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EC number: -
CAS number: -
Direct MTT Reduction
The test material was not able to directly
Test Material, Positive Control Material
and Negative Control Material
The individual and mean OD540
values, standard deviations and tissue viabilities for the test
material, negative control material and positive control material are
given in Table 1 (attached background material). The mean viabilities
and standard deviations of the test material and positive control,
relative to the negative control are also given in Table 1.
The relative mean viability of the test
material treated tissues was 96.6% after a 15-minute exposure.
The qualitative evaluation of tissue
viability is given in Table 2 (attached background material).
Following the 15-minute exposure the test
material treated tissues appeared blue which was considered indicative
of viable tissue.
The relative mean tissue viability for the
positive control treated tissues was < 40% relative to the
negative control treated tissues and the Standard Deviation (SD) value
of the % viability was < 20%. The positive control acceptance
criterion was therefore satisfied.
The mean OD540for the negative
contol treated tissues was > 0.6 and the SD value of the %
viability was < 20%. The negative control acceptance criterion
was therefore satisfied.
Introduction. The purpose of this
test was to evaluate the skin irritation potential of the test material
using the EPISKIN™ reconstituted human epidermis model after a treatment
period of 15 minutes followed by a post-exposure incubation period of 42
hours. The principle of the assay was based on the measurement of
cytotoxicity in reconstituted human epidermal cultures following topical
exposure to the test material by means of the colourimetric MTT
reduction assay. Cell viability is measured by enzymatic reduction of
the yellow MTT tetrazolium salt
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue
formazan salt (within the mitochondria of viable cells) in the test
material treated tissues relative to the negative controls. The
concentration of the inflammatory mediator IL - 1α in
the culture medium retained following the 42 hour post-exposure
incubation period is also determined for test materials which are found
to be borderline non-irritant based upon the MTT reduction endpoint.
This complimentary end-point will be used to either confirm a
non-irritant result or will be used to override the non-irritant result.
Triplicate tissues were treated with the
test material for an exposure period of 15 minutes. At the end of the
exposure period each tissue was rinsed before incubating for
approximately 42 hours. At the end of the post-exposure incubation
period each tissue was taken for MTT-Ioading. The maintenance medium
from beneath each tissue was transferred to pre-labelled micro tubes and
stored in a freezer for possible inflammatory mediator determination.
After MTT loading a total biopsy of each epidermis was made and placed
into micro tubes containing acidified isopropanol for extraction of
formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period
each tube was mixed thoroughly and duplicate 200 μl samples were
transferred to the appropriate wells of a pre-labelled 96 -well plate.
The optical density was measured at 540 nm.
Data are presented in the form of %
viability (MTT reduction in the test material treated tissues relative
to negative control tissues).
The relative mean viability of the test material treated tissues was
96.6% after a 15-minute exposure.
The quality criteria required for acceptance of results in the test were
The test material was considered to be non-irritant.
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