40D ! ° Prod Lict Testing m 

FINAL REPORT 

CLIEPi7[': 

ATTEN'£TON: 

Senior Scientist 

TEST: The blatTek Corporation EpiDetmT'' Skin Model 

In Yirro Toxicity Testing System 

TEST h,RTZC;LE: 

EXPERIiVXEt*1T 

12EF'EREIVCE NO.s 

Jt I -- ) (~~— r ofa. ls.~ 

5teven Nitka 

Viee President 

L.aboratory I?irector 

?his repor, is su6rnlied fot the exclus4ve u3a of tha persor., par;nershiy, or corporaticr. lo wham S is adtlressed, and neither the report nor rhe 

nane ol these 4a0otdlorios not ar,y member yi ils sta!I, enay he used In conne<tion wi:h the ativertising w sais of any prcduot or process 

tuitF.00, wrihan authprizaUon, 

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Qbjective: 

To evaluate the test artiele for irritancy potential utiliaing the MatTek Corporation EpiDerm in 

uiCro toxic'rty testin}; system. 

Introduction: 

"MatTek's patented EpiDerm Skin Model consists of nonnal, human-derived epidermal 

keratinocytes (N1iEK) which have been cultured to forin a multilayered, higlily differentiated 

modet of the human epiderrnis. k:cratinocytes are cultured on specially prepared, permeable cell 

culture inserts ..," This system "... closely parallels human skin. $piDerm consists of highly 

organized basal, spinous, granular and cornified layers analogous to those found fn vfvo. 

Epiderm cultured keratinocytes are mitotically and metabolically active."I 

EpiDerm, when used with the recommended cell metabolism assay, can ctuickly providc 

toxicological profiles. The procedure utilizes a water-soluble, yellow, tetrrdzoliurn salt (MTT t3- 

[4,5-dimcthylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, ), which is reduced by succinate 

dehydrogenase in the mitochondria of viable cells to a purple, insoluble formazan derivative. 

Substances which damage this mitochondrial enzyme inhibit the reduction of the tetrazolium salt. 

The amount of MTT reduced by a culture is therefote proportional to the number of viable cells. 

Test Artfcle: 

Reference Article: 1% Triton X 100 

I IvfatTek Corporation, 200 Homer Avenue, Ashland, Massachusetts 01721. 
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M1tethod: 

After the appropriate tissue preparation, 100 tnicroliters of the test, reference and negakive 

control articles were added to the Millicells containing the EpiDerm samples. The six (6) well 

plates containing the dosed EpiDerm samples were then incubated at 37°C, five (5)% carbon 

dioxide and ? 90°!o humidity. A.fter the appropriate exposure periods, each insert was 

individually removed from its plate and rinsed Nvith phosphate buffered saline (PBS) to remove 

any residual test material_ Each wet'e then rinsed a second time. Excess liquid was shaken off 

and each EpiDerm sample was placed into 300 microliters of iviTT solution. The EpiDerm 

samples were then returned to the incubator_ 

After the three (3) hour lbl'T"1' exposure, each insert was removed and gently rinsed with PBS to 

remove any residual 1vIT"]' solution. Exeeas PBS was shaken from eaeh of the inserts wluch werc 

then blotted on the bottom on paper towels, The inserts were then each placed into one (1) well 

of a 24 well extraction plate. Each insert was then immersed in two (2) milliliters of exttaction 

solution overnight. After the exposure, the liquid within each insert was decanted back into the 

well from which it was taken. The remaining extractant solution was then agitated and a 200 

microliter aliquot of each extract was removed for evaluation. A Dynatech MR 4000 Automatic 

Nlicroplate Reader was used to detezmine the absorbance of each extract at 570nm, With the 

absorbance of the negative control defined as 100°/d, the percent absorbancies of the test and 

reference articles were determinetl. Tlte percentages listed below directly correlate with the cell 

metabolism in the EpiDerm samples, 

Results: 

Test/Referenee rkriicle Percent Pereent 

f%o & Exposurel System '4'iabili lnh4bition 

r ._ EpiDerm 

(100% - 1 hr.) 113 -13 

(100% - 4.5 hr.) 75 25 

(100% - 20 hr.) 68 32 

Triton X 100 EpiDerm 

(1%- I hr.) 90 10 

(1"io-4.5hr.) 74 26 

(1 % - 20 hr.) 7 93 
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Discussian: 

For each article, a semi-iog scale was used to plot the pereent viabilities, on the linear y axis, 

versus the dosing times, on the Iog x axis. By interpolation and where possible, the titne at 

which the pereent viability would be 50% (ET-50) was estimated for each artiole. 

T'he test article, ___ elicited an ET-50 greater than: 24 hours. The 

reference article, i% Triton X 100, elicited an ET-50 of approximately 7.6 hours. 

According to MatTek Corporation, as a general guideline, the following groupings can be used in 

assigning expected in vivo irritancy responses based on the ET-SO results obtained using 

MatTek's EpiDenn: 

ET-50 (hrs) Expected In vEvo Irritancy Ezample 

<0.5 Severe, probably corrosive Conc. Nitric Acid 

0.5-4 Moderate 1°lo Sodium I3odecyl Suifate 

4-12 Modcrate to Mi3d I% Triton X-100 

12-24 Very Mild Baby Shampoo 

24 Non-irritating 10%Tween 20 

Conclusion: 

Under the conditions of this test, the results indicate that i -1 has an 

expected in vivo dermal irritancy potential in the non-irritating range. The reference article, 1% 

Triton X 100, has an expected in vivo dermal irritancy potential in the moderate to mild range.