in designated NAmSA archive files. ! not contain TSCA CB* Company Sanitized. Does Page 6 o f 7 NAmSA - 26 World U nder in Testing Services fo r llse MedicoI Dewitt Industry NPF22Aho6oXr1tnhewT oreody44.1R19Oo94a4H6d666*4*923496551456 AtXhoUM Bort (or duptteaticn of (hit report, accept in hol . rn i*e<1pending NAmSA* wrM vt approval TUO 13-800 H L O -1997-00026 Lab No. 96T 16836 00 TABLE 1 1ISP INTRACUTANEOUS TOXICITY OBSERVATIONS RABBIT NUMBER 99555 99557 99555 99557 EXTRACT SC sc CSO CSO TEST EXTRACT Scoring Interval (Hours) 24 48 72 ER ED ER ED ER ED 0 00 0 00 0 00 0 0 0 1 11 1 11 1 11 1 1 1 CONTROL BLANK Scotin g Interval (Hoius) 24 48 7 2 ER ED ER ED ER ED 0 000 00 0 <) 0 0 0 0 1 111 11 1 111 11 ER = Erythema ED = Edema SC = 0.9% sodium chloride USP solution CSO = cottonseed oil. NF Company Sanitized. Does n o t contain TSCA CBl M m SA - 27 World leader in Telling S e n ire i for Ihr Meditai Derice M a u r y 2NPF2Aho6oXr1tnhwTroaocdy44.1R199oO**a6H6d66664**923496551459 Authorizationtor duplication of this rport. axcapt m wtid. aiM arvad ponding NAm SA'a writtenapproval Page 7 o f 7 Confidential TU0I4-807 HLO-1 9 9 7 -0 0 0 2 6 Lab No. 96T 16836 00 P.O. No. LGX-593521 STUDY TITLE: USP MUSCLE IMPLANTATION STUDY IN THE RABBIT (1 Week) TEST ARTICLE: H-22240 (test item) IDENTIFICATION NO.: H-22240 Santtfced. Poes Com paq tSC A CBl SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK, DE 19714-0050 Northwood O H 43619 Phone 419*66*9455 PAX 419*66*3954 AuthorIzttion tor tfuphcainn of this report, except in whole, ts reserved pending N A m S A 't written epprovsl Page 1 o f 6 TUO14-807 SUMMARY ........... INTRODUCTION . . MATERIALS -----METHODS . . . . . . RESULTS ................. CONCLUSION . . . RECORD STORAGE TABLE OF CONTENTS H L O -1 9 9 7 -0 0 0 2 6 Lab No. 96T 16836 00 Page Number 3 4 4 4 6 6 6 I Company Sanitized. Does not contain TSCA CB1 - 29 - Worl L ta irr Ia Toiling S tr o ittl for Ihr U tilcal D trltt Indmlrf AKhom r t a 'Ionh iplicjn oooiim an pon . n e t mi F2NP2Aho6oXr1lnhwTroaocdy44.1R19Oo94a4H6d6884493946551459 pandtig NAmSAa n u n approval Page 2 o f 6 The test article. H-22240 (test item). H-22240. was implanted in muscle tissue of the rabbit. The nuucle tissue was evaluated for evidence of irritation or toxicity in accordance with the guidelines of the current USP. Implant samples and USP negative control strips were loaded into needles and sterilized by steam. Rabbits were implanted and were then euthanatized I. week later. Muscle tissues were excised and the implant sites were examined macroscopically. Under the conditions o f this study, the macroscopic reaction was not significant as compared to the USP negative control implant material. The implanted test article met the USP requirements. Study and Supervisory Personnel: Martha Oswanski. BS, LAT Merle J. Heineke, BS, LAT Jason A. Karsten Tina M. Cox, BS, LAT Approved by: Manager. Toxicology tim b e r 2 7 , M b Date Completed /mm Company Sanitized. Does not contain troa ''rit - 30 - World U a d tr in Ttsting Sw nirti fo r the Mrdicol Dtvicr Induiiry F2NPA2ho8oXr1tnhowTroaocdy44.1R19oO9-a46Hd66664**293946551450 Autom ation (or flmiraiion of ih it report, axeapt m wholo. it ratorvad parting NAmSA't wrttan approvai Page 3 of 6 HLO-1 9 9 7 - 0 0 0 2 6 Lab No. 96T 16836 00 INTRODUCTION The test article identified below was evaluated for biocompatibility in accordance with the guidelines of the !* dy w a r.0 e t a 0 potemW f o r . load M W or toxic rtsponrojo maiwial implanied i,, dOec. c o m with muscle tissue. The reel aniclc was r e e v e d 00 Decemhet X 1996. T h i " K m l l t a l e d on December 17. 1996. end muscles ere e,placed on December 24. 1996. MATERIALS The sample provided by the sponsor was identified and handled as follows: Test Article: H-22240 (test item) Identification No: Storage Conditions: Control Article: Preparation: Sample Disposition: H-22240 Room temperature USP plastic 1 mm diameter negative control reference strips purchased by NAmSA from the offices o f the US Phannacopeial Convention and cut into 10 mm sections. A "tinimnn. 0f six sections o f the test article per rabbit, each approximately 1 mm x 10 mm, were loaded into 16 gauge needles. The test article was a it into 1 mm x 20 mm lengths and then folded in half to obtain 1 mm x 10 mm sections. For each rabbit, a minimum o f four USP strips were loaded into the same size needles as used for the test article. Test and control articles uM>re sterilized bv steam. Any remaining sample was discarded. METHODS Test System: Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: Rabbit (Orvctolaeus cuniculus) New Zealand White Myrtle's Rabbitry, Inc. No particular gender was prescribed for this test 2.5 kg to 2.6 kg at implantation No particular age was prescribed for this test Minimum 2 days Two Ear tag or tattoo Justification of Test System: The rabbit is suwested as an appropriate animal model for evaluating polymer materials 2 ^ has been used historically recause the response to implanted material is easily graded and compared to a known negative control material. Animal Management: Husbandly: Conditions conformed to Standard Operating Procedures which are based on the 'Guide for the Care and Use of Laboratory Aninu ls." Food: PROLAB High Fiber Rabbit Diet was provided daily. tscacbi n0t contain Sanitized Does Company - 31 - HAiiiSA World Leader fa resting Services fo r Ik t Mrd u a l D trU t Industry 2NPF2Aho6Xrot1hnTwt roaocdy44.1R19Oo9a44Hd8668444349833194B Autom ation lor dupbcainn of thhi rapo, n a p * hola. rawtvod pondkig NAmSA'. raion approvai. Page 4 o f 6 TUO 14-807 Water: Contaminants: Housing: HLO-1 9 9 7 - 0 0 0 2 6 Lab No. 96T 16836 00 Freely available, municipal (Toledo, OH) water was delivered through an automatic watering system. Reasonably expected contaminants in feed or water supplies did not have the potential to influence the outcome of this test. Animals were individually housed in stainless steel suspended cages identified by a card indicating the lab number, animal number, lest code, sex, and date implanted. Environmental: The room temperature was monitored daily. The temperature range for the rabbit was 64-74F. The room humidity was monitored daily. The humidity range for the rabbit was 30-70%. The light cycle was controlled using an automatic timer (12 hours light, 12 hours dark). Facility: Personnel: Selection: NAmSA is an AAALAC accredited facility. Associates involved were appropriately qualified and trained. Healthy animals were selected. To reduce die number o f animals used for testing, andto comply with the directives o f the NAmSA IACUC, rabbits on this study may have been used previously in an unrelated test model. Any previously evaluated t o t or control articles did not cause a response in the animats. Complete history o f animd u ti e |u traceable in laboratory records. Animals used for previous evaluations are tdentuspd in the report. Experimental Procedure: The fur was clipped from the area over the paravertebral muscles and the rabbits we /e J a le d w i* acepromazine maleate (0.2 ml/kg dose). After the muscle area was wiped clean with 7 0 . paravertebral muscle was infiltrated with 1% lidocaine to achieve local anesthesia on both sides o f the vertebral column. - Needles containing te-t article were inserted into the muscle at a 45 angle. A and the needle was withdrawn, leaving the sample in the paravertebral muscle. Six test ^ 'c l e s ^ o n s were implanted in the right paravertebral muscle of each rabbit. In the opposite muscle, four USP negative control strips were similarly implanted. Rabbits were returned to their respective cages following the procedure. Rabbits were observed daily for general health. Body weights were recorded prior to implantation and at termination. At 1 week the rabbits were weighed and euthanatized by an intravenous injection o f a sodium based drug. The paravertebral muscles were dissected free and methodically cut to locate ^ J ^ sites and two o f the USP negative control sites in each rabbit. Capsule foimation or other evidence ofunmt" was scored using appropriate magnification and an auxiliary light source. Scores were assigned and recorded as follows: 0 No capsule, no adverse reaction (other than minimal hemorrhage) 1 Up to 0.5 mm capsule or reaction area 2 0.6 to 1.0 mm capsule or reaction area 3 1.1 to 2.0 mm capsule or reaction area 4 > 2 .0 mm capsule or reaction area n o t c o n ta in TSCA CB1 Company Sanitized. Does I BAmSff - 32 WarU U a i t t In T ilting Strrlets foe ih t M tiltm l D n itt Innttry 226t Tracy Road PFNAiolofXfnltewood44.1199O4*H66666*4943496551459 Aidhorttaaon tor duplication or dita report, eaeapt in vrtiola. la reaorvad pendei NAmSA's *iittan approval. Page 5 of 6 HLO-1 9 9 7 -0 0 0 2 6 TU014-807 Lab No. 96T 16836 00 jl .o.i; *a -- L"AVM>oe imcimconic scores for test implants were compared to average scores of the control sites. ,, minus control) was reearded as "not significant, 0.6 to 1.0 as a trace, 1.1 to 2.0 as align, . i w s .o as "moderate" and S 3 . 1 ^ 'marked." The requirements of the USP ^^ and control score averages (macroscopic) was not greater than 1.0. The requirements o fthe w e re n met iftfie difference betweenthe test and control score for two (or more) implant sites exceeded 1 for any anunJ implanted. KESVL33 g i TM . ! otw.rvatinns- All animals appeared clinically normal throughout the duration o f the study. Body weight dita for individual rabbits were considered acceptable. af r r v ir fWw.Tvatinns: The findings for the macroscopic evaluation are shown below. Rabbit 98944* 99122* Weishi (kg) Day 0 2.6 2.8 2.5 2.8 1 2 3 4 1 2 3 4 Average: Test 0 0 0 0 0 0 0 0 0.0 USP Negttive Control 0 0 0 0 0.0 Previous use history traceable in laboratory records. Macroscopically, there was no difference between the reactions of the test and control materials. This resulted in a macroscopic reaction of "not significant" tissue contact irritation. Results and conclusions apply only to the test article tested. No f^ rnev^ i"" ^ ^ ^ f UltS tsm M ieby NAmSA. Any extrapolation o f these data to other samples is the responsibility o f die sponsor. CONCLUSION Under the conditions of this study, the macroscopic reaction was not " f " ^ ^ " pared * ^ USP negative control implant material The implanted test article met the USP requirements. RECORD STORAGE Ail raw data pertaining to this study and a copy of the final report are to be retained in designated NAmbA archive files. ' .Company Sanitized. Does not contain TSwi C3 - 33 - W arii Leader in Tettimt S a n iert fo r l i t M etical Device lo ta tir j 2261 Tracy Rond Noftflwood, OM 43610 Phon# 419466-046S FAX 4194664064 lo i thira in t raranrarlA u tic rtu m i dupction cl p o il, c o p l Oiol#. poraXng N A m S A * n rttm ppprairal Page 6 o f 6