~ A 

¢~ V 

; ,`eP-,"i`_ 

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ASSESSMfiNT OF OCJL.aR ?RRIThNCY PQTENTIAL 

ON CUi.TURED PIBRQ$LASTS 

(REPORT 

: : ?,S, .`IGi'E:1BER :;~ 9L 
CC'NII`BTS 

Pages 

1. INTROnUCTFON 

2. AIIT1iE^17'ICATiON OF THE STtJ➢`i 

j.. EXPUZMENTAL PAOTOCOL 

3.1, PRTNCIC^iE 

3.2. F.XPERZME'dT'Ai. .viOnEL 

3.3. OFERP:TK:vG Pf?OGEBIJRE 

j.:l. Z?:TERPR1~TP.TZCN OF RESiSi;TS 

4. RESULTS - GOM1CLliSIO\ 

4n,31ESLiLTS 

i 

=} 

5 

c 

l 

7 

A 

7 

~ 

7 

J.'. t'!5`!^.LL'SSO*7 7 
1. zN':RaDOC"fSaN 

AC t'.ie refluest oP the Ca7IP4A4` f .. ., .. ,. 

.. , We navQ studied on rahbit cornea :L bccbLdst cultc:.res, 

the oc;li8c 's.rritancy Potenc'.ai Ci tile p"Coauct : 

':o periorT this tr=al, •.••E rer.e;.ved or, SePtember the 22nd, 1992, a saaple 

.:x.kz Che ?'ollow;ng c:liaracter_st:cs . 

Cantainer : plast:.c flask 

Quan..,tv : atout 1.00 ml 

ror+.o . : liqni.d 

Colour : translucent 

aii 

S ml ot' tnis se.vo'_e :aere mntrodured into a nermeticaSly stoapered glass 

r23si;. labpllea 92.1476, in order to be scored at room te.aperature. !'or 

v:11'ee ye,3r:s &i: :'1U3C, 

J 
2.AJ;':iENTSCA'Z'ICN OF THE STL'DI; 

1 the undensigned, Karo fl6UE-CR.ABt7T< Girector of the study, 

certiP,; tt:at ;he r+_ai (: rotocol. B'_C?GSR S.A. fte£. CCIO) wzs p=r.cor;nec 

eccorair.g to the 'nternai procednres, estBqlishecl on `_:Ye basis o`' Gcor,i 

Laboratory practices., ucdcr tne responsihility . 

Tech+. icai oP Genevieve Enft!vA.°DI'J, 

Scien.taf'ac secretary oi" lfari.e-Fasca?e nJU,.M"TE. 

TlYis study was e!:tirely com sietec on ii:e nremises oP the Ldboratoire 

s3IGG TiR. 5, A. (F 33611 Gn7I2vr.T CFDEX) . 

Ai1 the doau.men.Es related to t1.:..s study are stored u: der the rPferer.ce 

CF14 92.1476 i.n spcciai prem.ises cYf i.abor.3tJire BICGI'k 5.A, i'or three 

vears. 

r_t the e.nL1 of L:1_5 CQriod, tFe sCilCiy docUments wi.Ll Ce, a:ter 5pon8vr 

ajKreemen.t, e_.tFer trans:"erred in sponsor premi5es or destroyed. 

The :resent report is made eaP 7 pages numbered from 3 to ; and S taoie 

Ce.STAS, November che 20Ch, 1p 01 2 

Jearr-Pt"anGois PIi:ON 

4'cterin.n.rian Ph.[3 . 

SCIENTIPIC 0?REC'I't;i? 

r% 

v ;\:'En D_ftEuTCfi 

3*_•ig7t ~ e H:iLn1iI':I' 

QUni.:TY nSSuRANC:; 

[; 
3. EX;'ERT61ENT4G PROTOCqL 

':he trial «as Pet`Coracdbetween November the 4th and t'he 6ta, 1907, 

accor'ving to the experjaent31 protocol described below . 

33. PRIh'CIPLE 

T`:e teLt.nique is propo5eu', as an alternativa to tl;e URAIZE eye test and 

ailoias to aasess t<<e 'r^ita*:c;.= cotential of a p^odact depending on its 

cyta o;ieit; towaras cor:ea °'_5_^oblssLs. 

R?kerer.ces .. RUUi3-vfc-.PCT 1`?. 1 FIhGN (.;.,.`i et aI. Rul_- Soc. P1.<^_rm, 

EORaEAU}{. 19 ~~J, ?29, `:9-lu~ 

HOE?G-GRAEGT (}4.}, 't;Ai,nVI:1T (B=;, PTNGat (J.F.) kTLA, 20, 

307-312. 1992 

EOUE-cRABCT (ci, ), xAL.4VIAT (E. j, PZho?i (3. F. 4,°,T[.A, 20, 

445 -455, 1492 

srevet de eetha3e r: °807221e^ date du 21.12.89. 

3,2_ REACTIE EXPERSYtENTAL 

Culturqs >f SIRC st_rain ?ibrobAZsts 

°roYii 1'aiJ`rlit cor;Tefl, 

:ulture medium . PEM iMinirvm Essential Medium) supplemented witii 

foetal aa1£ ser::m (10 t zCS}. 

3.3. UPEtIATING ?R€ICECLF.x. , 

3 , 3.. },P i'r..l.ing a7i 9 . nlll LUre Ne.'.l.ci 

^'o:."JDl.95t.> sU_.7.".t.Veu ir_ <;uitaT'e Nedi::ul ^L'e _iaT.e: =. i7 T.C:1ns:EY.11 'i;iJ _ o ll 

cuiture ciaamuer in'vw°ts .3b i,UC ce?isiwe31} cor_sisting of a 0.4 i,!t nore 

si;e ui`=roEsorus mcmbrane ' i <<er ::;ealoc vo a wylindricalholdec^. 

.if t... ~ ,l^©']illg, t."to elil<u - B t'Y22mU6rS ,...L-` p.c20@d intb eiCh ..issue eUitl6kE,' 

pldt . ~ .. :=. 2 c n..,;:.~~n~.Tl,~ 'Sait ~ :.:... a~edi~+~7i , 9it.. f,t:e Iic:1OC'd,';° aflctioP. . Asr ~ St: 

tu e,!:e ~ ~ ;ie:.l EiQLt. I 7'.`'c:e 'u=iits "I ° . c.:t?atC1 at 37 9 !; f.':lli..Ilg 'dFl hUtlrs ':1: 

3 3 ; CO_ . 15 1 '..'_: .;LnesN ,er"c. 

5 
3.3.2. Appl.;cvtion o: product 

'r1i tBG i ncu0aL1.ot1„ `j Trw:+.sSvesl (R) Cel.l cik3-i:ui'C cf=2tnbet i!7serCs a?'e 

transferreci to a new cluster p'.ate, each well of wh;ch contained the test 

Pro=.uct_ 

T':ie platr is incubated at 374 for 30 minutes, 1 nour ar.d 4 hours {3 

treated cu'_ture chamberv pr.n ::ime}. 

I-r oarallet, j control culture c;`:'iac:bers transCsrrcd in clt:ster plates 

cotli,FininS Da'.

r:.fi ii: 6i]„ ttb3.c11 pCOVe:3 t9 be !1QP_-cyt(}tO:Z1C in prel].milic^,Pv: 

studi.i?s.. G7Gt'e .ini'r! bated for. 30 ful.-tutt.'8. 

30 1 cyxrJto:<ici.ty as5ay 

30 uir.ute5. _ hour ar>d 4 r3ours after product contact, tne ce11 viability 

in treated and control piates is assessec by 'siTz tesc (*) or Nestrai Red 

V*), iP oiaysiuochemic.a] iater.erence :accurs between product and M'ti`. 

Opcical density i00. 11 ot` ti,e oGGa=ned cclourec 6o'uti:n iv r!easureu ac 

570 za (P?TT test) or 540?i^ 1Neutral P,ed, on a spectropnotometer. 

ror eac:h cnntact tiLe, a cytotoxic:ty percentage ::s calculaued accordin g 

to the ^ar w :cla . 

;nea.n controi DD - me&n treated 4)D 

FU " (t ) - x 100 

mean Cuntrol OD 

A Meaz: C.tccc,xicc , 1^de:r ('r1C:) is .^.a_ca±ated a_on3ing to the For7llla . 

? ',30 mR) - 0. 01 X a (1 ?tC) t 0,05 :S ,A (4 :1=') 

(il,i = 

1 

kl MOSMANN iT.I. journal !}i =e,nu^c og.ca1 Me*_hod . o;, ~;P n' 0'3, '_4t~ j. 

F~E' ~E ~ : ::i: ~ 

., ~ , T,o ~~, ~ M3~'~. 

. . ~~ ;f; +r L',u G. r. c,. , c.a.tgy '.2ttecs. ~:~, ~av i_ . 

fi 
3.4. INTERPRETATION t1E RESULTS 

F'rom the value of 3iCI, an equivalent ocuiar irritation index (IO 

equivalent) was calculated according to the foi.lowing equatzons 

established Fr,om re5ults o6tained caith 150 cosmetia products : 

0 r MCI 5 lfi IO eyuivalent = 1.1? MCI 

10 <MCI s 30 10 equivalent = 0.46 h1ci t 7.14 

30 < h9CI s 74.2 10 2quivaicnt = 1.14 MCI - 14.74 

The ,irritancy rotential of t.he product was estimatad by the corresponding 

10 equivalent on th+: ha5is oP the classiYication proposed by the "Journal 

de la :epubl'_que Francaise" :iated Ju'_y the 10th, 1992, for assessment of 

priuiary ocuiar irritation. 

4. RESULTS - CANCLUSION 

4.1 RESULTS 

They a.re collected in Tabie 1. 

The product was studied 20 ;a diluted in culture medium deprived ot' fetal 

calf serum. The cell viability was assessed by 41TT test. 

4 contact time-dependant cytotoxicity saas recocded (4.7 to 66.7 ,Y of 

mortality). 

These values allowed to ootain a?7CI of 12.73, i.e. 10 equivalent of 12,9 

that allows `o consider i:he product as &LIGHTLY IRRITANT_ 

4.2 CONCLUSION 

Under experiinental c; ~ nclit'_ons oi the assay the results obtained with the 

product 1, allow us ta consider the risks of 

intolerance t•eaction, itt caSe o'z accidental cnntact wit:i oc;uiar mucosa, 

as oniy mild. 

`d 

p 

7 
DF,TEERMINrl?'IC?:! D^ LA. CX70TGX.''iCZT4 ^_'3bT_e8i2 I. 

I{{{ Gt °------------------------------- rTgb2® I) 

~ HSOGIR S..A_ y (EV.Bi,UAS"ION UF 'i:^'E CY,CTD:YICZTX) .... ... 

II code BL ogir S.A. 

(BSogi.r S.:i_ code) ` . . ...... 

Ref. 9roduit t7pezateur : G. I3ernardin 

 (product Ref.} ' ~ ~ (operator) . 

1"gpe Celiulaire ~ : Fibreb2aste5 s'ovche : S.S.B'.C. - cozn6e de 

(cellular type) (Bibrob2aets) ;Line) I.apin (]ta:bit Coz"nea) 

.. 511nemencemsnt- 'a# 7?ate : 4..11.1392 . (Piat,:ng) (Date) . 

........ Der.site Ce11q1a1s'a : 30 000 celSuies/puiCs 

(Cell.ular dens.ity) . (30,000 ceils/weli} 

Ik M.ilieu de cult.ure : MEAf + 10% S.v.=. 

(C;riture medium) + F.C.S. I0$) . 

. Traicement - $ Date : 6.11.1992 . --- 

(Treatme:lt) ;DareJ Psr I ' 

_ . . _ . . fiapl.ication produiL : (aa sucfe) ----- 

(aroduct application) ----- 

 D32u6 ~ x ~ A 20s (v/v) dans 

(D.iZuted) ----_ s..i.2ieu sans SYF 

i'lvec fi.li.t , ~ x M.T.T. X ~ 

~3 Protoco3s : (W1-th fi2m} ----- -- 

(FrotocoS) ---- f2ouc-a --- 

. Sar.s film ( ~ zveutre ~ C 

(Wythottt f11m) ---- (N.autra% --- 

lTed) 

.......... 

D.O. 2 

. ...... ..... 

Moye;ane (Mean) 

T6mczn 2 : Con"tart 30,10; : Contact 1H ~ Co7ttact 4H 

(Controi .) :(30r~a contact)r(I H contact} (Q H eontact) 

0.442 . 0.422 

~ 

...... .....:.:...... .....'.. ......=.__. ( ... -...,--.. 

t? 406 : 0.423 0.-72 1 0.129 

..................... ..._... ............._.._... Ij......... ... 

0.403 .. 0.347 , 0 ,22 4 

 0.417 0.397 . 0,207 0.?39 

------ ----------- 

pnpu1. ik 49.7 ~ 33.3 

.;iorra.'.ite 3 o.o . 4.7 . 50.3 ~ 66.7 

('s Morta2ity) 

Zndi.oe :loyen de Cytoeox±aice ;S.M.C.) 12.73 

(Mean cltotOxicity _Tndex - M.C.I.) 

1.0. SQt7zvRL. . 32.9 

OgS8RVr1T20NS :