v~

~

, 

## G ~~ k 4 QcM.P4NY :

".SSESSYE
~ T 4F IRRITANGY QN CFitlHI4AL1-AVYi'OIG t+i&+fBRM`E tlF` THE HEC7' S EGG PRODGCT : .(REPORT : ..

GGSTAS, ^}akFE?dBER 1092r .

~ E G ~ LH 

# C0NME S ^~

1. INTROD[7CTIQN


### <. AuTBE?dTIG3TIaN oE THE STUDY 3, EXPSRIM04T:9L PROTOCOt. 

### 3.1. PRINCIPLu 3.2. c.XPE.^nlh:Eiv'Ti9L MOUEt. 

### 3.3- CPER;ti,zQNAL PROCEDUR& 3.4. CLAS5IFICATIGN
4. RESL=LI`S - GoNCLI7sI0x 4.11 I;E5ULTS 

### 4_2. CCKCiu$I4N Fages

3 

### 4
7 

### 5 5 

### 5 '1

, 7 

### 7

21.

At the request oi ttie CQMPANY

we assessed on the cnoripallaa4toic membrane ot' the hen's ego, tze r,ntensity ot t.he irritat::cn induned by the product a

To perform tiu.s study, we rece:ved on September the 24nd 1992, a sample with. Cbe °ollowi.,zg charac teristics :

. Container : plastic Plask . Quar..tity : about. IQQ mI _ Form ; transluaid liquxd CoJ.our ; brown D: : 5.7 5 wl cf this saxple wer© intraduced into a hermetically stopaered glass rTzsk, labelled 92,1476, ia order to be stored at room temperature Por :.hree years at most.

~

### 3ean-FranQois PINON Veterinarian Ph,u_ 3CIF-NI7'IFIC DIRBCTOR

CENT`:fi 3f1.°, ~ i GR ; 

### f~ f

~ 

# . } 

# .--- ",li



# _. ~ 

# ---=---- ~ Brlgiti.e liE1L:iVIri1. QGf1LITY ASSU&1NCE 

# .._.. - , 2, AL'TIfENTICATION OF THE STU6Y° T the vndersiy*ned, Evelyne DARPIL'SIER, Study Dianager, attest that the study (Protocol REF. 8I6CIR S.A. 61CA) was perPormed according to the internal procedures estabiished on the basis of Good Laboratory P'ractices, iazder the responsibilitu :

Techr.ical of Gorin.ae SCHEi ➢ ER, Saentific s2cxetary of Catherine PRIAM, 1'iiis stut3•,• »as carried out in the premises of 9IOGIR S.A. Laboratory, (3?61l G ~ lnET GEDEX) . AI1 the doc3ments related to this study are sr.ared under the reference KGA $2.1476 in special premises of SIOuIF. S.A. La"coratory for tixree years. Ac thz er:d of this neriod, the study documents will 'ne aPter sponsor agreemert, either transferred an sponsor premises or destroyed. The present report is made of 7 pages numbe.red t'rom I to 7 snd 1 table.. CfiSTAS, November the lOth 1992



## 43- EXpERIWP1AL PROTQCOL

The study w2,s performed on Octo6er the 21st and the 22nd 1992, accordi:

~ g to the experimental protocol deseribed below ; 3.1. PRItiCIPLE :'he technique zs proposed as an alternative to the 'JRr1TZE test End allows to assess the irritancy power of a product after singie application on the chorzoallantoic membrane of tlze hen's egg. RcFBTtENCn ;N.P. LUEPK& - Hen's Pgg Cnorioa2lar.toic Rfemprane Test for

irritati,on poteatial (Pd,. cPaea. toxia. vo1. 24 n° 

### 61 F, pp 4 97'" 496, 1986). 3.2. EXPERI[+tEtr`T«3L biDDEI. 6 fertili.zzad eags froa Legaorn :

orzginating from BELLEJILLg 69200 (society NICHAUDET) weighiMg taetwee2 50 ano 60 g Frior to rhe trial, the eggs were incubated during '0 day9 in an i.ncubator L.M 12 MAYFh^v'6 GCLOSIO;ti, under controlled temperature and num;dity. 3.3, OP£RA'L'IONAL PROCEDURE 3 3... 1 -_Esa Freparatson-.. The sr,ei'_ eC the egg was careful } .y scratcned around the air cell and then oared off. After removal of the ;nner egg membranes the vascular chorioaliantoic membrane was exposed.3,3.2`—,AppLicatiori`-;of'=productThe prc:ducc svas applied 20 :' dilueed in a_elume of 0.2 ml onto tle enorioall.3ntoic membrane- A'ter 20 seconds it was washed out by irri, a a:i.oh 'Bit.7 r j m i l oF' s'aline. .



### 5337acroscopi 

### ~ exam a in tiozz ar+d cetilcciT 

### ~~ o r theLtrPex' 

### 333.0_ 4 manroscopic examination of the chorioallantoic meskirane was performed 30 seconds after product applieation ana 2 and 5 minutes later in order to appreciate at the level of the mexbrane, the follewing acents : Hyperaemia e Secondary vessels becomes visi.ble due to increased blood

i'lovr _ liaemorrhage : 3..tood flows out the b7.00d vessels. . Coagulation : Opacity of the membrane due to the proGein coagulaticn. and%or Zntravascuiar coagc:lation, uach pirameter has beer, scored 3ccarding to the following scale :

APPt1RTTI0N 'TIT7ES ( HYPER2E4IA 2L1P:"lORRtL?sGE 

# I COACL'L4TION 

### ~ 30 SECON'DS 5 7 9 2 MTVUTES j 7 7 

# ~ 5 MINGTES 3 

### ~ The scoties o:;tained for these 3 parameCers wei-e summed to give an individual index„ :9ie mean of these in_divicival indices allows to obtaln the primary irritancy index (PI-GAAi).

63.4. CLASSIFICtATIfl' sk Given the index obtained, the product can be classSfied as follows :

i PS-CAM CLASS 0 ta s 0.9 ,T6T IRRI,A-tti'T I f to 5 4.9 3LIGHTLk IRRITAh'i` 5 

# I to S 8.9 

# ~ M9DEfiATELX IR?tlTAtr'T 

# ~ 9 to s 21 t VERY IRRITANT

~
4. RESULTS - CONCLUSICN $.1. RESULTS They are shoam Sn tahle 1. Some hyperaemia together w=_th a coaTuiatian was observed an on]y one memnrane. These ohservatians ailowed to obtain an irritation 9ndex of 1,7. 4.2. CGNCLUS?DN AccoC@inggto tlze adopted classification and unde_r , our ekperimen.tal canditions, the groduct : aFPeared to 6e SLIGHTLX I[3RITA.NT when it was 3pni:ied 20 y. diluted on the chorioallantoic memhrane_

------, ,;ew--PranWis PTN0N 6xpart Pharmacclogist-Toxicola 

# ~ -- ut



### 7