BIOMAT., MED. DEV., ART. ORG., 4(3&4), 235-261 (1976)

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QUANTITATIVE CELL CULTURE BIOCOMPATIBILITY TESTING OF MEDICAL DEVICES AND CORRELATION TO ANIMAL TESTS

R. E. Wilsnack, D.V.M. Huntingdon Research Center D i v i s i o n o f Becton, Dickinson and Company Post O f f i c e Box 527 Brook1andvi 11e, Mary1and 21 022 ABSTRACT The b i o c o m p a t i b i l i t y o f a wide v a r i e t y o f b i o m a t e r i a l s was q u a n t i t a t i v e l y assessed, i n a p h y s i o l o g i c a l l y normal environment, as t o c y t o t o x i c i t y induced i n WI-38 c e l l s by c e l l c u l t u r e medium e x t r a c t s . M a t e r i a l s t e s t e d i n c l u d e d PVC p l a s t i c , rubber, s i l i cone rubber, polyethylene, polypropylene, a c e t a l , polyurethane, Teflon@, nylon, epoxy, and polystyrene. C e l l c u l t u r e t e s t r e s u l t s were c o r r e l a t e d t o U.S.P. animal t e s t s . P o t e n t i a l t e s t a r t i f a c t s , lead, barium, cadmium, and endotoxin were t e s t e d f o r c y t o t o x i c i t y i n WI-38 c e l l s . C e l l c u l t u r e methods y i e l d e d more p o s i t i v e t e s t s , p a r t i c u l a r l y rubber, PVC p l a s t i c and s i l i c o n e rubber compounds, than observed i n U.S.P. animal t e s t s . P o s i t i v i t y i n animal t e s t s d i d not correlate quantitatively t o cytotoxic t i t e r s i n c e l l culture. Discrepancies between c e l l c u l t u r e t e s t s and animal t e s t s , s p e c i f i c a l l y rubber compounds, were a t t r i b u t a b l e , i n some instances, t o d i f f e r e n t i a l s i n e l u t i o n e f f i c i e n c y between s a l i n e , cottonseed o i l , and complete MEM c e l l c u l t u r e medium. I n o t h e r instances, p a r t i c u l a r l y PVC p l a s t i c s , d i f f e r e n c e s between c e l l c u l t u r e and animal t e s t r e s u l t s were due t o an i n h e r e n t d i f f e r ence i n t h e two i n d i c a t o r systems t o respond t o s p e c i f i c t o x i c moi e t ies

.

I.

INTRODUCTION

An a r r a y o f i n v i v o and i n v i t r o b i o c o m p a t i b i l i t y t e s t s (1-25), f o r t h e a p p r a i s a l o f medical devices, has been evolved w i t h t h e presumptive goal o f p r e d i c t i n g and p r e v e n t i n g adverse

235 Copyright Q 1977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any rneahs, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.

MUSNACK

236 r e a c t i o n s d u r i n g c l i n i c a l a p p l i c a t i o n i n humans.

Previous t e s t

r e s u l t s have been d i f f i c u l t t o c o r r e l a t e and i n t e r p r e t because o f t h e n o n q u a n t i t a t i v e endpoint parameters i n h e r e n t t o many t e s t s , t h e l a c k o f s t a t i s t i c a l l y adequate numbers and generic Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by York University Libraries on 08/23/13 For personal use only.

v a r i e t y o f t e s t specimens, wide b i o l o g i c v a r i a t i o n s i n i n d i c a t o r systems , and d i v e r s e physical/biochemical ambience employed i n each t e s t system.

To t h i s end, we have attempted t o q u a n t i f y t h e biocompatib i l i t y o f s p e c i f i c f o r m u l a t i o n s o f b i o m a t e r i a l specimens i n a human (WI-38) c e l l c u l t u r e system (26) and have attempted t o p l o t t h e c o r r e l a t i o n t o U.S.P.

animal t e s t s (27).

The c e l l c u l -

t u r e t e s t system was designed t o encompass p h y s i o l o g i c a l l y normal biochemical and p h y s i c a l environments i n order t h a t i n s i g h t might be gained i n t o t h e short-term b i o a v a i l a b i l i t y o f leachable t o x i c m o i e t i e s which c o u l d be p e r t i n e n t t o c l i n i c a l biomedical device usage. 11. A.

MATERIALS AND METHODS

C e l l Cultures WI-38 c e l l s (28) were received from American Type C u l t u r e

C o l l e c t i o n and were propagated i n 32 oz p r e s c r i p t i o n b o t t l e s w i t h growth medium c o n s i s t i n g o f Minimum E s s e n t i a l Medium (Eagle) w i t h E a r l e ' s s a l t s (MEM), 10% f e t a l bovine serum (FBS) (BBL, D i v i s i o n o f Bioguest) (Table l), 2 mM glutamine, p e n i c i l l i n (100 u n i t s / m l ) ,

streptomycin (100 mcg/ml),

Fungizone

(2.5 mcg/ml), and Gentamicin ( 5 mcglml) ( t h e t o t a l m i x t u r e

r e f e r r e d t o as complete MEM medium).

A l l c e l l c u l t u r e s were

maintained i n a h u m i d i f i e d atmosphere (90% r e l a t i v e humidity) w i t h 5-7% carbon d i o x i d e a t a temperature o f 37°C.

A l l suspen-

sions were prepared w i t h t h e a i d o f 0.25% t r y p s i n s o l u t i o n s and were counted i n a haemocytometer.

Cells f o r c y t o t o x i c i t y scoring

were placed i n 60 nm p o l y s t y r e n e c e l l c u l t u r e dishes (Falcon P l a s t i c s ) a t 250,000 c e l l s p e r d i s h and u s u a l l y reached monolayer

BIOCOMPATIBILITY OF BIOMATERIALS

237

TABLE 1

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Chemical C h a r a c t e r i z a t i o n o f F e t a l Bovine Seruma Specific gravity Total protein A1 bumin Globulin Blood urea n i t r o g e n Total l i p i d s Total b i l i r u b i n Hemoglobin

1.078 4.1 g% 2.0 g% 2.1 g% 14.0 mg% 109.0 mg% 0.4 mg% 12.2 mg%

adata provided by BBL, D i v i s i o n o f B i oQuest

i n 5-7 days.

Alternatively, cells f o r cytotoxicity titrations

were p l a t e d i n Y i c r o t e s t I1 t i s s u e c u l t u r e p l a t e s ( 6 mm w e l l s ) (Falcon P l a s t i c s ) a t 20,000 c e l l s p e r w e l l and n o r m a l l y achieved monolayer i n 5 days.

141-38 c e l l s were n o t used beyond t h e 35th

passage. The c e l l c u l t u r e s were f r e e o f mycoplasma, as determined by a e r o b i c and anaerobic c u l t u r e (29), a t t h e beginning and a t t h e t e r m i n a t i o n o f t h e study.

6.

WI-38 C e l l C u l t u r e MEM E l u t i o n Test T e s t m a t e r i a l s were c u t i n t o pieces n o t t o exceed 20 mm i n

any dimension, o r specimens made up o f p a r t i c l e s l e s s than 20 mm i n c r o s s - s e c t i o n a l dimension were t e s t e d as received.

None o f

t h e t e s t specimens were s t e r i l e a t t h e t i m e o f t e s t n o r were specimens a s e p t i c a l l y handled.

M a t e r i a l s were placed i n t o (150

m l ) b o r o s i l i c a t e glass b o t t l e s c o n t a i n i n g MEM t 10% f e t a l bovine serum w i t h a n t i b i o t i c s (as described under c e l l c u l t u r e s ) (herea f t e r r e f e r r e d t o as complete MEM medium) a t a r a t i o o f 1 g t e s t m a t e r i a l t o 5 m l o f complete specimens and complete

MEM medium.

Bottles containing t e s t

MEM medium were incubated ( s t a t i o n a r y ) a t

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238

WILSNACK

37OC for 24 hours. After the 24-hour elution period, the mixtures were centrifuged a t 500 x g and supernates were decanted for testing. Two modes of testing were employed: cytotoxicity scoring and cytotoxicity t i t r a t i o n . To determine the cytotoxicity score, 5 ml of undiluted t e s t eluate was placed into each of two 60 mm c e l l culture dishes containing monolayers of WI-38 c e l l s from which complete MEM medium had been aspirated. The plates containing t e s t eluates were held a t 3 7 O C in a humidified (90% r e l a t i v e humidity) atmosphere containing 5-7% carbon dioxide for 24 hours, a f t e r which time a l l f l u i d s were decanted and c e l l s were fixed in methanol a n d stained w i t h Papanicolaou hematoxylin s t a i n (Fisher S c i e n t i f i c Co.). Culture dishes were microscopically (lOOX) scored as t o the degree of morphologic a l l y discernable cytotoxicity. Cytotoxicity was sub,jectively scored from 0 to 4 , 0 indicating a negative t e s t and 4 indicating v i r t u a l l y complete c e l l destruction. Cytotoxici ty t i t e r s were determined by preparing two-fol d d i l u t i o n s (2-128) of t e s t eluates i n complete MEY medium with subsequent application of 0.25 ml of each d i l u t i o n of t e s t eluate t o each of two wells in a Microtest I1 p l a t e which contained monolayers of WI-38 c e l l s from which a l l medium had previously been aspirated. Microtest I1 p l a t e s , containing monolayers o f WI-38 c e l l s and two-fold dilutions of t e s t eluates were incubated, fixed, stained, and morphologically scored as described under procedure f o r cytotoxicity score. The cytotoxic i t y t i t e r was defined as the reciprocal of the highest d i l u t i o n of t e s t e l u a t e t h a t induced morphologically discernable a l t e r a tion of WI-38 c e l l s a s determined by microscopic (100 X ) observation of stained c e l l preparations. C.

Systemic Injection Tests o f Saline and Cottonseed Oil Eluates i n Mice

Methods used in the performance of this t e s t have been delineated i n U.S.P. X I X ( 2 7 ) . Test materials were eluted w i t h

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BIOCOMPATIBILITY OF BIOMATERLALS

sodium chloride i n j e c t i o n , U.S.P. (Cutter Laboratories) (herea f t e r referred t o as s a l i n e ) and refined cottonseed o i l (VWR S c i e n t i f i c Co.) Random-bred female mice, ranging in weight between 1 7 and 23 g , were used as indicators of t o x i c i t y . Saline eluates were injected intravenously a t a dose of 50 ml/kg body weight. In a similar manner, cottonseed o i l eluates were injected i n t r a p e r i toneally a t a dose r a t e of 50 ml/kg body weight. All mice were observed f o r abnormal c l i n i c a l signs and death, a t 4 hours postinoculation and a t 24-hour i n t e r v a l s t h e r e a f t e r f o r a t o t a l of 72 hours. D.

Intracutaneous Tests of Saline, Complete MEM Medium, and Cottonseed Oil Eluates i n Rabbits

Test protocol was based on U.S.P. X I X (27) and u t i l i z e d female New Zealand white r a b b i t s , weighing 2.5-3.5 kg. Test eluates of s a l i n e (50 o r 70°C), complete MEM medium (37OC), and cottonseed o i l (50 or 70°C) were injected (0.2 ml) intracutaneously a t ten l i n e a r l y arranged s i t e s , parallel to the spinal column, on each of two rabbits. The procedure was modified from t h a t described i n U.S.P. X I X i n t h a t 7 t e s t eluates and 1 control were injected (10 injection s i t e s per e l u a t e ) on each r a b b i t . Injection l i n e s were demarcated by outlines prepared on the shaved skin w i t h p i c r i c acid. Injection s i t e s were examined a t 24, 48, and 72 hours postinoculation f o r evidence of erythema, edema, and necrosis and were scored according to the following numerical scale: 0 = no discernable response 1 = t r a c e response 2 = s l i g h t response 3 = mild response 4 = moderate response

E.

Intramuscular Implantation Test i n Rabbits

Female New Zealand white r a b b i t s , weighing 2.5-3.5 kg, were used as t e s t subjects. Test procedure was basically as described

239

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240

MILSNACK

i n U.S.P. XIX ( 2 7 ) . Test materials and U.S.P. negative controls were prepared i n strips 1 x 1 x 10 mn and inserted i n t o the paravertebral muscles, of each of 2 r a b b i t s , t h r o u g h a 15 ga needle, w i t h 4 t e s t s t r i p s and 4 control s t r i p s per r a b b i t . After 3-7 days, rabbits were euthanized, muscle surrounding the sample was excised, and the tissues circumscribing the implants were macroscopically examined f o r evidence of hemorrhage, inflammation, and encapsulation necrosis.

F.

Preparation and Quantitation of Heavy Metal Solutions

Solutions o f BaCl ( 9 9/25 ml), 3 CdS04.8H20 (28 9/25 ml) and Pb (NO3)2 (9g/25 m l ) were prepared in complete MEM medium and were allowed t o s t a b i l i z e f o r 24 hours a t 37OC. Solutions were then adjusted t o pH 7.2 w i t h 10N NaOH and p r e c i p i t a t e s were removed by centrifugation. Control media were prepared f o r each metal solution w i t h a comparable volume of 10N NaOH added with subsequent adjustment o f a l l control solutions t o pH 7.2 w i t h 10N H C l . Final concentration o f metal ions, i n each respective solution, was determined by atomic absorption spectroscopy on an atomic absorption spectrophotometer (Perkin-Elmer Model 303). Five-fold d i l u t i o n s o f each metal solution, as well as accompanying control solutions, were prepared i n complete MEM medium. Each appropriate d i l u t i o n (0.25 ml) was placed into each of 4 wells of a Microtest I1 p l a t e containing a monolayer of WI-38 c e l l s from which growth medium had been previously a s p i r ated. Plates containing monolayers o f WI-38 c e l l s and d i l u t i o n s o f metal solutions were held f o r 24 hours a t 37OC i n a humidified atmosphere (90% r e l a t i v e humidity) containing 5-7% carbon dioxide. Plates were then fixed, stained, and scored f o r cytotoxicity as previously described. G.

Preparation of Endotoxin Solutions

m)

was purchased (Mallinckrodt) in powder Endotoxin (E. form, and two-fold d i l u t i o n s were prepared i n complete MEM

BIOCOMFATIBILITY OF BIOMATERIALS medium.

241

Only those d i l u t i o n s w i t h o u t v i s i b l e p r e c i p i t a t e were

tested f o r cytotoxic effect. C y t o t o x i c i t y t i t e r was determined u t i l i z i n g procedures

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described f o r heavy metals.

H.

Test Specimens M a t e r i a l s f o r t e s t were s e l e c t e d a t random from a t o t a l

group submitted f o r v a r i o u s forms o f b i o c o m p a t i b i l i t y t e s t i n g . Test specimens i n c l u d e d f i n i s h e d products and raw m a t e r i a l s ; however, t h e m a j o r i t y o f t h e m a t e r i a l s t e s t e d were u n f a b r i c a t e d raw m a t e r i a l .

Raw m a t e r i a l s were t e s t e d i n a v a r i e t y o f c o n f i g -

u r a t i o n s i n c l u d i n g sheets, tubing, p e l l e t s , granules, blocks, c y l i n d e r s , and screens.

111. A.

RESULTS

C y t o t o x i c i t y o f Barium, Lead, and Cadmium i n HI-38 C e l l Culture Cadmium induced morphologic a l t e r a t i o n o f exposed c e l l s a t

concentrations as low as 25 ppm, barium was c y t o t o x i c a t a h i g h e r l e v e l o f 500 ppm, and c y t o t o x i c i t y was n o t observed w i t h l e a d concentrations up t o 1000 ppm (Table 2).

Higher c o n c e n t r a t i o n s

o f l e a d c o u l d n o t be maintained i n s o l u t i o n i n complete MEM medium w i t h o u t f o r m a t i o n o f a p r e c i p i t a t e . Barium, lead, and cadmium c y t o t o x i c i t y was q u a n t i f i e d s i n c e these heavy metals have h i s t o r i c a l l y been present i n e l u a t e s of some m a t e r i a l s used i n t h e f a b r i c a t i o n o f medical devices and c o u l d be a f a c t o r i n the i n g r e d i e n t - s p e c i f i c i n t e r p r e t a t i o n o f c y t o t o x i c it y

B.

.

C y t o t o x i c i t y o f Endotoxin i n WI-38 C e l l C u l t u r e C y t o t o x i c i t y was noted i n those t e s t p r e p a r a t i o n s c o n t a i n i n g

167 pg/ml o r more o f endotoxin

(g. a) (Table

3).

Endotoxin

242

ULLSNACK

TABLE 2 D e t e c t i o n L i m i t s o f Barium, Lead, and Cadmium i n 141-38 C e l l Cultures

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C y t o t o x i c i t y o f Compounds Tested Concentration, ppma 1,000 500 100 50 25 10

B a r i um

LeadC

Cadmi um

4b 4 0 0 0 0

0 0 0 0 0 0

4 4 4 4 2 0

aCompounds d i l u t e d i n MEM + 10% FBS b C y t o t o x i c i t y score ‘Higher concentrations (>1,000 pprn) could n o t be maintained i n solution without precipitation

c y t o t o x i c i t y was t i t r a t e d t o p r o v i d e a p o i n t o f reference i n t e s t environments i n v o l v i n g contamination o f a b i o m a t e r i a l w i t h Gramnegative b a c t e r i a .

TABLE 3 D e t e c t i o n L i m i t s o f Endotoxin (&. i n WI-38 C e l l C u l t u r e s

Concentration o f a Endotoxi n, u g h 1

a)

C y t o t o x i c i t y Score

167 83 41 aEndotoxin d i l u t e d i n MEM + 10% FBS

243

BIOCOMPATIBILITY OF BIOMATERIALS C.

O v e r a l l C o r r e l a t i o n o f WI-38 C e l l C u l t u r e t o I n Vivo Tests C e l l c u l t u r e - p o s i t i v e specimens were i d e n t i f i e d i n a l l

classes of t e s t m a t e r i a l except polypropylene and n y l o n (Table

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4). P o l y v i n y l c h l o r i d e (PVC) p l a s t i c s , rubber, and s i l i c o n e rubbers evoked t h e h i g h e s t p r o p o r t i o n o f p o s i t i v e responses i n c e l l culture tests.

A

t o t a l o f 1013 specimens were t e s t e d y i e l d -

i n g one c l a s s o f 115 m a t e r i a l s p o s i t i v e i n c e l l c u l t u r e and one o r more i n v i v o t e s t s and a second r e a c t i v i t y c l a s s c o n s i s t i n g o f specimens p o s i t i v e i n c e l l c u l t u r e and negative i n i n v i v o t e s t s . C y t o t o x i c i t y t i t e r s , an approximation o f t h e b i o a v a i l a b i l i t y o f t h e t o x i c moiety, were c l e a r l y h i g h e s t among t h e rubber compounds.

S i x hundred t h i r t e e n o f t h e specimens were n e g a t i v e

by a l l t e s t s . With t h e exception o f one specimen, none o f t h e t e s t samples n e g a t i v e i n t h e c e l l c u l t u r e t e s t were p o s i t i v e by an

-i n vivo

t e s t (Table 4 ) .

This specimen was a Teflon@ polymer,

and inadequate q u a n t i t y was a v a i l a b l e t o c o n f i r m a b o r d e r l i n e r e a c t i o n o f a cottonseed o i l e l u a t e i n t h e intracutaneous r a b b i t test.

As a measure o f t h e r e p r o d u c i b i l i t y o f t h e c e l l c u l t u r e t e s t , 50 specimens were s e l e c t e d by random number t a b l e t o be r e t e s t e d a t t h e conclusion o f t h e study.

On t h e b a s i s of c y t o -

t o x i c i t y score, none o f t h e r e t e s t s d e v i a t e d more than 2 u n i t s from t h e o r i g i n a l score (Table 5 ) .

D.

P o l y v i n y l C h l o r i d e (PVC) P l a s t i c s One hundred f i f t y - t w o PVC p l a s t i c specimens, r e p r e s e n t i n g

a wide range o f formulations, were t e s t e d y i e l d i n g 74 (49%) samples c e l l c u l t u r e - p o s i t i v e . S i x t y - t w o (84%) o f these specimens were n e g a t i v e i n a l l animal t e s t s (Table 6). Systemic i n j e c t i o n t e s t s on c e l l c u l t u r e - p o s i t i v e m a t e r i a l s were n e g a t i v e w i t h a l l e l u a t e s ; intracutaneous t e s t s were n e g a t i v e w i t h s a l i n e e l u a t e s and p o s i t i v e i n 9 o f 69 specimens w i t h cottonseed o i l

0-8

4

No. of specimens negative i n c e l l culture 1 untested i n animals

T i t e r range I n c e l l culture

0

74

5

No. o f specimens negative i n c e l l c u l t u r e L positive 3n animal t e s t s

negative i n c e l l culture L animal tests

No. o f speclmens

No. o f specimens porltive I n c e l l c u l t u r e & untested I n a n i w l tests

c u l t u r e h negat i v e i n animal tesb 62

7

No. o f specfmens positfve i n c e l l c u l t u r e & animal tats

No. o f specimens positive I n c e l l

152

Total nmber specimens tested

0-64

0

0

101

2

127

66

296

0

0

202

0

0

0

202

0-32

0

0

72

0

14

22

108

0-2

0

0

16

0

1

0

17

0-16

0

0

4

0

?1

9

24

0-2

0

0

19

0

3

0

22

PolyPolySilicone PVC Rubber propylene ethylene Epoxy Rubber Acetal

a- 1

0

1

9

0

0

1

11

0-2

0

0

6

0

1

1

8

0-2

0

0

19

0

1

0

20

0-32

0

0

36

0

25

2

63

0-32

0

0

46

0

28

7

81

4

1

613

7

237

115

1013

PolyPolyCanposi t i o n Nylon Teflon urethane styrene Misc. unknm Total

Sumnary o f Specimens Tested for Cytotoxicity i n HI-38 Cell Cultures

TABLE 4

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1w

5

245

BIOCOMPATIBILITY OF BIOMATERIALS TABLE 5

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R e p r o d u c i b i l i t y o f C y t o t o x i c i t y (MEM Eluate) i n WI-38 C e l l C u l t u r e s O r i g i n a l C y t o t o x i c i t y Score 0 1 2 3 4 Number o f samples y i e l d i n g same c y t o t o x i c i t y score on r e p e a t t e s t a

31

0

Number o f samples y i e l d i n g 1 u n i t d e v i a t i o n i n cytot o x i c i t y score on repeat test

0

0

2

0

0

Number o f samples y i e l d i n g 2 u n i t deviation i n cytot o x i c i t y score on repeat test

0

1

0

1

1

T o t a l Number o f Samples

0

1

1

3

50

aSamples s e l e c t e d f o r repeat t e s t by random number t a b l e

e l u a t e s (Table 6).

Intramuscular i m p l a n t t e s t s were p o s i t i v e i n

1 o f 22 t e s t s on c e l l c u l t u r e - p o s i t i v e PVC p l a s t i c s (Table 6). The p r o p o r t i o n o f c e l l c u l t u r e - p o s i t i v e m a t e r i a l s y i e l d i n g p o s i t i v e r e s u l t s on an i n v i v o t e s t i n g was n o t a f u n c t i o n o f t h e cytotoxic t i t e r , i.e.

t h e two PVC p l a s t i c specimens w i t h t h e

h i g h e s t c y t o t o x i c t i t e r (1:8) were negative i n v i v o (Tables 6 and 7).

None o f t h e c e l l c u l t u r e - n e g a t i v e specimens were p o s i -

t i v e i n vivo. P a t t e r n s and t i t e r s o f c y t o t o x i c i t y were f o r m u l a t i o n s p e c i f i c ; and d i f f e r e n c e s i n response, as measured by c e l l c u l t u r e and i n v i v o t e s t s , were a t t r i b u t a b l e t o t h e e l u t i o n and/or t h e t a r g e t phase o f t h e c e l l c u l t u r e t e s t .

The e l u t i o n phase o f

t h e t e s t was assessed by performing intracutaneous t e s t s w i t h complete MEM e l u a t e s and negative r e s u l t s were observed i n most instances (Table 8). C l e a r l y , t h e t a r g e t phase o f t h e c e l l

4 2

4

a

CSO

-

cottonseed oil

aNumerator = number specimens positive Denominator = number specimens tested

o/ 2

O/ 3

0118

19

2

1

0/65 a O/ 39

78 49

o/ 2

0161 0135 0117 O/ 3

Systemic Injection Tests Saline Eluate CSO Eluate

0

Cel’l Culture Cell Culture Number Titer Specimens

0/2

0139 Dl1 9 O/ 4

0/72

O/ 2

1/ 4

3/19

5/44

0174

Animal Test Results Intracutaneous Tests Saline Eluate CSO Eluate

a/ 2 o/ 3 a/ 1

1/16

0/23

Intramuscular Implant

Quantitative Correlation Between WI-38 Cell Culture Cytotoxicity Titers, Systemic Injection, Intracutaneous, and Intramuscular Implant Tests for PVC Plastics

TABLE 6

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1 1 1 2

2 2 4 4 4

8 8

74T-78

74T-488

74T-82

74T-500

74T-614

74T-349 74T-350 73T-74

74T-460

74T-458

CSO

-

cottonseed oil

74T-465

Cell Culture Titer

Sample Number

0

0

0 0

0 0

0

0

0

0

0

0

0

0 0

0

0

0

0

0

0 0

0

0

0

0 0

0

0

0

+

+

0

0

0

+

0

0

+ 0

+

Intramuscular Implant

-+

In tracu taneous Tests Saline Eluate CSO Eluate

0 0 0

0

Systemic Injection Tests Saline Eluate CSO Eluate

Correlation Between WI-38 Cell Culture Cytotoxicity Titers, Systemic Injection, Intracutaneous, and Intramuscular Implant Tests for Selected PVC Plastic Specimens

TABLE 7

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H

TABLE 8

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Formul ation-Specif i c Comparison of PVC P l a s t i c Cytotoxicity T i t e r s in WI-38 Cell Culture with Intracutaneous Tests on CSO a n d MEM Eluates Intracutaneous Rabbit Test MEM Eluate

Cell Culture Cytotoxicity T i t e r

C S0 Eluate

BB

cc

1 8 1

0 0 0

OD

2

DD

1

0 0

PVC Formulation

AA a

0 0 0 0 0

DD EE FF GG HH

0 0 0

0 0 0

0 0

0 +l

I1 JJ AA

0 0 0 0

KK LL

MM HH

AA

0

+1 0 0

0

0

0

0 0 0

+4

NN

+3 +3

00

+4

NTO b

aSamples w i t h same l e t t e r designations a r e multiple batches of the same formulation CSO - cottonseed o i l bNT - not tested

culture t e s t , WI-38 c e l l s , detects cytotoxic moieties eluted from PVC p l a s t i c formulations t h a t were not demonstrable w i t h the same e l u a t e i n vivo (Table 8 ) . E.

Rubber

Rubber formulations proved the most toxic compound group, i n terms of cytotoxic t i t e r as well as percentage of specimens

BIOCOMPATIBILITY OF BIOMATERIALS

249

p o s i t i v e , i n c e l l c u l t u r e as w e l l as i n v i v o .

One hundred

n i n e t y - f i v e (66%) o f 296 Specimens t e s t e d proved c y t o t o x i c i n c e l l c u l t u r e , and 127 (65% of t h e c e l l c u l t u r e - p o s i t i v e samples were n o t r e a c t i v e i n v i v o (Table 9).

The systemic i n j e c t i o n t e s t

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y i e l d e d negative r e s u l t s w i t h a l l s a l i n e e l u a t e s (162) and 130 o f 132 cottonseed o i l e l u a t e s from c e l l c u l t u r e - p o s i t i v e specimens (Table 9).

S i m i l a r l y , a l l 98 s a l i n e e l u a t e s were n e g a t i v e i n t h e

intracutaneous t e s t s corresponding t o p o s i t i v e c e l l c u l t u r e t e s t s . Intracutaneous t e s t s w i t h cottonseed o i l e l u a t e s proved t h e most s e n s i t i v e o f t h e i n v i v o t e s t s as evidenced by 72 (38%) p o s i t i v e t e s t s o f 192 c e l l c u l t u r e - p o s i t i v e specimens t e s t e d (Table 9 ) . The p r o p o r t i o n o f specimens p o s i t i v e w i t h t h e intracutaneous t e s t (cottonseed o i l ) e l u a t e d i d n o t vary as a f u n c t i o n o f t h e c y t o t o x i c t i t e r i n c e l l c u l t u r e (Tables 9 and 10).

Intramuscular

i m p l a n t a t i o n proved a r e l a t i v e l y i n s e n s i t i v e t e s t w i t h 5 o f 29 t e s t s p o s i t i v e , on m a t e r i a l s c y t o t o x i c i n c e l l c u l t u r e s , w i t h o u t q u a n t i t a t i v e r e l a t i o n s h i p t o c e l l c u l t u r e t i t e r (Table 11). o f t h e specimens negative

None

n c e l l c u l t u r e demonstrated r e a c t i v i t y

i n any i n v i v o t e s t . I n an attempt t o e l u c date t h e mechanism o f a c t i o n causing a discrepancy i n t h e t o x i c r e a c t i v i t y o f rubber f o r m u l a t i o n s v i v o and i n v i t r o , complete MEM e l u a t e s from c e l l c u l t u r e - p o s i t i v e specimens were i n j e c t e d i n t r a c u t a n e o u s l y i n t o r a b b i t s .

Twenty

complete MEM e l u a t e s w i t h c y t o t o x i c t i t e r s 16 o r greater, prepared from specimens negative i n t h e intracutaneous t e s t by cottonseed o i l e l u t i o n , y i e l d e d 14 p o s i t i v e intracutaneous t e s t s (Table 12).

Complete MEM medium leached t o x i c m o i e t i e s from

rubber f o r m u l a t i o n s t h a t were b i o l o g i c a l l y u n a v a i l a b l e by s a l i n e o r cottonseed o i l e x t r a c t i o n .

I n a d d i t i o n , t h e complete MEM

e x t r a c t i o n s were performed a t 37°C as compared t o 50°C f o r t h e s a l i n e and cottonseed o i l e x t r a c t i o n s . F.

Polyethylene Although 36 o f 108 p o l y e t h y l e n e samples t e s t e d were c y t o -

t o x i c i n c e l l c u l t u r e , none of t h e p o s i t i v e specimens repre-

1/9

o/ 2

0/39 0/52

0/9

o/ 2

45

67

17 3

64

CSO

-

cottonseed o i l

= number o f specimens p o s i t i v e anurnerator Denominator = number o f specimens t e s t e d

32

0/13 0/19

14 19

2

4 8 16 1/44

0/27

0/13 0/13

0/24

30

O / l 01

0/28

101

1

0/101 a

Systemic I n j e c t i o n Tests S a l i n e E l u a t e CSO E l u a t e

0

Cell Culture Cell Culture Number Titer Specimens

O/ 2

O! 7

0/35

0/19

2/ 3

12/17

30/66

3/19 13/44

2/14

0/10

o/a

0/101 10/29

0/17

0/101

Animal Test Results Intracutaneous Tests S a l i n e Eluate CSO E l u a t e

Q u a n t i t a t i v e C o r r e l a t i o n Between MI-38 C e l l C u l t u r e C y t o t o x i c i t y T i t e r s , Systemic I n j e c t i o n , Intracutaneous, and Intramuscular Implant Tests for Rubber

TABLE 9

o/ 1

31 5

1/15

o/ 1

o/ 1

1/3

o/ 3

O/ 28

Intramuscular Imp1a n t

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BIOCOMPATLBILITY OF BIOMllTERLALS

In

.r

E 0

7

c,

3

m

LL

0

E

n L a,

O

m J Y

2 f

n 3

'c 0

Y

:'c QI

a h 0 L

.r .r

c,

u

c, X

3 0 c, 0

251

0 0 0

2 2 4

74T-297

74T-568

CSO -

cottonseed o i l

64

73T-179 0

0

0

32

74T-159 0

0

0

0

0

0 0

16

73T-123

0

0

0

i.

0

0

0

0

8

16 16

0

0 0

0

73T-39 73T-93

0

0

0

0

0

+ +

0

0

t

0

0

0

0

Intracutaneous Tests Saline Eluate CSO Eluate

74T-351

0

1

74T- 20 1

73T-86

Systemic I n j e c t i o n Tests Saline Eluate CSO Eluate

C e l l Culture Titer

Sample Number

0

+

0

0

+

0

0 0

+

0

Intramuscular Implant

Correlation Between WI-38 C e l l Culture C y t o t o x i c i t y T i t e r s , Systemic I n j e c t i o n , Intracutaneous, and Intramuscular Implant Tests f o r Selected Rubber Specimens

TABLE 11

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N

N

cn

253

BIOCOMPATIBILITY OF BIOMATERIALS TABLE 12

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Formulation-Speci f i c Comparison o f Rubber C y t o t o x i c i t y T i t e r s i n WI-38 C e l l C u l t u r e w i t h Intracutaneous Tests on CSO and MEM Eluates Rubber Formulation

Cell Culture Cytotoxicity Titer

C a F C C

I F U A C F

Intracutaneous Rabbit Test CSO E l u a t e MEM E l u a t e

16 16 16 16 16

0 0 0

16

0

32

0 0

16 16 16

+2 +2 +3 +3

0 0

16 16 16 16 16

0 0 0

C

I I

+3 +2 +2

0 0

F F C C C A K

0 0

+2 0 0

+2

0

0

0

+2

16 16

0 0

+3

32 32 32

0 0 0

+2 +3 0 +3

aSamples w i t h t h e same l e t t e r designations a r e mu1 t i p l e batches o f t h e same f o r m u l a t i o n CSO cottonseed o i l

-

sented v i r g i n polymer.

A l l o f t h e t o x i c responses were a t t r i b -

u t a b l e t o pigments, adhesives, o r o t h e r a d d i t i v e s (Table 13). S a l i n e e l u a t e s from p o l y e t h y l e n e samples were n e g a t i v e i n systemic i n j e c t i o n t e s t s w i t h t h e exception o f one specimen c o n t a i n i n g a formaldehyde r e s i d u e (Table 13).

P o s i t i v e responses

were evoked b y cottonseed o i l e l u a t e s i n 1 o f 108 systemic i n j e c t i o n t e s t s and 22 o f 108 intracutaneous t e s t s (Table 13).

The

degree o f inflammatory o r p a r e n t e r a l response observed i n v i v o

WILSNACK

254 TABLE 13

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Correlation Between WI-38 Cell Culture Cytotoxiclty Titers and U.S.P. Tests for Various Polymers

Cell Culture

Animal Test Results Systemic Injection

Cell Culture T I ter

Number Specimens

Polyethylene 0 1

72

2

17 9

4

4

8 16

1

32

1 4

0 2

16 1

Saline Eluate

CSO Eluate

Intracutaneous Tests Saline Eluate

CSO Eluate

0/72 12/17 5/9 014 011

111 1/2

o/ 1

0/16

0116 011

0/19 012 01 1

O/ 2

4/4

Acetal 0 1 2

19

0 1

10 1

2 1

0/19

o/ 1 1/10

111

Polyurethane 0 1

2

6 1 1

0/6 0/1 o/ 1

016 01 1 011

0/6 01 1 011

Polystyrene 0

19

2

1

0/19

o/ 1

0119 011

o/ 1

0119 O/l

‘Numerator = number specimens positive Denomhator = number specimens tested

CSO

- cottonseed o i l

d i d n o t c o r r e l a t e t o c y t o t o x i c t i t e r i n c e l l c u l t u r e , a phenomenon p r e v i o u s l y observed w i t h PVC p l a s t i c and rubber f o r m u l a t i o n s .

No t o x i c i n v i v o t e s t s were noted among t h e group o f specimens n e g a t i v e i n the c e l l c u l t u r e t e s t .

BIOCOMPATIBILITY OF BIOMATWIALS G.

255

Epoxies The epoxy compounds t e s t e d were b i o l o g i c a l l y i n e r t w i t h t h e

exception o f a s i n g l e specimen p o s i t i v e i n c e l l c u l t u r e and nega-

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t i v e i n a l l i n v i v o t e s t s (Table 13). H.

Acetals Twenty-two a c e t a l s specimens were t e s t e d and found t o be

devoid o f t o x i c i t y i n v i v o as assessed by s a l i n e and cottonseed o i l e x t r a c t i o n (Table 13).

Three o f t h e 22 specimens evoked low

l e v e l c y t o t o x i c i t y i n c e l l c u l t u r e , and a l l o f t h e p o s i t i v e specimens were samples o f separate l o t s o f t h e same m a t e r i a l formulation.

I.

Teflons@ Consistent w i t h p a s t r e p o r t s o f b i o l o g i c i n e r t n e s s ( 1 7 ) , 2 o f

11 Teflon@ specimens e l i c i t e d t o x i c responses i n c e l l c u l t u r e o r A s i n g l e specimen was c y t o t o x i c i n c e l l c u l t u r e and r e a c t i v e i n t h e intracutaneous t e s t when assessed by animal t e s t s (Table 13).

cottonseed o i l e x t r a c t i o n .

The second b i o l o g i c a l l y a c t i v e speci-

men e x h i b i t e d a t o x i c i t y p a t t e r n a b e r r a n t t o t h e e n t i r e study; a cottonseed o i l e x t r a c t evoked an inflammatory response upon intracutaneous i n j e c t i o n ; and t h e c e l l c u l t u r e t e s t was negative. T h i s specimen was n o t r e p r e s e n t a t i v e o f t h e v i r g i n polymer and contained pigmented a d d i t i v e s .

J.

Polyurethane

Two o f e i g h t polyurethane specimens, uncategorized as t o p o l y e t h e r o r p o l y e s t e r type, were c y t o t o x i c i n c e l l c u l t u r e ; and one o f t h e two was recorded p o s i t i v e by v i r t u e o f cottonseed o i l e x t r a c t i o n and intracutaneous i n j e c t i o n (Table 13). i n j e c t i o n t e s t s were negative.

A l l systemic

None o f t h e specimens n e g a t i v e i n

c e l l c u l t u r e were p o s i t i v e i n vivo.

WILSNACK

256 K.

Polystyrene B i o i n c o m p a t i b i l i t y was noted i n a s i n g l e specimen o u t o f a

t e s t group o f 20 (Table 13).

The s i n g l e p o s i t i v e response was

r e p o r t e d i n c e l l c u l t u r e , and a l l i n v i v o t e s t s were devoid o f Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by York University Libraries on 08/23/13 For personal use only.

a t o x i c response. L.

Miscellaneous and Formulations o f Unknown Composition T h i s specimen category i n c l u d e d those f o r m u l a t i o n s f o r which

an inadequate number o f l o t s were t e s t e d f o r meaningful a n a l y s i s and a more d i v e r s e polymer group i n c l u d i n g those specimens subm i t t e d w i t h o u t d e f i n i t i v e i d e n t i f i c a t i o n as t o f o r m u l a t i o n . Sixty-two o f 144 miscellaneous and u n i d e n t i f i e d specimens were c y t o t o x i c i n c e l l c u l t u r e , some a t t a i n i n g c y t o t o x i c t i t e r values o f 32 (Table 4).

Nine o f t h e 62 c e l l c u l t u r e p o s i t i v e specimens

were r e a c t i v e i n i n v i v o t e s t s , a l l by cottonseed o i l e x t r a c t i o n and intracutaneous i n o c u l a t i o n . M.

S i l i c o n e Rubbers S i l i c o n e rubbers, undelineated as t o heat cure o r room

temperature cure, proved a d i v e r s e specimen group i n terms of biocompatibility.

Twenty o u t o f 24 s i l i c o n e rubber specimens,

r e p r e s e n t a t i v e o f more than one b a s i c m a t e r i a l manufacturer, were c y t o t o x i c i n c e l l c u l t u r e ; and 9 o f t h i s c y t o t o x i c group were t o x i c i n v i v o (Table 14).

Intramuscular i m p l a n t a t i o n was t h e

most d e f i n i t i v e o f t h e i n v i v o t e s t s f o r s i l i c o n e rubber, d e t e c t i n g t o x i c i t y i n 6 specimens n e g a t i v e by cottonseed o i l e x t r a c t i o n and intracutaneous i n j e c t i o n (Table 14).

This relationship

between t o x i c response detected by i n t r a m u s c u l a r i m p l a n t a t i o n and intracutaneous t e s t s i s unique t o s i l i c o n e rubber and has n o t been observed w i t h any o t h e r c l a s s o f polymers. The s e v e r i t y o f t h e intracutaneous t e s t response, as measured by t h e cottonseed o i l e x t r a c t i o n , was n o t d i r e c t l y p r o p o r t i o n a l t o t h e c y t o t o x i c t i t e r s as evidenced by t h e f a c t t h a t t h e 5 specimens evoking t h e h i g h e s t c e l l c u l t u r e t i t e r were n e g a t i v e by t h e i n t r a cutaneous t e s t w i t h cottonseed o i l e l u a t e s (Table 14).

BIOCOMPATIBILITl OF BIOMATERLALS

257

TABLE 14

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Quantitative Correlation Between WI-38 Cell Culture Cytotoxici ty Titers, Intracutaneous, and Intramuscular Implant Tests for Silicone Rubber Cell Culture Cell Culture Number Titer Specimens 0 2 4 8 16

4 1 5 5

3

Animal Test Results Intracutaneous Test Intramuscular CSO Eluate Implant

0/4 a o/ 1 O/ 5 21 5 1/3

0/2

o/ 1

4/5 3/4 2/ 3

aNumerator = number of specimens positive Denominator = number of specimens tested CSO - cottonseed oil

Analysis of test results for the silicone rubber group was impeded by the inability to identify specific formulations. It is reasonable to assume that silicone rubber formulations are not monolithic in terms of biocompatibility, but exhibit formulationspecific toxicity profiles as previously demonstrated for rubber and PVC plastic.

IV. DISCUSSION Cell culture methods have been utilized and reported for over a decade, in a wide variety of modes, to assess the bioincompatibility of medical devices and materials. Data presented in this report is intended to extend previous reports and to accommodate those variables most critical to the extrapolation of in vitro data to the clinical environment. Conceptual elements of the study include: (A) use of a normal human cell, unmodulated by virus (30) or mycoplasma contamination (311, as a target system since the products tested are intended for human use; ( B ) use of large number

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258

WILSNACK

of specimens t o measure biologic compatibility of a variety of formulations and multiple l o t s of s p e c i f i c formulations; ( C ) quantit a t i v e assessment of cytotoxicity t o allow correlation s t u d i e s ; ( D ) use of physiologically normal elution menstrua and temperatures t o permit an evaluation of the b i o a v a i l a b i l i t y of toxic moieties; and ( E ) provide f o r the correlation of a l l i n v i t r o data w i t h accepted in vivo systems i n U.S.P. animal t e s t s . The WI-38 c e l l c u l t u r e t e s t employed i n this study (26) defined a larger proportion of toxic specimens than any of the animal t e s t s . Cytotoxic p r o f i l e s were unique to classes of materials; and i n many instances, were quantitatively and q u a l i t a t i v e l y characteri s t i c o f s p e c i f i c material formulations. If a small number of formulations had been tested f o r each material c l a s s , an array o f cont r a s t i n g conclusions on t e s t methods could have been tendered depending on the p a r t i c u l a r formulation tested. Rubber specimens yielded the highest percentage of cytotoxic samples (65%) i n v i t r o and fewest discrepancies (43%) between fi vitro and in v i v o t e s t r e s u l t s . Disparities i n c e l l culture and animal t e s t r e s u l t s were a t t r i b u t a b l e , i n some instances, t o an elution d i f f e r e n t i a l ; wherein complete MEM medium eluted toxic moieties a t 37OC not manifested by elution with cottonseed o i l o r s a l i n e a t 50-60°C. Toxic molecules in rubber were the most readily leachable as evidenced by the cytotoxic t i t e r range. Cytotoxicity p r o f i l e s , i n v i t r o and i n vivo, were formulation-specific and reproducible from l o t t o l o t . PVC p l a s t i c s manifested a high percentage (45%) of c e l l culture-positive samples w i t h a concomitantly low proportion (1 l % ) of these specimens positive i n animal t e s t s . The d i f f e r e n t i a l i n t e s t r e s u l t s i s circumstantially ascribable t o the t a r g e t phase ( c e l l cultures o r animals) of the t e s t system since e f f o r t s t o delineate differences i n the elution phases were negative. P o s i t i v i t y i n animal t e s t s was not proportional t o cytotoxic t i t e r s i n c e l l culture; however, none of the materials negative i n c e l l c u l t u r e were positive i n animals.

BIOCOMPATIBILITY OF BIOMATFRIALS

259

Polypropylenes, polyethylenes, most epoxies, nylons, most Teflon@,

and most polystyrenes were b i o l o g i c a l l y i n e r t , i n v i t r o

and i n vivo, under t h e a s c r i b e d t e s t parameters.

P o s i t i v e speci-

mens i n t h e polyethylene group were a r t i f a c t u a l i n t h e sense t h a t Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by York University Libraries on 08/23/13 For personal use only.

none were v i r g i n polymer and t o x i c i t i e s were a s c r i b a b l e t o additives.

A Teflon@ sample proved t h e a b e r r a n t specimen o f t h e e n t i r e

study by v i r t u e o f a p o s i t i v e inflammatory response ( i n t r a c u t a n e -

ous) t o a cottonseed o i l e x t r a c t and a negative c e l l c u l t u r e response. O v e r a l l , t h e WI-38 c e l l c u l t u r e t e s t proved a s e n s i t i v e and q u a n t i t a t i v e l y reproducible test.

I n comparison, animal t e s t s

were r e l a t i v e l y i n s e n s i t i v e , p a r t i c u l a r l y systemic i n j e c t i o n t e s t s ( 6 o f 944 p o s i t i v e ) ; whereas t h e intracutaneous t e s t w i t h c o t t o n seed o i l e x t r a c t i o n proved t h e most d e f i n i t i v e i n v i v o t e s t . Intramuscular i m p l a n t a t i o n proved u n i q u e l y s e n s i t i v e (among v i v o t e s t s ) f o r t h e bioassessment o f s i l i c o n e rubbers, d e t e c t i n g more p o s i t i v e specimens than t h e o t h e r animal t e s t s .

The b i o l o g i -

c a l s i g n i f i c a n c e o f t h e c e l l c u l t u r e - p o s i t i v e t e s t s , unconfirmed i n animals, i s dependent i n p a r t on t h e intended use o f t h e t e s t e d product.

R e a l i s t i c a l l y , these c e l l c u l t u r e - p o s i t i v e and animal-

negative t e s t s cannot be shunted as b i o l o g i c a l l y f a l s e s i n c e they represent t o x i c m o i e t i e s e l u t e d by p h y s i o l o g i c a l l y normal menstrua under p h y s i o l o g i c a l l y normal environments, p h y s i c a l and biochemic a l , t h a t d e s t r o y normal human c e l l s d u r i n g t h e 24-hour exposure.

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Quantitative cell culture biocompatibility testing of medical devices and correlation to animal tests.

The biocompatibility of a wide variety of biomaterials was quantitatively assessed, in a physiologically normal environment" as to cytotoxicity induce...
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