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Diss Factsheets

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 19th to August 29th, 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted July 22nd, 2010
Principles of method if other than guideline:
The Maktek Corp EpiDerm™ reconstituted human epidermis Skin Model was used to assess the potential dermal irritation of the test substance. The MTT (3-[4,5 -dimethylthiazol- 2-yl]-2,5- diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular viability after exposure to the test substance for 60 min and 42 hrs post incubation exposure. The duration of exposure resulting in a 50 % decrease in MTT conversion in test substance-treated EpiDerm™ cultures, relative to control cultures, was determined (ET50).
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate
EC Number:
Cas Number:
Molecular formula:
4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
unchanged (no vehicle)
Details on test system:
- Model used: EpiDerm Skin Kit (MatTek Corporation).

Upon receipt of the Skin Kit (MatTek Corporation), the solutions were stored as indicated by the manufacturer. The tissues were stored at 2-8 °C until use. On the day prior to testing, the maintenance medium was set to room temperature prior to use. Nine-tenths ml of maintenance medium was aliquotted into the appropriate wells of 6-well plates. Each 6-well plate was labeled with the test substance, positive control, or negative control. Each tissue was inspected for presence of air bubbles. The tissues were transferred aseptically into the 6-well plates and incubated at 37± 1 °C in a humidified atmosphere of 5 ± 1 % C02 in air (standard culture conditions) for 60 ± 5 min. After 60 min, the tissues were transferred to appropriate wells containing 0.9 ml of fresh warmed (to 37 °C) maintenance medium. The plates were returned to the incubator for 18 ±3 hrs to acclimate the tissues.

The test substance was added to a 1.0 mg/ml MTT (Sigma) solution in warm Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 30 µl of the test substance were added to 1 ml of the MTT solution and the mixture was incubated in the dark at standard culture conditions for at least one hour. A negative control, 30 µl of sterile, CMF-DPBS, was tested concurrently. If the MTT solution color turned blue/purple, the test substance was presumed to have reduced the MTT. The test article was not observed to directly reduce MTT in the absence of viable cells.

NUMBER OF REPLICATE TISSUES: three for test substance, negative and positive controls.

The tissues were treated in triplicate with the test substance for 60 ±1 min. 30 µl of the test substance were applied to each of three tissues at 1 min intervals per tissue. Immediately after dosing, a sterile bulb-headed rod was used to spread the test substance over the surface of each tissue. Then the tissues were treated in triplicate with the positive or negative control for 60 ±1 min. 30 µl of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control administration onto the tissue, a nylon mesh was placed gently over the dose to spread the negative and positive controls. The plates with dosed tissues were kept in the laminar flow hood until the last tissue was dosed. After the last tissue was dosed, all plates were transferred to the incubator for 35±1 min at standard culture conditions. After 35 min, all the plates were removed from the incubator, placed into the laminar flow hood and kept at room temperature until the exposure period was completed for the first dosed tissue.

After 60 ± 1 min of test or control exposure, the tissues were rinsed with sterile, CMF-DPBS by filling and emptying the tissue insert 15 times. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMFDPBS was decanted. The bottoms of the tissue inserts were blotted on sterile paper towels and were transferred to new 6-well plates containing 0.9 mL of fresh warmed (to 37 °C) Maintenance Medium. The tissue surface was carefully blotted with sterile cotton-tipped applicators to remove any excess moisture, and the tissue surface was visually observed for residual test substance using a dissecting scope.

POST-TREATMENT EXPOSURE: the tissues were placed into the incubator at standard culture conditions for a post-treatment expression incubation of 42 ± 1 hrs. After an initial 24±1 hrs of incubation, the 6 well plates were removed from the incubator and the tissues were transferred into new 6 well plates pre-filled with 0.9 ml fresh Maintenance Medium warmed to approximately 37 °C. The tissues were placed back into the incubator at standard culture conditions for an additional 18±1 hrs for the remainder of the 42 ± 1 hr post- treatment expression incubation.

After the total 42±1 hours post-exposure expression incubation, the 6-well plates were removed from the incubator. Each tissue was blotted on a sterile paper towel and transferred to an appropriate well containing 0.3 ml of MTT solution. The 24-well MTT plates were incubated at standard culture conditions for 3±0.1 hrs. After the 3±0.1 hour incubation, the Epiderm tissues were submerged, gently swirled, and rinse media decanted in a beaker containing approximately 150 ml of CMF-DPBS three times. The tissues were then blotted on absorbent paper, cleared of excess liquid and transferred to a prelabelled 24-well plate containing 2.0 ml of isopropanol in each designated well. The plate was covered with parafilm and shaken for at least 2 hrs at room temperature to extract the MTT. The extract solution was mixed (homogenized by pipetting up and down three times) and two x 200 µl aliquots were transferred to the appropriate wells of a 96-well plate. 200 µl of isopropanol were added to the wells designated as blanks. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.

Corrected Individual OD570 =Individual OD570 - mean Blank OD570.
Mean corrected OD570 values were calculated for the individual test substance and control tissue from the duplicate aliquots. The group mean of the corrected OD570 values for the negative controls were calculated. The following percent of control calculations were made:
% of Control =(Final individual corrected OD570 of Test substance or control tissue)/ (corrected mean OD50 Negative Control)X100

- The test substance is considered to be irritant to skin if the mean tissue viability is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability is greater than 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount(s) applied: 30 μl.

- Amount(s) applied: 30 μl

- Amount(s) applied: 30 μl
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 hours

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Other effects / acceptance of results:
- Direct-MTT reduction: No.
- A bubble was removed from the surface of the second tissue treated with the positive control with a sterile cotton swab after rinsing. A dissecting scope was used to check for residual test article after rinsing.

ACCEPTANCE OF RESULTS: the mean OD570 of the negative control, CMFDPBS, was 2.103. The mean viability of the positive control, 5 % SLS, was 5.9 %. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 20 for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid.

In vivo

Irritant / corrosive response data:
The correlation of the results of this in vitro assay with expected in vivo has not been firmly established. However, considering the criteria by European centre for validation of alternative methods scientific advisory committee for prediction of skin irrtation, test substance is determined to be a non-irritant.

Any other information on results incl. tables


During the conduct of the study, a deviation occurred. The tissues were allowed to incubate at 37±1 °C in a humidified atmosphere at 5±100 % CO2 (standard culture conditions) for 14 hours and 52 minutes prior to dosing. The tissues were removed from the incubator and placed at room temperature in teh laminar flow hood for 9 min prior to dosing. According to the protocol, the tissues will be incubated overnight for 18±3 hours to acclimate the tissues and then the tissues will be placed at room temperature at least 5 minutes prior to dosing. Although the protocol does not specificlly state that the tissues will be incubated overnight in standard culture conditions its is implied that this will be the procedure followed, as the protocol states that the tissues will be incubated for 60 ± 5 min for the first pre-incubation period. Since the tissue have been aclimated only 8 min less than the time specified in the protocol (18±3 hrs), it is unlikely that this deviation had any significant impact on the quality and integrity of the study. The review of the data showed that the testing system performed as expected based on the positive and negative control OD valied and SD values, failing in the ranges specified in the criteria for determination of a valid test (viability of the positive control resulted in 5.9 % - i.e. < 50 %, the mean OD570 value of the negative control tissues was 2.103, i.e. 1000 -2500, the S.D. calculated from the individual % tissue viability of the three identically treated replicates of the negative control was 0.60 and 0.88 for the positive control, i.e. < 18 % for both controls)

Applicant's summary and conclusion

Interpretation of results:
other: not classified as skin irritant according to the CLP Regulation (EC) No. 1272/2008
Non-skin irritant
Executive summary:

The skin irritation potential of the test substance was evaluated in the in-vitro EpiDerm™ reconstituted human epidermis, according to the OECD Guideline 439.

EpiDerm™ tissues were exposed in triplicate to the test substance for 60 min (35 min at standard culture conditions and 25 min at room temperature). A positive (5 % SLS) and a negative (CMF-DPBS) control was also exposed in triplicate for 60 min. After the exposure period, the tissues were rinsed and subjected to a post-treatment expression incubation at standard culure conditions for 42 hours. The MTT determination (3 hours incubation with 1.0 mg/ml MTT solution), extraction with isopropanol and measurement of absorbance at 570 nm followed.

The mean viability of the positive control was 5.9 %.The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 20 for the positive control. Since the acceptance criteria were met, the assay was considered valid. 

The mean % viability for the test substance was established to be 98.6 %. Based on the OECD 439 classification criteria the test substance can be considered as non-irritating.