Document Yj4mEgNMb87DVXOGNevOz1Gbk
F 477
TRADE SECRET
Study Title H-25878: ISO Biocompatibility Testing
M IM DuPont-13216
Study M onitor: Carol Finlay, B.A.
Study Completed o n : August 26,2003
P erform ing La bo ra to r y : North American Science Associates, Inc. 2261 Tracy Road Northwood, Ohio 43619-1397
for
E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050
L abo rato ry Project ID : DuPont-13216
W ork Request Number:
Service Code N um ber:
Company Sanitized. Does not contain TSCA CB1 Page 1 o f 29
H-25878: ISO Biocompatibility Testing
DuPont-13216
TABLE OF CONTENTS
Page
ST U D Y IN F O R M A T IO N ............................................................................................................................... 3
S U M M A R Y ..........................................................................................................................................................4
A P P E N D IC E S .....................................................................................................................................................5 A. H-25878 CYTOTOXICITY STUDY USING THE ISO AGAROSE OVERLAY METHOD.................................6 B. H-25878 ISO INTRACUTANEOUS STUDY...............................................................................................................13 C. H-25 878 USP MUSCLE IMPLANTATION STUDY................................................................................................. 22
Company Sanitized. Does rrot contain TSCA CBI -2-
H-25878: ISO Biocompatibility Testing
STUDY INFORMATION Substance Tested: Svnonvms/Codes: H-25878 Haskell Number: 25878
DuPont-13216
Known Impurities: None
Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
Study Initiated/Completed: June 26, 2003 / (see report cover page)
Company Sanitized. Does not contain TSCA CBI
H-25878 : ISO Biocompatibility Testing
DuPont-13216
SUMMARY
Attached are reports (Appendices A - C) from North American Science Associates, Inc. The results for the H-25878 sample are:
Test Substance H-25878
Test Results
C ytotoxicity
ISO Intracutaneous
(Agarose Overlay)
SCa
CSOb
c dd
ISO M uscle Implantation (7 day)
NSe
a Sodium chloride extract b Cotton seed oil extract c No evidence o f cell lysis or toxicity d No evidence o f significant irritation.
Primary Irritation Index Characterization: Negligible e Not significant
Study Monitor:
ffi /U \< d i i l A l i , Carol Finlajf, B.A. Staff Toxicologist
Company Sanitized. Does not contain TSC CBS
H-25878: ISO Biocompatibility Testing
DuPont-13216
APPENDICES
Company sanif;2ed
eontalnTSCAcai
H-25878: ISO Biocompatibility Testing
DuPont-13216
APPENDIX A H-25878 Cytotoxicity Study Using the ISO Agarose Overlay M ethod
Company Sanitized. Does not contain TSCA CBS
H-25878: ISO Biocompatibility Testing
Confidential
DuPont-13216
Lab No. P.O. No.
STUDY TITLE: CYTOTOXICITY STUDY USING THE ISO AGAROSE OVERLAY METHOD
(SOLID)
TEST ARTICLE: H-25878
TEST FACILITY:
NAMSA 2261 Tracy Road Northwood, OH 43619-1397
SPONSOR:
CAROL FINLAY
DUPONT HASKELL LABORATORY
1090 ELKTON ROAD
PO BOX 50
NEWARK, DE
9714-0050
Page 1 of 6
NAMSA
Ensuring Mwikft! DeWc* m l CompRansf
C m Hdotrs: 2261 Tracy Road. Northwood. OH 619-1337/415.665.5455/Fax 419.666.2654 3<WCobb IntematiotiBl Blvti.. Kin n ssB w , GA 30152-7501' /770.427.31D1 / Fax 770.420.5092 9 Moigan. Irvine, CA 92818-2078 / 949,951,3110/ Fax 949^51^760 Aftfliaws: fran co * Germany * I s r a e l Taiwan Unhid Kingdom
Authorization for duplication of this report except in whole, is reserved pending NAMSA's written approval.
100311
Company Sanitized. Does not contain TSCA CB1
H-25878: ISO Biocompatibility Testing
SUMMARY.............. INTRODUCTION..... MATERIALS............ METHODS................ RESULTS.................. CONCLUSION......... RECORD STORAGE
DuPont-13216
TABLE OF CONTENTS
Lab No.
luPoat-1321
Page Nimber 3 4 4 4 5 6
6
ft***v
NAMSA
Entering M td is il Devio MMV lid CunplitnM"
Corp. Hdqtrs: 2281 Tracy ftaari. Northwood, OH 43618*187 MtB.98B.M55/ Fax 419.665.2354 3400 Cobb international Bli/1, Kannesaw, GA 30152-7601 / 770.427.3101 ! Fax 770.428.5632 9 Morgan, Irvine, CA92618-2C7B/949J51.3110/ Fdx 848.851.3280 Affiliates; Frsncn Gfltmnny Israel Taiwan United Kingdom
Authorization lor duplication of (his resort, except in whole, is reserved pending NAMSA's written approval.
Page 2 o f6
10O3U
Company Sanitized. Does not contain TSCA CBI -8-
H-25878: ISO Biocompatibility Testing
DuPont-13216
SUMMARY
An in vitro biocompatibility study, based on the requirements of the International Organization for Standardization (ISO 10993-5), was conducted on the test article, H-25878, to determine the potential for cytotoxicity. An approximate 1 cm x i cm portion of the test article, a negative control, and a positive control were each placed on triplicate agarose surfaces directly overlaying confluent monolayers of L-929 mouse fibroblast cells. After incubating at 37"C in 5% CO; for 24 hours, the cell culture was examined macroscopically for cell decolorization around the test article and controls to determine the zone of cell lysis (if any). The cultures were then examined microscopically ( IOOX) to verify any decolorized zones and to determine cell morphology in proximity to the articles.
Under the conditions of this study, the test article showed no evidence o f causing cell lysis or toxicity. The test article met the requirements o f the USP since the grade was less than a grade 2 (mild reactivity). The negative control and the positive control performed as anticipated.
Study and Supervisory Personnel:
Lionel D. Brock, BS Jennifer N. Moritz, BA Michelle Weingates-Furiate, BA Lisa A. Severbof, BA
Approved by:
p S ti
Martha Oswvansskki,, BS/, LAT JAchnical Reviewer
Ji f 3 t 2 6 * 3 Date Completed
/cal
NAMSA
Eaoiriig Madicil B avin tdhrty and Campllwwe
Corp. Hdqtrs: 2261 Tracy Road, Nort{lw(tOH4361S-I397 / 49.M E-9455/Fax 419.B6G.2354 3400 Cobb International Blvd., Kepnesaw,GA 30152-7801 / 770.4275101 i Fax 77C.42i.5692 9 Morgan, Irvine, CA 92618-2078/ 948.951.3110/Fax S46.95l.3230 Affiliates France Germany Israel Teiwnn * United Kingdom
Authorization for duplication of this reppn, except in whole, is reserved pending NAMSA'a written approval.
Page 3 of 6
1003U
sd Does not contain TSCA CBI
H-25878: ISO Biocompatibility Testing
DuPont-13216
Lab No.
.D uPont-m M ,
INTRODUCTION
The test article identified below was subjected to an in vitro cytotoxicity study for biocompatibility based on the
requirements of the International Organization for Standardization: Biological Evaluation of Medical Devices, Part 5: Test for Cytotoxicity in vitro Method. The test was performed to determine die potential o f the test article to cause cytotoxicity. The test article was received on June 25,2003. The cells were dosed on June 26,2003, and the observations were concluded on June 27,2003.
MATERIALS
The sample provided by the sponsor was identified and handled as follows:
Test Article:
H-25878
Identification No.:
Not Supplied
Storage Conditions:
Test Article Preparation:
Negative Control Preparation
Positive Control Preparation:
Test System Management:
L-929, mouse fibroblast cells, (ATCC CCL 1, NCTC Clone 929, of strain L, or equivalent source) were propagated and maintained in open flasks containing single strength Minimum Essential Medium supplemented with 5% serum and 2% antibiotics (IX MEM) in a gaseous environment of 5% carbon dioxide (COj). For this study, 10 cm2wells were seeded, labeled with passage number and date, and incubated at 37C in 5% C02 to obtain confluent monolayers of cells prior to use. Aseptic procedures were used in die handling o f foe cell cultures following approved NAMSA Standard Operating Procedures.
Preparation of Agarose Overlay:
The culture wells were selected which contained a confluent cell monolayer. The growth medium in each well was replaced with 2 ml o f equal amounts o f double strength Minimum Essential Medium supplemented with 10% serum and 4% antibiotics (2X MEM), supplemented with neutral red, and 2% agarose (final concentration 1% agarose, IX MEM). Hie MEM-agarose mixture (2 ml) was then placed in the cell culture wells and allowed to solidify over foe cells to form the agarose overlay.
NAMSA
Enturlog MtHftal OavlM
S vta y *nd CsnpttiM**
Corp. Hriutr*: 2251 Tracy R o ai.N o rttiw o oi. OH 43619-l397 / 419.566.9455/Fa*4l9.6E6.2S54
K ,3 4 Cobb Inletnalional B lv d , biimmw M 30152-7301 / 770.427.3101 /Fa 773.426.5892
9 Morgan. Irvine, CA 92618-2078 / 949,951.3110/ Fa 949.951.3290
Affiliates: France * Germany Israel Taiwan United Kingdom
Abthoriatfon for duplication of this report, except in whole, is reserved pendiftfl NAMSA's written approval.
Page 4 o f6
1003U
Company Sanitized. Does not contain TSCA CBI -10-
/**as**`\
H-25878: ISO Biocompatibility Testing
DuPont-13216
Lab No.
,DuPont-13216
Experimental Procedure:
The test article was placed on the solidified agarose surface in three separate cell culture wells. Similarly, the negative control and the positive control were each placed on the solidified agarose surface in three cell culture wells. The wells were labeled with the corresponding iab number and dosing date, and incubated at 37C in 5% C02for 24 hours.
Following incubation, the cultures were examined mactoscopically for cell decolorization around the test article and controls to determine the zone of cell lysis (if any). After macroscopic examination, the cell monolayers were examined microscopically ( 100X) to verify any decolorized zones and to determine cell morphology in proximity to the article.
Scoring for cytotoxicity was based on the following criteria:
Grade
.0 1 2 3 4
Reactivity
None Slight Mild Moderate Severe
Condition of Cultures No detectable zone around or under specimen Some malformed or degenerated cells under specimen Zone limited to area under specimen and up to 4 mm Zone extends 5-10 mm beyond specimen Zone extends greater than 10 mm beyond specimen
NOTE: This chart (direct excerpt from USP) fails to accommodate 1 mm - 4 nun zones. The USP was notified of this. They responded that I mm - 4 mm zones should be categorized as mild (2).
For the suitability o f the system to be confirmed, the negative control must have been a grade of 0 (reactivity none) and the positive control must have produced a zone of lysis (reactivity moderate to severe). The test article passed the test if all three monolayers exposed to the test article showed no greater than a grade of 2 (reactivity mild). The test would have been repeated if the controls did not perform as anticipated and/or if the test wells did not yield the same conclusion (e.g., one well passed and the other two wells failed).
The scores obtained were as follows:
Test Article:
ARTICLES
Negative Control:
Positive Control;
RESULTS
ZONE OF LYSIS (mm)
(1) 0 (2) 0 (3) 0 (1) 0 (2) 0 (3) 0 (1) 6 (2) 5 (3) 5
GRADE 0 0 0 0 0 0 3 3 3
REACTIVITY None None None
None None None
Moderate Moderate Moderate
NAMSA
EmmlngAbdicai Davit*
iafrtir airi CampHiiic#-
Corp. Hdqtrs: 2261 Tracy Road, Northwood, OH43B19-!M7/41S.S08.3455/Fuic4JS.6B6.2954 3400 Cobb Inwn&tioittl Bivd., Kannasaw, GA 30152-7601 / 770427.3101 / Fax 770.426.5SM 9 Morgan, Irvine, CA 92018-2079 / 949.551.3110 f Fex 949.951,3280 Affiliates: Franca Germany Israel Taiwan United Kingdom
Authorization for duplication of this report, except in whole. 1$ reserved pending NAMSA's written approval.
Page 5 of 6
I003U
C o n r a n ,, S m lllze d , D o e s
- 11 -
?SCA ^
H-25878: ISO Biocompatibility Testing
DuPont-13216
rI
_ DuPont-13216 ^
(jm m j
ubN-
Results and conclusions apply only to the test atticle 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. All procedures were conducted in conformance with good laboratory practice and ISO 17025.
CONCLUSION
Under the conditions of this study, the test article showed no evidence of causing cell lysis or toxicity. The test article met the requirements o f the USP since the grade was less than a grade 2 (mild reactivity). The negative control and the positive control performed as anticipated.
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.
NAMSA
Enuring Mndie>IOvte Safety m il Compilation'-
Cam. Hdqtrs: 2261 Tracy Rosd, Nonhwood, OH 43618-1397 /419,666.9455/ Fa* 419.666.2S54 3400 Cobb International Bind.. Kernes**, SA 30152-7601 / 770.4273101 / Fa* 770.4265632 9 Morgan, Irvine. CA 92518-2070 7840.9513110/ Fa* 649.8513260 Affiliates: Krance Germany Israel Taiwan * United Kingdom
Authorization for duplication of this report, except in whole, is reserved pending NAMSA'3 written approval.
Page 6 o f6
1003U
-12-
TSCA OBJ
H-25878: ISO Biocompatibility Testing
DuPont-13216
APPENDIX B H-25878 ISO Intracutaneous Study
Corapany - 13 -
D o ! Mnttl)n ISCA CB|
H-25878: ISO Biocompatibility Testing
Confidential
DuPont-13216
Lab No. P.O. No.
iuPont-13216
STUDY TITLE: ISO INTRACUTANBOUS STUDY
EXTRACT
TESX ARTIO M : H-25878
TEST FACILITY:
NAMSA 2261 Tracy Road Northwood, OH 43619-1397
SPONSOR:
CAROL FINLAY
DUPONT HASKELL LABORATORY
1090 ELKTON ROAD
PO BOX 50
NEWARK, DE
19714-0050
NAMSA
Emmlng MailiMl Ocvloa Solely mil Compliance*
Corp.Hdqtrs: 226! Tracy Road. Northwood, OH 43619-1397 / 419.668.945S/ Fax 419.666.2954 3409 Cobb Intarnatiofiaf B lv l . Kerne ssw , S A 30162-760! / 770.4273101 / Fa< 770.426.S692 9 Morgan, Irvin, CA92618-2078 / 94fi.fl51.3710/Faic 946.9513280 Affiliates: Franca Germany Israel * Taiwan * United Kingdom
Authorization for duplication of this report, except in wfioie, ie reserved pending NAMSA's written approval.
Page i o f 8
10O3U
Company Sanitized. - 14-
contain TSCAC
H-25878: ISO Biocompatibility Testing
DuPont-13216
TABLE OF CONTENTS
SUMMARY............................................................... INTRODUCTION...................................................... MATERIALS............................................................. METHODS................................................................. DEVIATION.............................................................. RESULTS................................................................... CONCLUSION.......................................................... RECORDSTORAGE................................................ APPENDIX
1 - ISO INTRACUTANEOUS OBSERVATIONS
Lab No.
iPont-13216
Page Number 3 4 4 4 6 6 7 7
8
NAMSA
Ensuring M sdlctl O tric i
Safety lid C raiplisner"
Ccrp, Hdqtra: 2261 Tracy Road, N cntwood, OH 43613-1397 1419.E66.B46S / Fex 419.666.2954 3400Cobh International 6lvd.( Karnasavv. GA 30152-75I /770.4Z7.310T/fax 770.426.5982 9 Moroan, Irvins, CAB2618-2078/949.951.311D/FK 949.951.3280 Affiliates: Franca Germany Israel Taiwan * United Kingdom
Aulhoiuaiiuii fot duplication of this report. im pu pi in w h o le,is resawed ponding NAMSA's written approval.
Page 2 of S 1O03U
0g not a a s
-15 -
H-25878: ISO Biocompatibility Testing
DuPont-13216
SUMMARY
The test article, H-25878, was extracted in 0.9% sodium chloride USP solution and sesame oil, NF. These extracts were evaluated for intracutaneous reactivity based <mthe requirements of die International Organization for Standardization 10993: Biological Evaluation of Medical Devices, Part 10: Tests for Irritation and Sensitization.
A 0.2 ml dose o f the appropriate test article extract was injected by the intracutaneous route into five separate sites on the right side ofthe back o f each rabbit. Similarly, the corresponding reagent control was injected on the left side of the back of each rabbit. The injection sites were observed immediately after injection. Observations for erythema and edema were conducted at 24,48, and 72 hours after injection.
Under the conditions of this study, there was no evidence of significant irritation from the extracts injected intracutaneously into rabbits. The Primary Irritation Index Characterization for the extracts was negligible.
Study and Supervisory Personnel:
Brian T. Dougan Mark J. Menzel, RVT Brian Brooks Deedee M. Davis, BA Suzanne E. Bliss Debra S. Dunn Martha Oswanski, BS, LAT
Approved by:
_______
Jdshua D. Moninger, B 9 f / Technical Reviewer
dly %zce
Date Completed
/cal
NAMSA
Ecm lap MMiles) Dtvlss
Safety end Canplisset"
Corp. Hdqtrc 3 1 Tracy Road, Nortbwcod. QR 436TS-1S97 f 41R.66B.84551Fax 4t9.666.2354 3400 Cobb International Bivd., Kdiwesaw, GA30152-7BC! / 770.427,3101 /F a x 770.426.5S92 9 Morgan, frvinfl, CA82818'2076/W0.951 <31107 Fax S49J51,3MQ Affiliates: Prunes Germany Israel Taiwan * United Kingdom
Awhorizatwmfor duplication of this report except in whole, is reserved pending NAMSA'swrltten approval-
Page3ofS
100311
H-25878: ISO Biocompatibility Testing
DuPont-13216
INTRODUCTION
The test article identified below was extracted and the extracts were evaluated for biocompatibiiity based on the requirements of the International Organization for Standardization 10993: Biological Evaluation o f Medical Devices, Part 10: Tests for Irritation and Sensitization. The purpose ofthe study was to determine whether leachabies extracted from the material would cause local dermal irritant effects following injection into rabbit skin. The test article was received on June 25,2003. The animals were injected on June 30,2003, and the observations were concluded on July 3,2003.
MATERIALS
The sample provided by the sponsor was identified and handled as follows:
Test Article:
H-25878
Identification No:
Not Supplied
Storage Conditions:
Room temperature
Vehicles:
0.9% sodium chloride USP solution (SC)
Preparation;
Condition of Extracts:
Test SC: clear with particulates SO: clear
Control clear clear
methods
Test System:
Species: Breed: Source:
Sex: Body Weight Age: Acclimation Period: Number of Animals: Identification Method:
Rabbit {Oryctolagus cuniculm)
New Zealand White Myrtle's Babbitry, Inc. Female 2.0 kg at selection
Young adult Minimum 5 days Two Ear tag
Justification of Test System:
The intracutaneous injection test in rabbits is specified in the current ISO testing standards and has been used historically to evaluate biomaterial extracts.
NAMSA
Enuring Madieiri Davie* t nitty tnd Campitene*"
Corp.HUqtrs: 2261 Tracyftaad.N anhwood,O H 43613-1397/419.666.9423/ F a x 4l9.66fi.29S4 3400 Cobb International Blvd.. Kannaeaw, GA3G152-76C1 /770 4273101 / Fax 770.426-5592 ^Morgan, Irvins. CA92BU-2078 /9 *9 .9 5 U n O /F e x 649.951.32fO Affiliates; France * Germany Israel Taiwan * United Kingdom
AuUiorJiBtlcn for Oup/ication of this report, except in whole, is reserved pending NAMSA's written approval
Page 4 o f 8
1Q03U
TSCA'Cb /
- 17-
H-25878: ISO Biocompatibility Testing
DuPont-13216
r-- *
Lab No.
`ont-1321
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.
Water:
Freely available, municipal (Toledo, OH) water was delivered through an automatic watering system.
Contaminants:
Reasonably expected contaminants in feed or water supplies did not have the potential to influence the outcome o f this test.
Housing:
Animals were individually housed in stainless steel suspended cages identified by a card indicating the lab number, animal number, test code, sex, and date dosed.
Environmental'
The room temperature was monitored daily. The temperature range for the room was within a range of 6)-72F.
The room humidity was monitored daily. The humidity range for the room was 30-77% (see DEVIATION).
The light cycle was controlled using an automatic timer (12 hours light, 12 hours dark).
Facility:
NAMSA is an AAALAC International accredited facility and is registered with the United States Department o fAgriculture. Additionally, NAMSA maintains an approved Animal Welfare Assurance on file with the National Institutes o f Health, Office for Laboratory Animal Welfare.
Personnel:
Associates involved were appropriately qualified and trained.
Selection:
Only healthy, previously unused, thin-skinned animals free of mechanical irritation or trauma that could interfere with the test were selected.
pxperimenta) Procedure:
Within a 4 to 18 hour period before treatment, each rabbit was weighed and clipped free of fur from the bade and both sides o f the spinal column to yield a sufficient injection area. The dipped area ofthe back was wiped wife a 70% alcohol soaked gauze pad just before injection and allowed to dry. Two rabbits were prepared per pair of extracts. A 0.2 ml dose oftest article extract was injected intracutaneously into five separate sites on the right side of the back o f each rabbit; 0.2 ml of the reagent control was injected into five separate sites on the corresponding left side o f the back, injections were spaced approximately 2 cm apart The appearance of each injection site was noted immediately aft injection. The animals were returned to their respective cages following the procedure.
NAMSA
Enuring Modici! Burice U le t* ml CwnpttMsr
Coip. Hdqtri: 2361 T r it y fnnri, Northwood. OH 43619-139J741&6fl6.a455/f: ax 419.66S.29S4 3400 Cobb inwmationsl B i v i , KeimS8W,6A 30152-7801 / 770.427.3101 / Fax T7 0 .8 . 9 2 9 Morgan, Irvine, CA 8261B-ZD78/ 949.95U110/ Fax M 9.ffI.32B0 A ffilia i : Franca * Ganraivy * Israel * Taiwan fc Unta! Ktoodom
Authorization for duplication of this report, except In whole, is reserved pending NAMSA's written approval
Page 5 o f 8
tow u
Company Sanitized. Does not onfiift T&C SS
H-25878: ISO Biocompatibility Testing
DuPont-13216
Lab No.
'ont-13216.
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 erythema (barely perceptible) 2 Well-defined erythema 3 Moderate erythema 4 Severe erythema (beet redness) to eschar
formation preventing grading of erythema
EDEMA (ED)
I
0 No edema
I
1 Very slight edema (barely perceptible)
|
2 Well-defined edema (edges of area well-
defined by definite raising)
3 Moderate edema (raised approximately 1 mm)
4 Severe edema (raised more than 1 mm, and
extending beyond exposure area)
For each animal, the erythema and edema scores obtained at each time interval were added together and divided by
the total number of observations. This calculation was conducted separately for each test extract and reagent control. The score for the reagent control was subtracted from the score for the test extract to obtain the Primary Irritation Score, The Primary Irritation Score of each animal was added together and divided by the total number of animals to obtain the Primary Irritation Index (PH). The Primary Irritation Index was characterized by number and description as follows: 0-0.4 (negligible), 0.5-1.9 (slight), 2.Q-4.9 (moderate), 5.Q-8.0 (severe). Any adverse reaction noted in the test extract was compared to the corresponding reagent control.
PE-VIA.HQN
During the course of the study, the relative humidity was found to be 30-77% in the room housing the animals. Although this lies above the range o f 30-70% specified by NAMSA Standard Operating Procedure (SOP), this increase would not be expected to Impact animal health and would have no impact on the conduct of the study. As a result, this temporary rise in relative humidity did not affect the integrity o f the study.
RESULTS
All animals appeared clinically normal throughout the study. Results of scores for individual rabbits appear in Appendix 1. All injection sites appeared normal immediately following injection. The Primary Irritation Index (PH) and Characterization for each extract are summarized below:
Extract
SC SO
Animal Number
34742 34744 34742 34744
Test Score Average
0.0 0.0
0.5 03
Control Score Average
0.0 0.0
0.3 0.1
Primary Irritation
Score
0.0 0.0
0.2 0.2
PrimBiy Irritation Score Total
0.0
0.2
Primary Irritation Index Characterization
Negligible
I
1 I
Negligible
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 ofthe sponsor. All procedures were conducted In conformance with good laboratory practice and ISO 17025.
NAMSA
Ensuring M idien)Davie*
Safety if Compliance-
Corp.Hdqtrfc 2261 TrecyRo ac.N o n.hw osd,3H 43619-1397/il9.65fi.B455/FBx^3.M6^S5-5 3400 Coob International Blvd.. Kennesaw. GA 30152-7601 / 770.427.3101 /F a x 770.426.5692 9 Morgan, Irvine, CA82518-2078 1948.95U11D t Fa* S49.95l.32aa Affiliates: France Germany * Isral Taiwan United Wnjdom
Authorization for duplication oftfiis report, except in whole, is reserved pending MAMSA's written approval.
Page 6 of 8
1003U
c H p a n y g am
- 19-
not t s c a CBI
H-25878: ISO Biocompatibility Testing
DuPont-13216
CONCLUSION Under the conditions of this study, there was no evidence of significant irritation from the extracts injected intracutaneously into rabbits. The Primary Irritation Index Characterization for the extracts was negligible.
RECORDSTORAGE
All raw data pertaining to this study and a copy o f the final report are to be retained in designated NAMSA archive files.
NAMSA
Eniurlnp Madlccl Davie Safa* and Cwnplnne"
Corp. Hdqtrs: 2261 TiacyRoBd, NMItwood, OH 613-1337/419566.3455/Fax 419.685.2954 3400 Cobb Internationa! Blvd., K e n n a a m GA 30182-7801 /770.427.3101 i raK77a.42S.S632 9 Morjian, Irvins, CA 92618-2078/949.951.3110/Fax 949.951.3200 M flliatav France * Bamrawy ' Israel * Taiwan * Unrad Kingdom
Aathorizatfco for duplication ol tills pore, except in whole, is reserved pending NAMSA's written approval
Page 7 ofS
1003U
" '"' ' seses,
-20-
H-25878: ISO Biocompatibility Testing
DuPont-13216
Lab No. APPENDIX 1 ISO INTRACUTANEOUS OBSERVATIONS
`ont-13216,
Ma 8
Rabbit Number/ Gender
34742 2.0 Female
34744 2.0 Female
34742 2.0 Female
34744 2.0 Female
Extract
sc sc so so
24 Hours
Test Control
ER ED ER ED
00 00 0 0 Q0 0000 0000 0000
00 00 0000 0000 0 D0 0 0000
I 0 10 I 0 10 10 10
0 10 0000
0000 0000 00 10 0000 00 00
Scoring Interval
48 Hours
Test Control
ER ED ER ED
000 0 000 0 000 0 000 0
000 0
000 0 000 0
000 0 000 0 000 0
10 1 0 000 0 100 0 000 0 000 0
000
000
00 0 00 0 0 00
0 0 0 0 0
72 Hours
Test Control
ER ED ER ED
O0
00 00 00 00
00 00 00
00
00
0 0 00
0 0 00 0 0 00
0 0 00 0 00
0 00 0 000 1 0 00
0 000 0 0 00
10 00
1 0 00 1 0 00 1 0 00 1 0 00
ER = Erythema ED " Edema
SC " 0.9% sodium chloride USP solution SO = sesame oil, NF
NAMS
fanning MitTieal Dmp SafetyM i CowptUiwr*
Page fco f 8
Corp. Hdqtrs' 2281 TrecyRoad, Mortftwood, 0H43619-97 f 4t9.866.M 55/Fax *19.666.2654
3403Cobh InternirtiOGal Blvd., Kem w sa . GA.30U2-76Q1 / 770,4273101 / Fax 770.428,5692 9 Morgan, iivinfi, CA92818-27/949.85U110/Fex S49 .K U 2 B 0
Affiliatasi France Germany * tsrBBl Taiwan * United Kingdom
C & m P S a n itiz e dDoes not contain TSCA CBt
H-25878: ISO Biocompatibility Testing
DuPont-13216
APPENDIX C H-25878 USP M uscle Implantation Study
o , not
cmntain Ts c a
H-25878: ISO Biocompatibility Testing
Confidential
DuPont-13216
STUDY TITLE: USP MUSCLE IMPLANTATION STUDY
7 DAY
TEST ARTICLE H-2S878
/""v
TEST FACILITYi
NAMSA 2261 Tracy Road Northwood, OH 43619-1397
SPONSOR
CAROL FTNLAY
DUPONT HASKELL LABORATORY
1090 ELKTON ROAD
PO BOX 50
NEWARK, DE
19714-0050
NAMSA
Enuring Medical Device Safetyend Compline-
CotD.HifqtiK 22B1 Tracy Road,Nartfiwood, 0H436?3'1397/419.66S.M 55/FaK419.66U354 3400 Cobb Internationa; Blvd., Kennesaw, A 30152-7601 i 770.427.3101 /F a x 770.426.56S2 9 Morgan. Irvins. CA 92618*2073/ 949.651-3110/ Fas 949.951.32312 Affiliates: France Germany Israel Taiwan United Kingdom
Authorization for duplication of this report, except in whole, is reserved pending NAM SA's written approval
Page J o f?
1003U
Company Sanitized. Does not c o n ta i TSC CBI -23 -
H-25878: ISO Biocompatibility Testing
DuPont-13216
Lab No.
TABLE OF CONTENTS
Paee Number
SUMMARY.............. .............................................................................................................................
3
INTRODUCTION................................................................................................................................... MATERIALS..........................................................................................................................................
4
4
METHODS..............................................................................................................................................
4
RESULTS................................................................................................................................................
6
CONCLUSION.......................................................................................................................................
6
RECORD STORAGE.............................................................................................................................
6
APPENDIX I - BODY WEIGHTS AND MACROSCOPIC OBSERVATIONS....'..............................................
7
Pag 2 o f7
NAMSA
Ensuring Mwfleif Davis* Safety and CempKanca*'
Corp. Hdtflf 2261 T'acyHoad, Nwtftwood, OK 43616-138? M 1& W 6.84/Fex4l9.666.2S64
3400 Cobb Incernsiional Blvd., Kennssaw. S A 30152-7861 ( 770427,3101/ F a x 770.4265692
5 Morgan, Irvine. CA 926>fl'Z078/94a8&1.311O/Fax 949.951.3280
Affiliates Franca * Germany
k Taiwan * United Kingdom
Authorization for duplication of this report except in whote.is reserved pending NAMSA'a written approve!.
1O03U
Company Sanitized. Does no! contain TSCA CBJ -24-
H-25878: ISO Biocompatibility Testing
DuPont-13216
Lab No.
The test article, H-25878, was implanted in muscle tissue of the rabbit The muscle tissue was evaluated for evidence o f irritation or toxicity in accordance with the guidelines of the current USP.
Negative control samples were sterilized by steam. Rabbits were implanted and were then euthanized 1 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 negative control implant material, The implanted test article met the USP requirements.
Study and Supervisory Personnel:
Meridith A. Baugher, RVT, ALAT Christine Szych, RVT Julie Hom, RVT, LAT Deedee M. Davis, BA Martha Oswanski, BS, LAT
Approved by:
Joshua . Moningej, BS Technical Review
JJy /, t#*3>
Date Completed
/elm
Page 3 o f7
NAMSA
Enw lng Medic!Device Safetyad C tnpliince*
Cotp. Hdtfrs; 2261 Tracyflosd, Northwootf, OH 436)9*1337 / 413.66S^4F5/rax 413JS6.23M 3400 Cabb littematlonal B lv O e n n e g a w , GA 30182-7801 i 770.42731011Fax 770.42fl.5692 9 Morgan, Irvin?, CA 32618*20791943.651311&/Fax 949.951.3230 AffiSstas: Franca * Germany * Israel Taiwan United Kingdom
Authorization tor duplicatfan of this report except in whole, is reserved pendino NAMSA's written approval.
10O3U
Company Sanitized. Does not contain TSCA CBI -25-
H-25878: ISO Biocompatibility Testing
DuPont-13216
r *1
ip v
INTRODUCTION
^u-Poirt-53216 u in "
The test article identified below was evaluated for biocompatibility in accordance with the guidelines o f 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 was received on June 25,2003. The animals were implanted on July 1,2003, and muscles were explanted on July 8,2003.
The sample provided by the sponsor was identified and handled as follows:
Test Ardete:
H-25878
Identification No:
Not Supplied
Storage Conditions:
Negative Control Article:
Preparation:
Test System:
MBfflQPS
Species: Breed: Source: Sex: Body Weight Range: Age: Acclimation Period: Number of Animals: identification Method:
Rabbit (Oryctolagus cumculm) New Zealand White Myrtle's Rabbitiy, Inc. Female 3.0 kg to 3.8 kg at implantation No particular age was prescribed for this test Minimum 5 days Two Ear tag
Justification of Test System:
The rabbit is suggested as an appropriate animal model for evaluating polymer materials by the current USP testing guidelines. The muscle tissue has been used historically because the response to implanted material is easily graded 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."
Food:
PROLAB High Fiber Rabbit Diet was provided daily.
Page 4 o f7
NAMSA
Enuring M ade*! Device SitMy inti C o m fllin w
Carp.Hdqtrs: 2 2SITiacyRo ad,N o n hw n o d.OH 43!9-t39?4 1 2 .B 6 6 M 5 /Fax 449.888.2954 3AOQG0bb Inrernstiunsi B lv c .,Kennflsaw, G A 30152-7601 /77P.4Z7.3101 / F a x 773.426.5932 8 Morgan, Irvine, CA f61fl-M78 /U & 9 S U U 0 i fa x 949.S5t.32BQ Affiliates: France * Germany Israel Taiwan United Kingdom
Authorization tor duplication of this report except in whole, is reserved pending NAMSA's written approval.
1003U
Do fteti -26-
H-25878: ISO Biocompatibility Testing
DuPont-13216
Water:
Lab No.
Freely available, municipal (Toledo, OH) water was delivered through an automatic watering system.
Contaminants:
Reasonably expected contaminants in feed or water supplies did not have the potential to influence the outcome of this test.
Housing:
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 room was within a range of61-72F.
The room humidity was monitored daily. The humidity range for the room was 30-70%.
The light cycle was controlled using an automatic timer (12 hours light, 12 hours dark).
Facility:
NAMSA is an AAALAC International accredited facility and is registered with the United States Department o f Agriculture. Additionally, NAMSA maintains an approved Animal Welfare Assurance on file with the National Institutes of Health, Office for Laboratory Animal Welfare.
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 were 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 are identified in the report
Experimental Procedure:
Rabbits were clipped free of fur over ihe paravertebral muscles. An intramuscular Injection of a combination of ketamine hydrochloride and xylazine general anesthetic was administered to each animal at a dose o f 0.6 ml/kg. Each rabbit was then injected subcutaneously with 0.02 mg/kg buprenorphine. After the anesthetic had taken effect the back was scrubbed with a germicidal soap, wiped with 70% isopropyl alcohol and the surgical site was painted with an antiseptic solution.
One incision was made on each side of the back through the skin and parallel to the lumbar region o f the vertebral column. A stylet was placed in Ihe hub of a loaded needle. The skin was moved to the desired location and the needle was inserted through the incision into the muscle at a 45 angle. The needle was withdrawn over the stylet, leaving the sample in the paravertebral muscle. Six test article sections were implanted in the right paravertebral muscle ofeach rabbit. In the opposite muscle, four negative control sections will be similarly implanted. The sections were placed at appropriately spaced intervals. The skin was closed with tissue glue. The rabbits were returned to their respective cages and monitored for recovery from the anesthetic.
Rabbits were observed daily for general health. Body weights will be recorded prior to implantation and at termination.
Page 5 o f7
NAMSA
Enrn ia g M idlcal Davie Safetyand Cwiplfme1
Carp. Hdqtrs: 2261 T r a c y R N o r t h w o o d . DH4351S-1397M 19;555.55/Fax 419-665-2954 34MCobl> International B ivd , Kennwaw. BA 30152-7601 / 770.427.3101 / Fax 770.426.5692 9 Morgan, Irvine, CA 92618-207/ 949.9513110/Fa x M9.951J280 Affiliates; fran ca * Germany * Israel * Taiwan * Uniiefl KtoQdem
Aulfiorizatien for duplication of this report except in whole, is reserved pending NAMSA's written approval.
1003U
-27-
C o m p aq S an fffe e o e
rcot Contain TSCA c a t
H-25878: ISO Biocompatibility Testing
DuPont-13216
f ____*1
--Du-Pont-132^
LI r % l
JL a b n o . m m m m m m t
At 1 week, the rabbits were euthanized by an intravenous injection of a sodium pentobarbital based ding, The paravertebral muscles were dissected free and methodically cut to locate four test article sites and two negative control sites in each rabbit Capsule formation or other evidence of irritation was scored using low magnification and the scores were recorded as follows:
0 No capsule, no adverse reaction (other than minimal hemorrhage) 1 Lip to 0.5 mm capsule or reaction area 2 0.6 to 1.0 nun capsule or reaction area 3 1.1 to 2.0 mm capsule or reaction area 4 >2.0 mm capsule or reaction area
Evaluations and Statistics:
The average macroscopic score for test implants was compared with the average score of the control sites. Calculations were rounded off to the nearest 0.1. A Reaction Index difference of 0.0 to 0.5 in scores (test minus control) was regarded as "not significant," 0.6 to 1.0asa"trace," 1.1 to 2.0 as "slight," 2.1 to 3.0 as "moderate" and
3.1 as "marked." The requirements of the LISP test were met if the difference between test and control score averages 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 o f the study. Body weight data for individual rabbits were considered acceptable.
reaction of "not s Appendix 1.
g: There was no visible reaction at any test or control site. This resulted in a macroscopic it" tissue contact irritation. The findings for the macroscopic evaluation are shown in
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. Ail procedures were conducted in conformance with good laboratory practice and ISO 17025.
CONCLUSION
Under the conditions o f this study, the macroscopic reaction was not significant as compared to the negative control implant 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.
Page 6 o f7
NAMSA
EraoriAfl Medic! Davie Safety nd Compliance-
Cojp. Hdqtrs; 2261 Tracy Road. Norttwuad, QH43619-1337 1413.666S455 f Fax 419,666,2954 3400 Cobb Intarnetlwiel BlwJ., Km m m w , GA 3&1S2-7AQ1 /7 7a Z 7J1 0i / Tax 773.426 5632 9 Morgan. Irving, CA82fi18-M78/m9&1.3110> Fax 9.551.3280 Affifiaias: France Germany Israel * Taiwan * Unlied Kingdom
Authorization for duplication of this report, except in whole, is reserved pending NAM$A' written approve!
1O03U
-28-
C o m p an y S an itized . D o es n o t c o n ta in T SC Jt C 8
H-25878: ISO Biocompatibility Testing
DuPont-13216
APPENDIX.!
BODY WEIGHTS AND MACROSCOPIC OBSERVATIONS
Page 7 o f 7
NAMSA
itiMring Medicei Devio Sriety tad CwnpIffM***
Corp. Hdotrs: 22=1 Tracy Road, Northwood, OH 43313-1337 { 418.68B.5455/Fax 41S.fi6S.2954 3400 Cobb lilem ationai B lvd, Kafinesaw, GA 3015Z-76D1 / 77&427.3101 / F a x 770.426.5B92 9 Morgen, Irvine. CA 82811-2078 / 943,95151 / F sx B40.85U26O Affiliate*: France * Germany ' Israel Taiwan United Kingdom
AuHwriaation for duplication oi this report except In whole, is reserved pending MAMSA's written approval.
1003U
-29-
Company Sanitized. Does not
SEBMbjscm ,cm
