Document dYa65QX2KprDvdJ4xg3kEm34B

AR226-3061 study Title TOP Bioeompatibility Testing of H-22072, H-22073, and H-22074 Study Completed on October 4, 1996 rvmtractlnq Laboratory Morth American Science Associates, Xnc. 2261 Tracy Road NOrthwood, Ohio 43619-1397 for B. Z. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714 stu d y w p ttn t Susan A. MacKenzie, V..M.D Ph.D., D.A.B.T. Medical Research Project Np_. laboratory Project.JE Haskell Laboratory Report No. 907-96 Page 1 of 53 t Sanitized. Does not contain TSCA CBI Company S ubstance T e s ta i Svnonvnia /O ther_goe la : GENERAL I M F D M I 9 M Tet_SuhgfaMXgf> #3< H-22072 DuPont HLO 907-96 MmSA L ab. N o . : P h y s ic a l Form: P u rity : C om position: 96T -12210-00 Blue fa b ric Not provided by sponsor C ontam inants : CAS R e g i s t r y N o , ; s C om pany Sanitize JQ'S fiOt QZ 2- m DuPont HLO 907-96 QgNEKAL IHFOBMATIQW(Continued) Teat Substance 82 Substance Tested: Svnonvma/Other Codea: B-22073 rotosa Lab, No, : tei< sai_ g 2 as: P u g ity = QOWOQBtism-- 96T-12211-00 Blue fabric Hot provided by sponsor Contaminante CAS R e g i s t r y M o , ; Teg^ed: Svnonvms/other Codee: TeBt Substance #3 K-22074 -3- Company Sanitized. Dos DuPont HLO 907-96 NAmSA Lab. No,: Physical Form: 96T-12212-00 Blue-green fabric * 4 '^sffiipansp Sanitized. Does not contain TSCA CB! DuPont Central Research and Development Haskell Laboratory for Toxicology and Industrial Medicine DuPont HLO 907-96 October 4, 1996 DuPont Non-Woven Old Hickory, Tennessee DSP Biocompatibilitv Testing of H-22072. B-22073. and H-22074 Attached you will find reports from North American Science Associates, Inc. The results for the H-22072, H-22073, and H-22074 samples are: Test Suhnt.anoe H-22072 H-22073 H-22074 T ggt EffgVlEP Cytotoxicity DSP Intracutaneous (Aaarose Overlav) ___SS&______ CSS* Non-toxic Pass Pass Non-toxic Pass Pass Non-toxic Pass Pass SP Muscle Implantation (7 dav) NSC NS NS a Sodium chloride extract b Cotton Seed oil extract c Not significant I o N fa ir Susan A. MacKenzie, V.M.D. ,v Ph.D., D.A.B.T. Research Toxicologist Company Sanitized. D oes not contain TSCA CBI m 5 KAiiiSAWorld Leader in Tenting Services for the Medical Device Industry DuPONT HLO 907-96 2261 Tracy Road Northwood, OH 43619 Phone 419*666*9455 FAX 419*666*2954 LAB NO. 96T 12210 00 P.O. NO. LGX 592565 E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK, DE 19714-0050 ATTN: DR. SUSAN A. MACKENZIE ID NO. H-22072 CYTOTOXICITY - AGAROSE OVERLAY, SOLID Test Article: H-22072 (control item) Test Article Description: Sheet - 1 cm? piece 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 0.5 cm piece of 725871H as a positive control, and a 1.0 cm length piece of UP-1 as a negative control were placed on the solidified overlay surface. Following incubation for 1 24 hours, the culture was microscopically examined lor evidence of cell____ decolorization to determine die zone of cell lysis. Arty decolorized zone present was examined microscopically to confirm cell lysis. Score Nontoxic (N) Toxic (T) Observations . .1 Mnrmnl cell morpholozv in proximity to test sample. r.ih .iar death and degeneration associated with the area beneath the test sample and possibly extended beyond the perimeter of the test sample. Where s Eon, of lysis was observed, die distance from the edge of the sample to the edge of die zone was measured and reported in millimeters (mm). . C om pS!^/Sanitized. Does not contain TSCA CB1 Ub C o m p l e t e d _ 5 i i i r 3 k _ A pproved. - &~ MG03O-110 Page l of 2 A u th o riza tio n tor d u p lica tio n ol th is rep o n ex ce p t in w hole, is re s e rv e d p e n d in g N A m S A 's w ritten ap p ro val NAmSAWorld Leader in Tenting Services for the Medical Device Industry 2261 Tracy Road Northwood, OH 43619 Phone 419*666*9455 FAX 419*666*2954 LAB NO. 96T 1221000 CYTOTOXICITY - AGAROSE OVERLAY, SOLID Results: TestyControl Articles Scote Test Article Negative Control: UP-1 NAmSA Positive Control: 725871H 0 0 7 Conclusion: Comments: Regniti 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 die responsibility of the sponsor. The above test article was nontoxic for L-929 mouse fibroblast cells under the above described test parameters. Placed die 'bluest' side of fabric down on agarose. . . . _ All raw data pertaining to this study and a copy of the final report are to be retained m 1NAmSA archive files. Test Article Received: 9-4-96 Date Prepared: 9-10-96 Date Terminated: 9-11-96 Com pany Sanitized. D oes not contain TSCA CBI u . Completed_____ --_____ Approved--------------------fSce ----------------------------------- p.ge 2 of 2 ,,,,o n ,.,.. n dup lication W . NA" S A * Confidential TUO13-800 DuPont HLO 907-96 Lab No. 961 12210 00 ' P.O. No. LGX 592565 STUDY TITLE: USP INTRACUTANEOUS TOXICITY STUDY IN THE RABBIT (Extracts) TEST ARTICLE: H-22072 (control item) IDENTIFICATION NO.: H-22072 SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 8- - Com pany Sanitized. Does not c ontain T & S B l ? NAinSA World Laadtr in Tasting Servie*! fo r I k t Medical Davie* Industry 1 2261 Tracy Road Norlhwood. OH 43619 Ptiona 419466-9455 FAX 419466^954 A u U m iz a M n Iw dupuettlon of m u rapali, rapi In molt. Is r a w v a a pondmg N A n SA' rttu n p p ro w i TUO13-800 TABLE OF CONTENTS SUMMARY .............................................................................. INTRODUCTION...................................................................... MATERIALS ........................................................................... M E T H O D S................................................................................. RESULTS ................................................................................... CONCLUSION ......................................................................... RECORD STORAGE................................................................. TABLE I - USP INTRACUTANEOUS TOXICITY OBSERVATIONS D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12210 00 Paye Number 3 not contain TSCACBI c o m p a n y S a n it iz e d . D o e s - 9- WorU L lld tr In Tilting S tn lc tl fo r Ikt M id i'll D iw l'i Inquiry Noittiwood. OH 43619 Phono 419-666-9455 FAX 419488-2954 Aumortutlon to. duptcouon <4 M o port. .CW m hot., I. K v o d P * * * * N A m S A 'i n tt* .ppnw.1 Page 2 o f 7 DuPont HLO 907-96 TUO13-800 Lab No. 96T 12210 00 SUMMARY m - i . v u r m n am niracted in 0.9% sodium chloride USP solution and The test article, H-22072 (control item). H-27&I . toxicity in accordance with the guidelines cottonseed oil, NF. These extracts were evaluated for intracutaneous toxtcny in areTM* e of the current USP. A 0.2 ml dose o ,ih . -5 ^ J Z Z , w conducted a 2 . . and 72 h a d . a t e in n .n d a .o u . u . Unde, d . condition, of du, a n d ,. Utm no M dm . of 1 ^ 11 ^M edanandaneously into rabbits. Each tea article eslract m a d . USP requuemein*. Study and Supervisory Personnel: Donald P. Baida, LAT Karen D. Coburn, ALAT Barbara M. Monroe Tina M. Cox, BS, LAT I llC h J L ; ^ fvl-tu. voX/bi Timothy Jansen. BLS * Section Manager, Testing Services Q0 DaoCompleicd ias f - 10 - Qoffilpaity Sanitized. Does net contain TSC CBf Page 3 of 7 SAiiiSA World U td tr In Truing Strrleti for Ihr Mtdlcol D trict Indnllrj 2261 Trhcy Road Northwood. OM 43619 Phon 419-666*9455 FA X 419666*2964 A iiA n c t o a tio n d u o t e a t a t d * port. * * * " * * " ' P *"*" N* " SA '* * M '*n ` * m ` ' TUO 13-800 INTRODUCTION D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12210 00 The . .Cl. Ideh.iW b e ta . t o t & ! ! ? ' local deimal in or to ri. d f a n fellw o y n h b h jio .. Tb. * article was received on September 4. 1996. The animals were injected on September 10. 1996. and the observations were concluded on September 13, 1996. The sample provided by the sponsor was identified and handled as follows: Test Article: H-22072 (control item) Identification No: H-22072 Storage Conditions: Room temperature Vehicles: 0.9% sodium chloride USP solution (SC) cottonseed oil, NF (CSO) Preparation: A ratio of 120 cmJ:20 ml (surface area of test article to volume of vetocle) was used for each preparation. The test article was extracted in SC and CSO at 121C for 1 hour. The extraction vehicles without test article were similarly nrenared to serve as control blanks. Condition of Extracts: SC: clear with test article particulates CSO: dear with test article particulates Control clear clear Sample Disposition: Any remaining sample was discarded. METHQBS Test System: Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: ahhit fOrvctolagus cuniculus) ew Zealand White [ohican Valley Babbitry o particular gender was prescribed for this ttst o particular body weight range was prescribed for this test o particular age was prescribed for this test linimum 2 days wo per pair of extracts ap ta o a p tattflO Justification of Test System: The nirauuneous injection lew in rabbits is specified In .he ciment USP id has been used hisKwiedly to evaluate biomaterial extracts. A"imal Management: Husbandry: Conditions conformed to Standard Operating Procedures which are based on the 'Guide for the Care and Use of Laboratory Animals." Food: PROLAB High Fiber Rabbit Diet was provided daily. Com pany S anitized. Does not contain TSCA CB1 Page 4 of 7 U - = ---- KAmSA World Lender In Tilling Strticti or ld i Mdical D a rin Industry AuBw raM on 10. d M * <* I** "*> " S ' " * N Am S* '` 2261 Tracy Road Normwood. OH 43610 Phon 410*666*9455 PAX 4 1fH66*2054 TUOI3-800 Water: Contaminants: Housing: DuPont HLO 907-96 Lab No. 96T 12210 00 Freely available, municipal (Toledo. OH) waier 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 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 10-70%. 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 a s - A 0.2 ml dose of tt* article extract was injected intracutaneously into five separate sites on die right side of the backof each rabbit, 0.2 ml of the control blank was injected into five separate sites on the corresponding left side of * e 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 sliglit (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 perc^otible) 2 Slight (edges of area wui-defu*ed by definite swelling) 3 Moderate (raised approximatr*.; 1 n_n) 4 Severe (raised more than 1 mm, may extend beyond the area of exposure) 1 I rhe cumulative average erythema and edema score for each test extract and coree^ondmg cramol blnk was alculated. For each extract, a difference in average scores (test minus control blank) of 1.0 or less was nsidered to be acceptable. A difference of 0.6 to 1.0 indicated a slight, but acceptable, reaction. A____ iifference > 1.0 was considered to be unacceptable. Additionally, the average score for each test extract and flank was calculated for each interval. Any adverse reaction noted in the test extract was compared to the rresponding blank. - 12 - Somoanv S anitized. Does n o t contain TSCA CBi 1 - Page 5 o f 7 K A i i i S f f World Ltadtr In Tilting Sinici* fo r t k t Medieoi D o lic i IndmUry 2261 Tracy Road Nodhwood. OH 43619 Phone 419466*9455 FAX 419466*2954 Authortuttoi lor Oi^dciDon ol ml* 'port. topi m nolo, a h m m I po'*" NAmSA* wrtinn approvtl Vf> TUO 13-800 D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12210 00 RESULTS Results of scores for individual rabbits appear in Table I. The findings are summarized below: I sc B cso Average Control Difference == Assessment 0.0 0.0 0.0 Passed 1.5 1.7 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 c v jd ^ io n ^ R e n a u lt is made by NAmSA. Any extrapolation of these data to other samples is the responsibility of the sponsor. CONCLUSION Under the conditions of this study, there was no evidence of s ig n if i t a m imtarion or toxicity from die extracts injected intracutaneously into rabbits. Each test article extract met the USP requirements. 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. Company Sanittzsd. Doss not c - 13 NAmSA W a r li L t o d ir in Toittm t S o rtie rt [ o r Ih r M e d ico ! D o tie r Im d attrj 2261 Tracy Road N orthw ood. O H 4 3 6 19 Phon 419666*9455 FAX 419666*954 A u ttio rtu ttc I dupfacatkjn <D tto. " p o rt, c a p i In Koto. I> noareoO p n k i g N A m S A '. w r i i a p p im a l Page 6 TUO 13-800 DuPont HLO 9 0 7 - 9 6 Lab No. 96T 12210 00 TABLE 1 iicp in tra c u ta n eo u s t o x ic it y OBSERVATIONS R A B B IT N UM BER 97209 omns 97209 EXTRACT SC SC CSO CSO TEST EXTRACT CONTROL BLANK Scoring Interval (H ours) Scoiin g Interval (H oiu s) 24 48 ER ED ER ED 0 00 0 0 00 0 2 22 1 2 22 1 72 1 2i1 ER ED ER ED 00 00 11 11 00 0_ 0 22 22 48 ER ED T2 ER ED 0 0 00 0 0 00 2 12 1 2 1 Li- 1 ER = Erythema ED = Edema SC = 0.9% sodium chloride USP solution CSO = cottonrced oil, NF Com pany Sanitized., Does not cerne: ' - 14 - 2201 Tracy Road Northwood. OH 43619 K AinSA World Leadir in Tailing Stnricai or Ikt M tiical D rain Indulto phono FAX 419*660*9455 4194664954 uvod ponefing NAmSA'a wrXlan approval Autf.ora.Mn lor d u clcilo n d IhU upon. o u a p l * < ' Page 7 o f 7 Confidential TU014-807 D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12210 00 P.O. No. LGX 592565 STUDY TITLE: USP MUSCLE IMPLANTATION STUDY IN THE RABBIT (1 WEEK) TEST ARTICLE: H-22072 (control item) ID'JULL3[CATION NO.: H-22072 SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 Company Sanitized. Does ' 2261 Tracy Road Northwood. OH 43619 SSAiiiSA World lAadtr in Ttttlnfi Strricri fo r Ih* Mrdlcnl D triet Industrf Phona FAX 419*666*9455 419*666*2954 . ^ i t m r v w t pvicttrkg N A m S A ' wrtRn approval. A ueiorttsbw t lor duplication ol th u pon , TU014-807 SUMMARY ............. INTRODUCTION . . . MATERIALS .......... METHODS ................ RESULTS .................. CONCLUSION -----RECORD STORAGE . OF CONTENTS D u P o n t HLO 9 0 7 - 9 6 ta b No. 96T 12210 00 Pllg*- N u m b e r 3 4 4 4 6 7 7 Com pany Sanitized. Does not contain TSCA CBI - 16 - 2261 Tracy Aoad NAiuSjI World Looior io T oltili Sonlcoi fo r tko M oilcot Dorico Imdmltry Northttood. O H 4361# Phon 419*666*9455 FAX 41*466-2954 Au^ofluSon(wd*#llon* fttoport. -O' ** " TM" 'v*d" rtn' 1N*mSA'' wnttan approval. Page 2 o f 7 TUO14-807 SUMMARY D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12210 00 'he test article. H-22072 (control item). H-22072, was implanted in muscle tissue o ftte ra to it. Themusde 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 " d WCTe S a n te d a n d 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 m 10 * * USP negative control implant material. The implanted test article met the USP requirements. Study and Supervisory Personnel: Christina M. Tolles Jason A. Karsten Paul A. Bell, HT(ASCP) Tina M. Cox, BS, LAT Approved by: Timothy J: Section M festing Services 9 - Z* - U Date Completed /mm /w .q - 17 - Com pany Sanitized. Does not contain TSCA CB! Page 3 of 7 NjI iiiS WorU U a itr la Turin Strtlcti fo r I k t M t d l f t D trlet Imdmstij 2261 Tracy Road Northwoad. O H 43619 Prion* 419*666*9455 FAX 419466*2954 Auttwttmtlon I dopdcnllon * W . repon. oPt m M n . ta rewnred p * ~ ig NAmSA'. m m m w * * * * ' TUO 14-807 INTRODUCTION D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12210 00 The test article identified below was evaluated for biocontpat.btl.ty m accordance T * current USP. The purpose of the study was to evaluate the potential for a local material implanted in direct contact with muscle tissue. The test article was reive* The were implanted on September 6. 1996. and muscles were planted on September 13. 1996. MATERIALS The sample provided by the sponsor was identified and handled as follows: Test Anide: H-22072 (control item) Identification No: H-22072 Storage Conditions: Control Article: Room temperature USP plastic 1 mm diameter negative control reference strips purchased by NAmSA from the offices of the US Pharmacopeia! Convention and cut into 10 mm sections. Preparation: A minimum of six sections of the test article per rabbit, each approximatdy 1mm x 1 0 mm. were loaded into 16 gauge needles. For each rabbit, a m i n im u m of four USP strips were loaded into the same sire needles as used for the test article. Test and control articles were sterilized by steam. Sample Disposition: Any remaining sample was discarded. METHODS Test System: Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: Babbit IQrvctolagus cuniculus) New Zealand White Mohican Valley Rabbitry No particular gender was prescribed for this test 2.7 kg to 2.8 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 suggested as an appropriate animal model for evaluating polymer materials by the current USP testing g liid e liS The muscletissue has been used historically because the response to unplanted material ts easily graded and compared to a known negative control material. Company Sanitized. Does not c o n f f f f i NAmSA' World U rd ir Im Toning Sonicoi fa r I k t Medical Dorico ladatlry 2261 Tracy Road Nortftwood. O H 43619 Phooa FAX 41 >666*9455 419666*2954 AugiortM lon fw duplication of this foport. oxcopt tfl vho lo . it ra a a rn pmdfciQ N SinSA't written cp p rw il. . TUO14-807 DuPont HLO 907-96 Lab No. 96T 12210 00 Animal Management: Husbandry: Conditions conformed to Standard Operating Procedures which are based on the 'Guide for the Care and Use of Laboratory Animals.' Food: Water: Contaminants: Housing: PROLAB High Fiber Rabbit Diet was provided daily. ' 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 die outcome of this test. Animals were individually housed in stainless steel suspended cages identified by a card [writrafifig the lab number, animal number, test 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 the number of animals used for tearing, andto comply with the directives of the NAmSA IACUC. rabbits on this study may have been used previously in an unrelated test model. Any previously evaluated to t or control articles did not cause a response in the animals. Complete history of animal usage is traceable in laboratory records. Animals used for previous evaluations are identified m the report. Experimental Procedure: m e fur was clipped from the area over the paravertebral muscles and the rabbits maleate (0.2 ml/kg dose). After the muscle area was wiped dean with 70% pmvertebnd muscle was infiltrated with 1% lidocaine to achieve local anesthesia on both sides of the vertebral column. Needles containing test article were inserted into the muscle at a 45 angle. A stylet was placed in the needle and the need!- was withdrawn, leaving the sample in the paravertebral muscle. Six test article sections were n L l , 7 Z * rabbit. I . the < w o ta mmcle. t a r USP * a. e COM* 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. Com pany Sanitized. Does not contain TSCA - 19 - K A m S A World Lender in Tilting Services fo r t k t Medicai Dewier Industry 2261 Tracy Road Nodhwood. OH 43619 Ph on 419-66*94$S FAX 41B-666-29S4 Autftortzban ior digiilcnuon of IN r*potl. cxcapt bt wbtol, is rvMtv^d pendino N>mSA'* writtbn approval. Page 5 o f 7 D uPont HLO 907-96 TUO14-807 Lab No. 96T 1221000 I At 1 week, the rabbits were weighed and euthanatized by an intravenous injection of a sodium pentobarbital based drug. The paravertebral muscles were dissected free and methodically cut to locate four of the test article sites and two of the USP negative control sites in each rabbit. Capsule formation or other evidence of irritation 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 Average macroscopic scores for test implants were compared to average scores of the control sites. Calculations were rounded off to the nearest 0.1. A Reaction Index difference of 0.0 to 0.S in scores (test minus control) was regarded as 'not significant,* 0.6 to 1.0 as a 'trace,* 1.1 to 2.0 as 'slight,* 2.1 to 3.0 as "moderate' and >3.1 as 'marked.' The requirements of the USP test were met if the difference between test and control score averages (macroscopic) was not greater than 1.0. The requirements of the test were not met if the difference between the test and control score for two (or more) implant sites exceeded 1 for any animal implanted. RESULTS Clinical Observations: All animals appeared clinically normal throughout the duration of the study. Body weight data for individual rabbits were considered acceptable. Macroscopic Observations: The findings fin- the macroscopic evaluation are shown below. 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 further evaluation of these results is made by NAmSA. Any extrapolation of these data to other samples is the responsibility of the sponsor. {ggmpgn^ Sanitized. Does not contain TSCA CBI _ 20 - Page 6 of 7 2261 Tracy Road NAm Sil% Noflhwood. OH 43619 World Loader in Totting Strvices Phone 419*666-9455 fo r the Medical Device Industry FAX 419466-2954 Authorization for duplication of this report, accept m whole, is reserved pending NAmSA'a written approval. M m S A WorldLeader in Tenting Servtcen for the Medical Device Industry D u P o n t HLO 9 0 7 - 9 6 2261 Tracy Road N orthw ood, OH 43619 Phone 419*666*9455 FAX 419*666*2954 LAB NO. 96T 1221100 P.O. NO. LOX 592S6S E. I. du loot de Nemours St Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 ATTN: DR. SUSAN A. MACKENZIE u jn o . ; CYTOTOXICITY - AGAROSE OVERLAY, SOLID Teat Article: H-22073 (test item) Test Article Description: Sheet - 1 cm? piece Procedure: A monolayer of L-929 mouse fibroblast cells was grown to canfluency sod overlaid with M inim um Essential Medium rupplemented witii serum, antibiotics, neutral red, and 2% agnose. The test article, a 0.5 cm x 0.5 cm piece o f 725871H as a positive control, and a 1.0 cm length piece of UP-1 as a negative control were placed on the solidified overlay surface. Following incubation for 24 hours, the culture was macroacopically examined for evidence of cell decolorization to determine the zone of cell lysis. Any decolorized zone present was **<--<t microscopically to confirm cell lysis. Score Toxic (T) Normal cell motphoiosv in proximity to test sample. feiiniar dararti and degeneration associated with the area beneath the test sample and possibly extended beyond die perimeter of die test sample. Where a 7nfw of lysis was observed, die distance from the edge of the sample to tfos edge of the zone was measured and reported in millimeters (mm). i 1 Company Sanitized. Does nolcontain TSCA CB! u> Completed Approved. - 22 - MG03O-110 Page 1 of 2 Authorization lor duplication ol this report, except in whole, is reserved pending NA m SA's written approval. NjIiiiSjIWorldLtadtr in Tenting Servirei for the Medical Device Induitry D u P o n t HLO 9 0 7 - 9 6 2261 Tracy Road Northwood, OH 43619 Phone 419*666*9455 FAX 419*666*2954 LAB NO. 96T 1221100 CYTOTOXICITY - AGAROSE OVERLAY, SOLID Teat/Cnniroi A rtel SSStB ^rOIKPf IrVHI (MB) T o t Article Negative Control: UP-1 NAmSA Positive Control: 725871H N N T 0 0 7 Reaulls aad ccocluaicas apply only to die teat article tested. No further evaluation of these m ulta if nade by NAmSA. Any extnpobtioa o f Aeae data to oOer ampies ia the responsibility of the apanaor. The above teat article waa bsbJsb S for L-929 mouse fibraMast cell under die above deacribed Ownmewt: Placed "Muest* side of fidiric down on agaioae. .. .. All raw data p**in"g to thii study and a copy of the final report are to be retained m A m m NAmSA archive files. Test Article Received: 9-4-96 Date Prepared: 9-10-96 Date Terminated: 9-11-96 Com pany Sanitized. Does not contain TSCA CB! Us Completed. A p p ro v e d __________(SajSU S-il - 23 - Page 2 of 2 Authorization tor duplication ot this report, except in whole, is reserved pending NA m SA's written approval Confidential TU013-800 D u P o n t HLO 9 0 7 - 9 6 Lib No. 96T 12211 00 P.O. No. LGX 592565 STUDY TITLE: USP INTRACUTANEOUS TOXICITY STUDY IN THE RABBIT (Extracts) H-22073 (test item) 3.CATION NO.: H-22073 D oes not contain TSCA CBI SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE 19714-0050 - 24 - SSAiiiSA WtrU Umir la T iltint S m in i f r Ik t U rtim i O n to * In im ilrj M t r tr ta t tor <M*ctloA rt W rapoil. asm * ,* im m d p trtn g NAm SA' 2261 Tracy Road N oftlm ood. O H 43619 Phone 41066644M FAX 4196664944 Page 1 o f 7 TU013-800 TABLE OF CONTENTS SUMMARY .............................................................................. INTRODUCTION........................................ ............................. MATERIALS ............................................................................ METHODS.................................................................... ............ RESULTS .................................................................................... CONCLUSION ......................................................................... RECORD STORAGE.................................................................. TABLE I - USP INTRACUTANEOUS TOXICITY OBSERVATIONS D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12211 00 Page Number 3 4 4 4 6 6 6 7 Com pany Sanitized. D oes not contain TSCA CBI - 25 - XAm SX W a rU Laadar Ut T ild n f S tr r lc a i f a r It* H t d iia l D arin h U m tlrj 2261 Tn e t Road Northw ood. OH 4361 PkO M 416666*455 FAX 4166662954 Align n il--fcni f-- c l (hi> fapoit aaoapl kt a4w*a. la rm arra pGndbiQ NAinSA'a M ila n approval. Page 2 o f 7 TUO13-800 SUMMARY D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12211 00 _ . - ,, vmvt'i riMt iiMni H-22Q73 was in 0.9% sodium chloride USP solution and fr h - -- - - H n -- * * ** e -- - of the current USP. a m TMi a m - n f th* anomoriate test article extract was injected by the intracutaneous route into five repantfe sit on the right side of back of each rabbit. Sitnilariy, the o o tre sp o n ^ blank v^ ^ faqeaed on S T s S of ie b a c k 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 K ar D. Coburn, ALAT Barbara M. Monroe Tina M. Cox. BS. LAT Approved by: Timothy Jans, BLS Sfytinn Manager, Testing Services CM Q -Q Co Date Completed 26 - BSAinSA WorU Ltadtr in Tttllug S tn ie tt fa r Ik t Utdicml D o rf tmdmttr, Aut ie im a on foe d u p ie tlla n of W o port. o tto n *> ppopany Sanitized. Does not contain TSC CB! 2361 Teoey Road Nofthw oo. O H 43619 Phono 4 1 M M 4 4 S S FAX 4194639S4 NAm SA'o n u n oppravol Page 3 o f 7 TU013-800 INTRODUCTION D u P o n t HLO 9 0 7 - 9 6 Lab N o. 96T 12211 00 -v - tid e was received on September 4. 1996. Theammals were injected on September 10.1996. and tbe observations were concluded on September 13, 1996. MATERIALS The sample provided by the sponsor was identified and handled as follows: Test Article: Identification No: Storage Conditions: Vefcides: H-22073 (test item) H-22073 Room temperature 0.9% sodium chloride USP solution (SC) cottonseed oil, NF (CSO) Preparation: A ratio of 120 cm2^ ml (surface area of test artide was used for * preparation. The test artide was extracted in SC and CSO at 121C for 1 hour. The extraction vehicles without test article were sim ilarly prepved to serve as control blanks. Condition of Extracts: Sample Disposition: SC: dear with test artide particulates CSO: dear with test article particulates Any remaining sample was discarded. METHQBS dear dear Test System: Spedes: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: ahhit fOrvctolam cuniculus) ew Zealand White [ohican Valley Rabbitry o particular gender was prescribed for th u te o particular body weight range was prescribed for this test o particular age was prescribed for this test linimum 2 deys wo per pair of extracts nr tap or tattoo Justification of Test System: The intracutaneous injection test in rabbits is specified in the current USP and has been used historically to evaluate biomatenal extracts. Animal Mqnayement; Husbandry: Conditions conformed to Standard Operating Procedures which are based on the 'Guide for the Care and Use of Laboratory Animals.* Food: ROLAB* High Fiber Rabbit Diet was provided daily. (tiomoanv Sanitized. D oes not contain TSCA CB1 - 27 - NAiiiSA W irU U m itr Im T tH if S trticu f n It* t t i t U i l D i t i " fit ia u r j t o d u e * * * <* M port. tn hole. M M d pm dhi* NAm S*1* wmm 2261 Tracy Ro*d Northwood. OH 43619 Phone 41*6864465 FAX 4194664964 Page 4 o f 7 TUO13-800 Water: Contaminants: Housing: Environmental: DuPont HLO 907-96 Lab No. 96T 12211 00 Freely available, municipal (Tolfedo. 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 ,,/Kiting the lab number, animal number, test code, sex, and date dosed. The room temperature was monitored daily. The temperature range for the rabbit was 64-74"F. 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 far from the back ^ SST aSde yield a sufficient injection area. Two rabbits were prepared per pair of ^ t s A O J m l #ose o f W fade extract w a fa'ected intracutaneously into five separate sites on the right side of the control blank was injected into five separate sites on the corresponding left side of the bade. Injections 2cm apart. T h e S l s were returned to their respective cages followmg the procedure. O b*TM , for c o t a . and to ctedtetad # 2 4 .4 8 , and 72 h ten .ate r injaednn. scored on a 0 to 4 basis. Any reaction at the injection sues was also noted. The reactions were evaluated according to the following subjective rating scale: ERYTHEMA (ER) 0 No erythema 1 Very 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) _ l* S u f o S teerval. Anyadverse ,, io n noted in Ite test act waa compared to the rresponding blank. Sanitized. D oes not contain TSCA CBI - 28 - * ,,. World L t a d t r in T o ttin g S*rrte*t fo r Ik* M t d it o i D*wie* industry 2261 Tracy Road Northwood, OH 43619 Phone 41*666-9455 fA X 41M66-2954 . Aum ertzstion to, ttu p tc lto r. o ' <M. p o o . o n e * * M o . ' " d ponding N A m S A 'o raton op p io . Page 5 of 7 TUO13-800 RESULTS DuPont HLO 907-96 Lab No. 96T 12211 00 Results of scores (r individual rabbits appear in Table l. The findings are summarized below The d a t a - ! . -- -- d-- scores did not exceed 1.0 at any observation interval. The M b e * Results and conclusions apply only to the test article testedNAmSA. Any extrapolation of these data to other samples No further evaluation of these results is the responsibility of the sponsor. is made by CONCLUSION RfCORD STORAGE All rate data peitaiiiliig to this study and a copy of the fiuti repon ate to be retained in designated NAtuSA archive files. Com pany S anitized. Does not contain TSCA CB1 - 29 - KAinSff World Lomdor in Tatimg S o n ia s _ _____ ________ f e r th Modicat D o r ia Industry Northwood. O H 43619 Phone 419*666*9455 FAX 419466*954 ___ _ ,, f t i i r v i l nandina N A m S A 's w rit* ppfOVRl Aulhortutiat tor dupfcSAtton of the* r*rt. * * tote, r*#*vna Page 6 of 7 TUO 13-800 D u P o n t HLO 9 0 7 - 9 6 Lab N o. 96T 12211 00 TABLE I 1. iNiTBArur AMFoiis TQXiriTY o bser v a tio n s ER = Erythema ED = Edema SC = 0.9% sodium chloride USP solution CSO = cottonseed oil, NF - 30 - lA m S A World Ldmitr in Trilliti Strrictt fo r I t i M tdlcal O trier lld m tlij Authofttaton loi duptcalloo d m u m a rt. *> " '**'v*a *anKed. Does not contain TSCA CBI Page 7 of 7 2261 Tracy Road Northwood. OH 43619 Phone 419466*9465 FAX 4194664964 u a m C A 'b written BOOrovai. Confdentia] TU014-807 D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12211 00 P.O. No. LGX 392565 STUDY TITLE: USP MUSCLE IMPLANTATION STUDY IN THE RABBIT (1 WEEK) TEST ARTICLE: H-22073 (test item) H-22073 SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours St Co. HASKELL LABORATORY ELKTON NEWARK - 31 contain TSCACBI Page 1 of 7 World Lsadir in Titling S i n i c a fo r tk i Mdical D ivici Industry Northwood. O H 43619 Ph on 419-666-9455 PAX 4104864954 Authorization for duplication of (M* roport. a p t in who!, it rtMvd pondinp NAwSA't m *ttn approval TUO14-807 SUMMARY .......... INTRODUCTION . . MATERIALS . . . . M ETH O D S............. RESULTS . . . -----CONCLUSION . . . RECORD STORAGE TABLE OF CONTENTS D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12211 00 Paf e Number 3 4 4 4 6 7 7 Ssm lized. D oes n o t contain I SCA CBI - 32 - ________ .. N A ih S jI World Leader in Truing S t n ic n fo r ike Medical Derice Indaitrf 2261 Tracy Hoad NOTtftWOOd. O H 43619 P hono 419-666-9455 FAX 4194664954 A u D w n u tia i iw d u p tcu o n <* thl* report. . c * X In a M a . It f#tt<vtcl (KWSnO N A m S M w rtttn tw ro v tl. Page 2 o f 7 TUO 14-807 SUMMARY D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12711 00 The test article, H-22073 (test item), H-22073. was implanted in nmscle tissue of ^ ^ b ^ I ^ imucle 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 implantedand were then euthanatized 1 week later. Muscle tissues were excised and the implant sites were examined macroscopteally. negative control implant material. The implanted test article met the USP requirements. Study and Supervisory Personnel: Christina M. Tolles Jason A. Karsten Paul A. Bell. HT(ASCP) Tina M. Cox, BS, LAT Approved by: 7 Timothy Jansen, Section Managt ting Services Date Completed /nun Sanitized. Does not contain TSCACB1 fMfl - 33 - SSiiiSA World Leader in Testing Services fo r Ike Medical Derice Industry 2261 Tracy Road Northwood. OH 43619 Ph on e 4 ` 9666*9d55 FAX 41fr666-29$4 A u ttxytu H a n tor d u p B C W . ot th l. port. o p H n h o * . I. M TM * * * '* '*>N A m S A '* w rtn,n t p 0 m t l Page 3 o f 7 TUO 14-807 D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12211 00 INTRODUCTION The test article identified below was evaluated for biocompatibility in accordance with the guidelines of the current USP. The purpose of the study was to evaluate the potential for a local irritant or toxic material implanted in direct contact with muscle tissue. The test article was received on September 4> 1996. The were implanted on September 6, 1996. and muscles were explained on September 13, 1996. MATERIALS The sarnie provided by the sponsor was identified and handled as follows. Test Article: H-22073 (test item) Identification No: Storage Conditions: Control Article: Preparation: H-22073 Room temperature USP plastic 1 mm ampler negative control reference strips purchased by NAmSA from the offices of the US Pharmacopeial Convention and cut into 10 mm sections. A mini.., of six sections of the test article per rabbit, each approximately 1 mm x 10 mm, were loaded into 16 gauge needles. For each rabbit, a of four USP strips were loaded into the same sb* needles as used for the test article. Test and control articles were sterilized by steam. Sample Disposition: Any remaining sample was discarded. Test System: Species: Breed: Source: Sex: Body Weigkt Range: Age: Acclimation Period: Number of Animals: Identification Method: Justification of Test System: METHODS Rabbit tOrvctolagus cuniculus) New Zealand White Mohican Valley Rabbitty No particular gender was prescribed for this test 2.8 kg at implantation No particular age was prescribed for this test Minimum 2 days Two Ear tag or tattoo .IntSC A C B l - 34 - NorthWOOd. OH 43619 P h o n s 419*606*9455 I B C T M M fo r ik t trdlcol Orfico Inuxtry FAX 419466-2954 dlAuthorization tot ducOcaUon th b p ort, . c o p i h o b . Io roootvod ponding N A m S A '. iritton op p to v.l Page 4 o f 7 TUO14-807 D u P o n t HLO 9 0 7 - 9 6 Lab No. S6T 12211 00 Animal Management: ' 1 Husbandry: Conditions conformed to Standard Operating Procedures which are based on the `Guide for the Care and Use of Laboratory Animals.* Food: PROLAB High Fiber Rabbit Diet was provided daily. Water: Contaminants: Housing: 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, test 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: NAmSA is an AAALAC accredited facility. Personnel: Associates involved were appropriately qualified and trained. Selection: Healthy animals were selected. To reduce the number of animals used for testing, and to comply with the directives of the NAmSA IACUC, rabbits on this study may have been used previously in an unrelated test model. Any previously evaluated test or control articles did not cause a response in the animals. Complete history of animal usage is traceable in laboratory records. Animals used for previous evaluations a it identified in the report. Experimental Procedure: The fur was clipped from the area over the paravertebral muscles and the rabbits were sedated with acepromazine maleate (0.2 ml/kg dose). After the muscle area was wiped clean with 70% alcohol, each paravertebral muscle was infiltrated with 1% lidocaine to achieve local anesthesia on both sides of the vertebral column. Needles containing test article were inserted into the muscle at a 45 angle. A stylet was piiced in the needle and the needle was withdrawn, leaving the sample in the paravertebral muscle. Six test article sections 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. --____ j n>e n o t contain TSCA, CBi - 35 - NAihSjI World Loader im Testing Services fo r the Medical Device Industry 2261 Trtcy Road N orlhwood. OH 43619 P h o n e 419*666*9455 FAX 41 <666*2954 Aultiortulttn lor duplication ol this report, eneepl n wnole. is reserved pending NAm SA1 *mt1n approvil Page 5 of 7 TUO14-807 D u P o n t HLO 9 0 7 - 9 6 l a b No. 96T 12211 00 * . t tlu, ,,Khirc were weiehed and euthanatized by an intravenous injection of a sodium pentobarbital r - follows: r s s s s - t s z i s z z z . -- 0 No capsule, no adverse reaction (other than minimal hemorrhage) 1 Up to 0.5 mm capsule or reaction area 2 3 0.6 l.i to to 21..00 mmmm capsule capsule or or reaction reaction area area 4 > 2.0 mm capsule or reaction area p-- -- Calculations were rounded off to the nearest . - t i to 2.0 as 'slight.* 2.1 to 3.0 as miM. motoace and 23.1a` X ^ o ta m e L of tte USP lea wm met If thedifferenceIMweenteat n*mrd*i IjO. Heletpdtemetilsofdmteatwetenotmet d S ^ c o L f s. for wo (or mo*) imp., aim. implanted. RESULTS fn -t-t ah animals appeared clioically normal ibrougbom tbe duratioo of Ibe udy. Bod, weight data for individual rabbits were considered acceptable. Previous use history traceable in laboratory records. Macroacopically, mere was no difference between dm teacdona of dm ma, and eondol mmedala. Tbit -- M In iS ro a c o p ie reaction of 'n o . aifnide- daane eondW a . . . - 36 - HAm SA World Loadtr in TtUimi S irrlcti _ __ fo r Ih t Medico/ Derice ! odoury ' Z Z Z Z u * d i.p l . > of M . port. ( * ta M ompany Sanitized. Does not contain TSCA CBl Page 6 of 7 2261 Tracy Road Northwood. OH 43619 Ph on e 419*666*9455 FAX 419-666*2954 P M S NAm SA-. * " XjIiiiSAWorld Leader in Testing Services for the Medical Device Industry D u P o n t HLO 9 0 7 - 9 6 2261 Tracy Road Northwood, OH 43619 Phone 419*666*9455 FAX 419*666*2954 LAB NO. 96T 1221200 P.O. NO. LGX 592565 E. 1. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK, DE 19714-0050 ATTN: DR. SUSAN A. MACKENZIE ID NO. H-22074 CYTOTOXICrrY - AGAROSE OVERLAY, SOLID Test Article: H-22074 (test item) Test Article Description: Sheet - 1 cm2piece 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 0.5 cm piece of 725871H as a positive control, and a 1.0 cm length piece of UP-1 as a negative control were placed on the solidified overlay surface. Following incubation for 24 hours, the culture was macroscopically examined for evidence of cell decolorizalion 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 ceU moroholouv in proximity to test sample. _ r"v|itiinr Hmnth and degeneration associated with the area beneath the test sample and possibly extended beyond the perimeter of the test sample. Where a 7npa of lysis was observed, the distance from the edge of the sample to the edge of the zone was measured i npany Sanitized. Does not contain TSCA CBI la s Completed 1^ Approved. - 38 - MG030-110 Page 1 of 2 Aiit'Hiii/ation lor rjuplniiitto.. m mn. report. wm tn whole, is reserved ponding N A m S A s written approval P World Leader in Tenting Services, m m SX for the Medical Device Industry DuPont HLO 907-96 2261 Tracy Road Northwood, OH 43619 PhO' ? 419*6669455 FAX 419*666*2954 LAB NO. 96T 1221200 Results: CYTOTOXICITY - AGAROSE OVERLAY, SOLDO TestyControl Articles Scote 7rme nf l ysi ftnnri Test Article Negative Control: UP-1 NAmSA Positive Control: 72587IH N N T Conclusion: Results and concluaiona 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 of the sponsor. The above test article was nontoxic for L-929 mouse fibroblast cells under the above described test parameters. Comments: Placed the "tealest* side of test article down on agarose. _ 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: 9-4-96 Date Prepared: 9-10-96 Date Terminated: 9-11-96 wnpany Sanitized. Does not contain TSCA ( I Ut Completed. Approved. -39- (See pace 1) Page 2 of 2 Aullirjri/alio.i for dupHc.il,on ul this repurl. e x c v l whole. ,s reserved ponding N A m SA's wrrtten approval Confidential TUO13-800 D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12212 00 P.O. No. LGX 592565 STUDY TITUS: USP INTRACUTANEOUS TOXICITY STUDY IN THE RABBIT (Extracts) TEST ARTICLE: H-22074 (test item) IDENTIFICATION NO,: H-22074 SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont cte Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX 50 NEWARK. DE Northwood. OH 43619 Ph on 419*666*9455 FAX 419666-2954 Authortutton I dupScelon al mia port. * * m whols. I v o i paroSng NAmSA'a written approvel. TUO13-800 TARI E OF CONTENTS SUMMARY .............................................................................. INTRODUCTION...................................................................... MATERIALS ............................................................................ METHODS................................................................................. RESULTS .................................................................................... CONCLUSION ......................................................................... RECORD STORAGE.................................................................. I . USP INTRACUTANEOUS TOXICITY OBSERVATIONS D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12212 00 Page Number 3 4 4 4 6 6 6 7 gggotgMmgfSanitized. Does not contain TSC& OBI KAiiiSA* World Ltadtr in Totting S tn ictt fo r t k t Modtcnl Doolto In d n tu j Au9xxtt, w (w <hp*con d port. * * * * * raW Y8d NAm SA'* *PProV* ' Page 2 o f 7 TUO13-800 SUMMARY D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12212 00 . . . . OTM' /._ h rtirJA was in 0.9% sodium chloride USP solution and IS S Z Z J F K E S ? -- L * of the current USP. -- -- * - * 1 *" *"" * , , , . ___ . .. .,, .I. eitrac. was injected by the intracutaneous route into five separate 'Z ^ J S ^ % T n * c n t> 2 4 .4 8 , and 72 to u * - I-- -- - je c m m . U o ta d . r i U ~ of this study, (here was no " " injected intracutaneously into rabbits. Each test article extract met the USP requirements. Study and Supervisory Personnel: Donald P. Baida, LAT Karen D. Cobum, ALAT Barbara M. Monroe Tina M. Cox. BS, LAT .. Approved by: IIVia L to . IsJ* - Timothy f a i U 'i T T * Section M anner, Testing Services Q -)Q - |lc O m an** gpm poqf Sanitized. Dc-^s /las - 42 - KAiiiSA World U adtr In Titling S tn lc tt fo r Iht H idlcal D trict M a u r y 2261 Tracy Road Northwood. OH 43619 P h o n e 419*666*9455 FAX 419*666*2954 ( ,, . a n c l part. d.P< m W .. rcccrroC pending N M n S * . m lm nppmvnt. .CE Page 3 o f 7 TUO 13-800 INTRODUCTION D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12212 00 lo c - 'd i-- 1 im * or w k r f t e . following 1. a* articlewas received on September 4. 1996. T h e rm a ls were mjected on September 10, 1996, and tbe observations were concluded on September 13, 1996. MATERIALS The sample provided by the sponsor was identified and handled as follows: Test Article: Identification No: Storage Conditions: Vehicles: Preparation: Condition o f Extracts: H-22074 (test item) H-22074 Room temperature 0.9% sodium chloride USP solution (SC) cononseed oil, NF (CSO) A ratio of 120 cm':20 ml (surfacearea of test tideijc was used for each preparation. The test article was extracted m SC and CSO at 121C for 1 hour. The extraction vehicles without test article were similarly nrepared to serve as control blanks. SC: <)with test article particulates CSO: clear with test article particulates Control clear clear Sample Disposition: Any remaining sample was uiscarded. METHODS Test System: Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: labbit fOrvctolagus cuniculus) Mew Zealand White tfohican Valley Babbitry Mo particular gender was prescribed for this test Moparticular body weight range was prescribed for this test Mo particular age was prescribed for this test Viburnum 2 days rwo 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 historically 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: PROLAB High Fiber Rabbit Diet was provided daily. Pgmjjjansf Sanitized. Does not contain TSCA CBS - 43 - KAniSA World U a d tr la T rilla i S m i n i fo r Ihr M tdicaI D tiic t In d o lir, 226' T ilcyR ud ' t'OflhWOOd. OH 43619 Pt.on 419466*94SS FAX 419466-29S4 Auihonullon lot dup&eatton rf'Di* repon, oicepl In noi., a m a n a d pamSng NAmSA'i reren .o p ro v .l Page 4 o f 7 TUO 13-800 Water: Contaminants: Housing: Environmental: D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12212 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 imtirating the lab number, animal number, test code, sex, and date dosed. 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 free of mechanical irritation or trauma that could interfere with the test were selected. c * rn "T m fl Pmrftfh>re: Prior to treatment, each rabbit was clipped free of fur from the back and both sides of the spinal yield a sufficient injection area. Two rabbits were prepared per pair of A 0 2 ml tom o ften te extract was injected intracutaneously into five separate sites on the right side c ffo c totk of the control b*"k was injected into five separate sites on the corresponding left side of the back. Injections w e r e 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 hoursafter 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) No eiythema Very slight (barely perceptible) Well-defined (pink) Moderate to severe (red) Severe (beet redness) to slight eschar formation (injuries in depth) EDEMA (ED) 0 No edema 21 Very slight (barely perceptible) Slight (edges of area well-defined by definite swelling) 3 Moderate (raised approximately 1 mm) 4 Severe (raised more than 1 mm, may e x te ^ b e ^ o n ^ h e a re ^ o ^ x g o ro re ^ ^ rhe cumulative average erythema and edema score for each test extract and corwponding control!ImJ alculated. For each extract, a difference in average scores (test minus control blank) of 1.0 or less was nsidered to be acceptable. A difference of 0.6 to 1.0 indicated a slight, but acceptable, reaction. A lifference > 1.0 was considered to be unacceptable. Additionally, the average score for each testextrart and ilank was for each interval. Any adverse reaction noted in the test extract was compared to the Responding blank. M - 44 - u d i World Loader iff Totting Sorvieot fo r the Mdical Device Indmttrj 2261 Tracy Road Northwood. OH 43619 Phono 41946*4455 FAX 419486*29*4 Authorization tor duplication of m u raport. axcapt In whola. la raaarvod ponding NAmSA'a wridan approval cm Page 5 o f 7 D u P o n t HLO 9 0 7 - 9 6 TUO13-800 Lab No. 96T 1221200 RESULTS | Results of worea for individual nbbitt appear in Table 1. The finding, are summarized below: th e difference in average scores was considered acceptable, The difference between test and control average scores did not exceed 1.0 at any observation interval. k> m l tn the test article tested. No further evaluation o f these results is made by rnuC L U S IO N i n S d intracutaneously into rabbits. Each test article extract met the USP requirements. All raw dar. Tenanting to ds study and a copy of the final tepon ire to be retained in detipuied NAmSA archive files. Company Sanitized. Does not contain TSCA CB1 - 45 - ^ HAiiiSA , W # rU Iid * r to aTM ' f , r t k t M titc m l D t r U i M m t t r j 2261 Tract Rood Northwood. OH 3618 Phono 41M664455 FAX l i M U i t i d d Ihb m ood. u M 0 m w hoU . b ro soive d pstxSng N A m S A 'i wrtnon Ppnwot. Page 6 o f 7 TUO13-800 D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12212 00 TABLE 1 USP 1NTRACUTANEOUS TOXICITY OBSERVATIONS RABBIT NUMBER 97215 97216 97215 97216 TESTEXTRACT Scoring Iomvxl (Houn) CONTROLBLANK Seulringbanval (Huon) EXTRACT 24 48 ER ED ER ED 72 ER ED 24 ER ED 48 ER ED 72 ER ED SC 0 0 0 0 0 0 0 0 0 0 sc 1! 0 0 0 0 0 0 0 0 0 0 0 cso 2 2 2 1 1 1 2 2 2 1 1 2 cso 1 2 2 2 2 112- 2 2 1 2 1 = Erythema = BiIhim a 0.9% sodium chloride USP solution = cottonseed oil. NF gs es .Company Sanitized. Does not contain TSCA CBI - 46 - HAiiiSff World L rtitr Ik T tulnt Strrlcti fo r tk t U tiic a l D rrict Ixdmtlry 2261 Tracy Road N oilhw ood. O H 43819 Pfiond 4 1 M 6 6 4 4 S 5 FAX 41M 6849S4 AuBierta i ln i 1 dupteulon of ml port, n e e d Ko*, la raaaivad land ing N A m SA 'i wrttan approval. Page 7 o f 7 Confidential TU014-807 D u P o n t HLO 9 0 7 - 9 6 Lab No. 96T 12212 00 P.O. No. LGX 592565 STUDY TTTLE: USP MUSCLE IMPLANTATION STUDY IN THE RABBIT (1 WEEK) TEST ARTICLE: H-22074 (test item) u Mea*ia CATION NO.: H-22074 SPONSOR: DR. SUSAN A. MACKENZIE E. I. du Pont de Nemours & Co. HASKELL LABORATORY ELKTON RD. P.O. BOX SO | 1 "* * * * R S w w m i z , * . Does not contain TSCA CBi Page 1 of 7 SBAniM WtrU Ltmdtr In Tfttlmg StrrU n f i r tk i H i U il D irle* M u t r y 2261 Tree? Road Worthwood, O H 43619 Pftoo* 4194664455 FAX 4194664954 A tiiT im -- rw tar dupScettan of W a raped, e n a p t in dieta, la raaaivad pandfco NAmSA*a "ttan aoprw al. TUO14-807 SUM M ARY.......... INTRODUCTION . . MATERIALS ____ M ETH O D S............. RESULTS ............... CONCLUSION . . . RECORD STORAGE TABLE OP CONTENTS D uPont HLO 907-96 U b No. 96T 12212 00 7 Com pany Sanitized. D oes not contain TSCA CB| - 48 - M iiiS J t hWmrULsmdtr Tnllhi Stnrtta fmr Ikm k!miltml Dtvlcm tmimltry 22*1 Tracy R a M Nanm raod. OH m i b Ph ew FAX AuBwOuBai h i dupktllan ol M ( npad. n p l h MM. MMMnwd p v * MAmBAI wMn Bppioml. Page 2 of 7 TU014-807 D uPont HLO 9 0 7 - 9 6 Lab No. M 7 1221200 SUMMARY The test ankle, H-22074 (test item), H-22074. waa implanted in muick tiaaue o f the rabbit. The Hade tiaaue waa evaluated for evidence of irritation or toxicity in accordance with the guidelines of the current USP. Implant lamplea and USP negative control itripe were loaded into needka and steriliad by amain. Babbits wane implanted and were diea euthanatized 1 week later. Muacte ttaauea were eadaed and the implant abca men mmantimii njacroscopicaUy. . Under the conditions of thia atudy, the macroacopic rtactko waa not ai(Biflcant aa compared to the USP negative control implant material. The implanted teat aitick met the USP requirements. Study and Superviaory Penotm d: Christina M. Tollea Jaaon A. Karaten Paul A. Bell, HT(ASCP) Micfead A. Safton, AAS (HT) Tina M. Cox, BS. LAT Approved by: /mm - 49 - M m SJK W trld L n it r (a Ttuim g S t r ic t ! f t t k t M ttic m l D t ic t lu d m u rf 2 2 ft Tracy Road N orthaood. OH N il Phen FAX t iM M M I I <11 m 1184 NAmaA'tA tlh o rka to n lo r duptem on d tu* n p e it a n bi a M A m a n a a panda a a m u p fo M l. Page 3 of 7 The test snide identified below was evaluated for biocompatibility in accordance with the guidelines of the current USP. The purpose of the study was to evaluate the potential for a local irritant or toxic response to material implanted in direct contact with muscle tissue. The test article waa tecdved on September 4,1996. The animals were implanted on September 6,1996, and muscles were explained on September 13,1996. MATERIALS The sample provided by the sponsor was identified and handled as follows: Test Article: H-22074 (test item) Identification No: H-22074 Stonge Conditions: Room temperature Control Article: USP plastic 1 mm diameter narrive control reference strips purchased by NAmSA from the offices o f the JS Pharmacopeia] Convention and cut into 10 mm aecdons. Preparation: A minimum of six sections of the test article per rabbit, each approximately 1 mm x 10 mm, were loaded into 16 gauge needles. For each rabbit, a minimum of four USP strips w oe loaded into foe same aize needles as used for foe test stride. Test and control articles were sterilized by stem . Sample Disposition: Any remaining sample was discarded. METHODS lE tt-S m cm : Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: Identification Method: Rabbit (Orvctoianis cuniculus) New Zealand White Mohican Valley Rabbitry No oartinular gender was prescribed for this test 2.9 kg to 3.0 kg at implantation No particular age was prescribed for this test Minimum 2 days Two Ear tag or tattoo g f-IW l gW tfflF The rabbit is suggeueu as an appropriate animal model for evaluating polymer materials by foe current USP testing guidelines. The muscle tissue has been used historically because foe response to implanted material is easily graded and compared to a known negative control material. - 50 - Company Sanitized. Coes not eon#!, Page 4 oi m m S WorU Lsaior fu T atting Stnrtcaa f t A M itic a ! D ii i c i tmaatry 2261 Tracy Road Northwood, O H 3619 Phona 19466*9455 FAX 419466*954 Autfwrteatton for duplication <J ( h it raport. tx c a p t in whoia. It ra ta tv a d p t n d h g N A m S A 't w M ttn approval. TU014-807 DuPont HLO 907-96 Lab No. 96T 12212 00 Animal Management: Husbandry: ' Conditions conformed to Standard Operating Procedures which are based on the 'Guide f^or the C^ are anda U*se_ o_#f Laboratory AAaniaimanalals.*" PROLAB* High Fiber Rabbit Diet was provided daily. 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 staiidess steel suspended cages identified by a card {rating the lab number, animal number, test 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). Personnel: Selection: NAmSA is an AAALAC accredited facility. /Avviate involved were appropriately qualified and trained. Healthy animals were selected. To reduce the number of animals used for testing, and to comply with the directives of the NAmSA IACUC, rabbits on this study may have been auUrsitmeidc1le*pUsrl^edvVii.ldUouUnsWol"ytf ci*anwuawsenu au*nreresplaoten'ds' et--e.i--ns--t-t-.-mh---e-o--da-_--ne--li--._m__aAl_sn.*y C^proemvipoluetsel"ykhleiasvStaoMlruyuatoef.daatnnei.simmt ioid,lrnucasoaansgtareoiilst traceable in laboratory records. Animals used for previous evaluations are identified in the report. Experimental Procedure: column. strips were similarly implanted. Rabbits were returned to their respective oages following the procedure. Rabbits were observed daily for general health. Body weights were recorded prior to implantation and at termination. - 51 - KAm SA f t r t k t k itils m l D trie t M n t r j ft fft K fc a m ua rtupamian at tN a la a a t. site* In W . H rw w v tf (Sompany Sanitised. Does not contain TSCA C I Page 5 of 7 N orthwood. O H <38 IB Phon* 19*86*455 FAX <19*884984 N*mS' -- " p p w l DuPont HLO 907-96 Lab No. 96T 12212 00 At 1 week, the rabbits were weighed and euthanatized by an intraVfcnout injection of a odium pentobarbital based drug. The paravertebral muscles were dissected free and methodically cut to locate four of the test uticle sites and two of the USP negative control sites in each rabbit. Capsule formation or other evidence of irritation was scored fag appropriate magnification and an auxiliary light source. Scores were assigned and recorded as luiiows: 0 No capsule, no adverse reaction (other than minimal hemorrhage) 1 Up to 0.S mm capsule or reaction area 2 0.6to 1.0mm capsule or reaction area 3 l .l to 2.0 mm capsule or reaction area 4 > 2.0 mm capsule or reaction area Average macroscopic scores for test implants were compared to average scores of the control sites. Calculations were rounded off to die nearest 0.1. A Reaction Index difference of 0.0 to 0.5 in scores (test minn rantmi) was regarded as "not significant,* 0.6 to 1.0 as a "trace,* 1.1 to 2.0 as slight, 2.1 to 3.0 as 'moderate* and fe3.1 as "marked." The requirements of the USP test were met if the difference between test and control score averages (macroscopic) was not greater than 1.0. The requirements of the test were not met if the difference between the test and control score for two (or more) implant sites exceeded 1 for any animal implanted. RESULTS r fircjffll Ahucrwrinn; All animal appeared clinically normal throughout the duration of die study. Body weight data for individual rabbits were considered acceptable. Macroscopic Observations: The findings for the macroscopic evaluation are shown brtow. Rabbit Number m 96517* Weight (kg) Day 0 Day 7 3.0 2.9 96477* 2.9 2.8 Site I 2 3 4 1 2 3 4 Average: Test 0 0 0 0 0 0 0 0 0.0 USP Negative 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 further evaluation of these results is made by NAmSA. Any extrapolation of these data to other samples is the responsibility of the sponsor. Company Sanitized. Does not contain TSCA CE I Page 6 of 7 H An& K World Leader In Telling Serticsi fa r Ike U edicel Derice la d eitrj 2201 Tracy Read Nonhwood. OH 43619 Phono 4194004455 FAX 4194004954 A u d w rtxa O a i lo r d u p k o tle n c 1 0 m report, oxce ol m M o . a rta o rv e d p o m m a N Am SA'o wrote o p p m a l. TUO 14-807 DuPont HLO 907-96 Labi . 96T 12212 00 CONCLUSION Under ihe conditions of this study, the macroscopic reaction was not significant as compared to the USF negative control implan' material. The implanted test article met the USP requirements. RECORD STORAGE All raw data pertaining to this study and a copy o f the final report are to be retained in designated NAmSA archive files. Company Sanitized. Does not contain TSCA Cj _ 53 - i World Leader in T tiling Services fo r the M edical Device Industry 22fit Tracy Hoad `torthw ood OH 43fit$ P hone 49-6fiiv`)45>5 FAX 4iy^6(.-?9b4 Authorization tor duplication ot this report, eic e p i tn whole. <s reserv ed pending NAmSA's written approval Page 7 of 7