Ethyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 9th to 15th, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

EC Number:

700-636-5

Cas Number:

5413-49-0

Molecular formula:

C10H1804

IUPAC Name:

ethyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin model.

PREPARATION OF THE SKIN MODEL
Upon receipt of the kit, the solutions were stored as indicated by the manufacturer. The EpiDerm tissues were stored at 2-8 °C until use. On the day of dosing, the Assay medium was warmed to approximately 37 °C. Nine-tenths ml of assay medium were aliquotted into the appropriate wells of 6-wells plates. Each culture was inspected for air bubbles between the agarose gel and Millicell insert priot to opening the sealed package. Cultures with air bubbles covering greater than 50 % of the Millicell area were not used. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70 % ethanol. The cultures were then incubated at 37±1 °C in a humified atmosphere of 5±1 % CO2 in air (standard culture conditions) for at least one hour. The medium was aspirated and 0.9 ml of fresh Assay Medium were added to each assay well below the Epiderm skin samples. The plates were returned to the incubator until treatment was initiated. Upon opening the bag, any remaining unused tissues were briefly gassed with an atmosphere of 5 % CO2/95 % air and placed back at 2-8 °C for later use.

ASSESSMENT OF DIRECT TEST ARTICLE REDUCTION OF MTT
- Incubation medium: MTT solution in warm Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mM L-glutamine (MTT addition medium)
- MTT concentration: 1.0 mg/ml.
- Test article concentration: 100 μl to 1 ml of MTT solution.
- Incubation time: approximately one hour.
- Incubation conditions: standard culture conditions.
- Controls: negative control, 100 μl of sterile deionised water tested concurrently.

MTT ASSAY
- Test item amount: 100 μl to each Epiderm construct.
- Incubation time: 1, 4, 8, 24 h.
- Incubation conditions: standard culture conditions.
- MTT solution: 1.0 mg/ml solution of MTT in warm MTT Addition Medium was prepared no more than 2 hours before use.
- Removal of test material and controls: extensively rinsed with Calcium and Magnesium-free Dulbecco's Phosphate Buffered Saline and the wash medium was decanted.
- Viability assay: three-tenth ml of MTT reagent were added to designated wells in a prelabeled 24 -well plate and the EpiDerm cultures were transferred to their appropriate wells. The plates were incubated for approximately 3 hours at standard culture conditions. The EpiDerm cultures were blotted on absorbent paper and then transferred to a prelabeled 24 -well plate containing 2.0 ml isopropanol in each designated well. The plates were covered with parafilm and stored in the refrigerator until the last exposure time was harvested and then the plates were shaken for at least 2 hours at room temperature. The liquid in the Millicell inserts was decanted into the well from which the Millicell was taken. Extract solution was mixed and 200 µl transferred to appropriate wells on a 96 -well plate. 200 µl isopropanol were placed in 2 designated wells as blanks. The absorbance at 550nm (OD50) was measured for each well with a Molecular Devices' Vmax plate reader.

NUMBER OF REPLICATE TISSUES: two.

DATA PRESENTATION
- Corrected mean OD550 values of negative controls was determined by substracting the mean OD550 value of the blank wells from their mean OD550 values
- Corrected mean OD550 values of the test article and positive controls was determined by substracting the mean OD550 value of the blank wells from their OD550 values
- Determination of ET50: two consecutive points in the exposure time response curves (% of control - test article or positive control exposure time) were selected, where one exposure time resulted in a relative survival greater than 50 % and one exposure time resulted in less than 50 % survival. The two selected expsures were used to determine the sloper and the y-intercept for the equoation y=m(x)+b. To determine the ET50, the equation was solved for y=50.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 100 µl.

POSITIVE CONTROL
- Amount(s) applied: 100 µl.

NEGATIVE CONTROL
- Amount(s) applied: 100 µl.

Duration of treatment / exposure:

4 exposure times for test article: 1, 4, 8 and 24 hours
2 exposure times for positive control: 4 and 8 hours
3 exposure times for negative control: 4, 16 and 24 hours

Number of replicates:

Test article, positive and negative control all tested in duplicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: ET50 (hours)

Value:

2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Expected a moderate in vivo irritation according to the MatTek guideline

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 1 h exposure

Value:

75.1

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 4 h exposure

Value:

33.8

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 8 h exposure

Value:

8.2

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 24 h exposure

Value:

7.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: no.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the ET50 value for the positive control, 1 % Triton-X-100, fell within two standard deviations of the historical mean (4.22 and 6.99 hours), thereby meeting the acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

other: not classified as skin corrosive according to the CLP Regulation (EC) No.1272/2008

Conclusions:

non skin corrosive

Executive summary:

The in-vitro skin irritation potential of the test substance was determined using the EpiDermTMskin model based on the colorimetric MTT viability assay. The skin tissue cultures were tested in duplicate with the test substance at four exposure times of 1, 4, 8 and 24 h. The exposure time of the positive control was also exposed in duplicate for 4 and 8 h. The ET50 value for the positive control, 1% Triton-X-100, was found to be 5.86 and was within two standard deviations of the historical mean (4.22 to 6.99 h), thereby meeting the acceptance criteria.

The ET50 for the test substance was established to be 2.8 h. The tissue viability after 1, 4, 8 and 24 hours was 75.1, 33.8, 8.2 and 7.7 % respectively.

Based on the classification established by MatTek Corporation (2005), the test substance can be considered as a moderate skin irritant in the MTT time course assay. However, besides the ET50 available, the determined tissue viabilities can be used for the evaluation of the classification of the substance. According to the OECD 431 (2004) the test substance is considered to be non-corrosive to skin: i) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15 %. Considering the aforementioned critetia the substance is considered as non-skin corrosive.