### ~ A 

### ¢~ V ; ,`eP-,"i`_

• f . 

### 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?OGEB I JRE 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,



### J2.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 T iR. 5, A. (F 33611Gn7I2vr.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 0 1 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 PROTOCq 

### L



### ':he trial «as Pet`Coracd between 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: ° 807221 e^ date du 21.12.89. 3,2_ REACTIE EXPERSYtENTAL Culturqs >f SIRC st_rain ?ibrobAZsts

°roYii 1'a i

J`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

9 ..l.ing a7i
. nlll LUre Ne.'.l. c i ^'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 , 9i t.. 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. 1 1 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 percenta g 

### e ::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. 0

1 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 7DF,TEERMINrl?'IC?:! D^ LA. CX70TGX.'' iCZT4 ^_'3bT _e8i2 I. I {{{ G

t °------------------------------- 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, 1 0; : 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

..................... ..._... ............._.._... I j.........
... 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 :