Ethyl 3-[4-(hydroxymethyl)-2-methyl-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:

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)

Principles of method if other than guideline:

The 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 metabolism after exposure to a test substance for various exposure times. 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:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-[4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl]propanoate

EC Number:

700-637-0

Cas Number:

902272-78-0

Molecular formula:

C10H18O5

IUPAC Name:

ethyl 3-[4-(hydroxymethyl)-2-methyl-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: 4, 8, 16, 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 500nm (OD50) was measured for each well with a Molecular Devices' Vmax plate reader.

NUMBER OF REPLICATE TISSUES: two.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze killed tissues.
- Procedure used to prepare the killed tissues: freeze killed tissues were prepared by placing untreated EpiDerm constructs in the - 20 °C freezer overnight, then they were allowed to thaw and then they were placed back into the - 20 °C freezer overnight. The killed tissues were then stored in the freezer until use.
- N. of replicates: single killed tissues treated with the test substance and the negative control for 4 and 24 hour exposure times in the same method as descirbed for the MTT assay.
- Method of calculation used: the background MTT reduction from residual NADH and associated enzymes was compared to the MTT reduction in the test article-treated killed-control tissues. The raw OD550 value of the negative control-treated killed control was substracted from the raw OD550 values for each of the test article-treated killed controls. The net OD550 represent the amount of reduced MTT due to direct reduction of test article residues.

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 equoation 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: 4, 8 16 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:

9.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Expected moderate to mild in vivo irritation

Remarks:

According to the MatTek guideline

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 4 h exposure

Value:

80.4

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 8 h exposure

Value:

57.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 16 h exposure

Value:

9.9

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 24 h exposure

Value:

11.3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: yes
- Killed controls (KC): there was little or no direct MTT reduction in the test article-treated killed controls compared to the negative control-treated killed controls. Therefore, the MTT reduction observed in the test article-treated viable tissue was ascribed to the viable cells.

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 for skin corrosion according to the CLP Regulation (EC) No.1272/2008

Conclusions:

The test substance can cause an in vivo moderate to mild irritation.
Tissue viability (4 h) = 80.4 %
ET50 = 9.3 hrs

Executive summary:

The aim of the study was to evaluate the potential toxicity of the test substance as measured by the conversion of MTT by EpiDerm tissues after the substance exposure for four different exposure times. The study was performed similar to the OECD Guideline 431. The ability of the test substance to directly reduce MTT was initially tested via incubating 100 µl with 1 ml MTT for approximately one hour, which showed that the substance can directly reduce MTT in the absence of viable cells. The EpiDerm cultures were tested in duplicated in the MTT assay with 100 µl test material at four exposure times – 1, 4, 8 and 24 hours. Positive control (1 % Triton-X-100) and negative control (sterile deionised water) run concurently. Since the test substance was found to directly reduce MTT, a killed-control experiment was conducted, which revealed that there was little or no direct MTT reduction in the test article-treated killed control compared to the negative control-treated killed controls and the MTT reduction in the test article-treated viable tissue was ascribed to the viable cells.

The ET50 value recorded is 9.3 hours. The correlation of the in vitro assay with the expected in vivo irritation has not yet been fully established, but MatTek suggests a guideline for appointing the expected in vivo irritation profile based on the ET50 scores using the EpiDerm (EPI-200) model. Based on this model, the substance is expected to be a moderate to mild in vivo irritant.

The tissue viability obtained after 4 hours of exposure was 80.4 %. As the study was performed similar to the OECD Guideline 431 the criteria for classification indicated in the guideline can be considered: even if the tissue viability after 3 minutes and 1 hours is not known, is expected to be much higher than the tissue viability observed at 4 hours and thus it is much higher than the threshold for classification as skin corrosive.