Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 5 june 2012 to 11 june 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 439) and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): MK-0954 Free Acid
- Molecular formula (if other than submission substance): C22H23ClN6O
- Molecular weight (if other than submission substance): 422.91
- Structural formula attached as image file (if other than submission substance): see reference substance
- Physical state: white powder
- Analytical purity: not provided
- Lot/batch No.: not available
- Expiration date of the lot/batch: 24 april 2013
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Test system

Type of coverage:
open
Preparation of test site:
other:
Controls:
other: negative and positive control
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
3 tissues per test substance with negative and positive control
Details on study design:
MK-0954 Free Acid was checked for possible direct MTT reduction before the study was started. To
assess the ability of the test substance to reduce MTT, 18.4 mg of the test substance was added to 2
ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative
control, sterile Milli-Q water was tested concurrently.

The test was performed on a total of 3 tissues per test substance together with negative and positive
controls. The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to
ensure close contact of the test substance to the tissue and the solid test substance (11.1 to 12.0 mg;
with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues
were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control)
respectively. The positive control was re-spread after 7 minutes contact time. After the exposure
period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to
remove residual test substance. After rinsing the cell culture inserts were each dried carefully and
moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and
rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium and were
transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were
incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues.
Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix
and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck,
Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The
amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the
TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control
tissues. Skin irritation potential of the test substance was classified according to remaining cell viability
following exposure of the test substance.

Results and discussion

Any other information on results incl. tables

table 1 mean absorption in the in vitro skin irritation test with MK-0954 Free Acid

   A (OD570) B (OD570  C (OD570)  Mean (OD570)  standard deviation (+/-)
 Negative control  0.925  0.954  0.899  0.926  0.028
 MK-0954 Free acid  0.964  0.950  0.783  0.899  0.101
 Positive control  0.043  0.020  0.039  0.034  0.012

OD = optical density; Triplicate exposures are indicated by A, B and C

table 2 mean tissue viability in the in vitro skin irritation test with MK-0954 Free Acid

   Mean tissue viability (percentage control)
 Negative control  100
 MK-0954 Free Acid  97
 Positive control  4

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
In conclusion the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report. The result is considered to be reliable.