4-fluoro-1,3-dioxolan-2-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Report date: 17 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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 (incl. QA statement)

Remarks:

(13 February 2003)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

483-360-5

EC Name:

-

Cas Number:

114435-02-8

Molecular formula:

C3H3FO3

IUPAC Name:

4-fluoro-1,3-dioxolan-2-one

Test material form:

other: Solid with a low melting point (i.e. liquefied solid)

Details on test material:

Name of the test susbtance: Monofluoroethylene carbonate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epithelial and formed into a stratified, cornified epithelium

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 6616 Kit D
- Date received: 3 August 2005
- Supplier: MatTek Corporation, Ashland, MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

ASSESSMENT OF DIRECT TEST MATERIAL REDUCTION OF MTT
A test substance may directly reduce MTT, thus mimicking the dehydrogenase activity of the cellular mitochondria. This property of the test substance is only a problem if, at the time of the MTT test (after the chemical has been rinsed off), there are still sufficient amounts of the test substance present on (or in) the tissues. To identify this possible interference, the test material was checked for its ability to reduce MTT directly.
Using a positive displacement pipette 100 μL of the test material was added to 900 μL of a 1.0 mg/mL MTT solution freshly prepared in assay medium and incubated at 37°C for 60 minutes. A negative control, 100 μL of sterile distilled water, was tested concurrently. If the MTT solution colour turned blue/purple (darkening) relative to the negative control, the test material was presumed to have reduced the MTT. Water insoluble test materials may show direct reduction only at the interface between the test material and the medium.

PREPARATION OF EPIDERM
The assay medium was pre-warmed to 37°C. Using sterile techniques, 900 μL of this assay medium was pipetted into the appropriate wells of pre-labelled 6-well plates for both the 3-minute and 60 minute exposures. Each well was labelled with details of the material to be applied to the tissue, and the exposure time to be used. EpiDerm tissues were transferred into the 6-well plates containing the pre-warmed assay medium 1 hour before dosing. This transfer was done under aseptic conditions in the biological safety cabinet, using sterile forceps. Care was taken to remove all maintenance agarose sticking to the outside of the cell culture inserts containing the EpiDerm tissues. The 6-well plates containing the EpiDerm samples were placed in an incubator at 37°C, 5% C02 for approximately 1 hour before dosing.

MAIN TEST
Before pre-incubation was complete, a 24-well plate was prepared for use as a "holding plate" for both the 3 minute and 60 minute exposures. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or 300 μL of a 1.0 mg/ml MTT solution (MTT loading plate) was dispensed into each well using a pipette. The two plates were placed in the incubator in a humidified atmosphere (37°C, 5% C02) until required.

After pre-incubation was complete, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. For the 60-minute exposure, 50 μL of sterile distilled water (negative control) was added to the first two tissues and the timer started. For the test material and the positive control material (8.0 N Potassium Hydroxide) 50 μL was also applied to the corresponding tissues.

The plate was placed back in the incubator in a humidified atmosphere (37°C, 5% C02) until the 60-minute exposure was complete.

When dosing for the 60-minute exposure was complete, the same procedure was repeated for the 3-minute exposure. Because the exposure time was so short, the tissues were dosed at regular intervals to allow for the time taken to rinse each insert following exposure and to ensure that each tissue received an equal exposure time. Rinsing was achieved by filling and emptying each tissue insert approximately 20 times using a constant soft stream of PBS to gently remove any residual test material. Excess PBS was removed by blotting the bottom of the insert with tissue paper. Each insert was placed in the prepared holding plate until all inserts were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated for 3 hours (37°C, 5% C02). The same rinsing and MTT loading procedure was repeated for the 60-minute plate when the exposure was complete.

After the 3-hour incubation was complete, the inserts were blotted and transferred to pre-labelled 24-well plates for MTT extraction. 2 mL of MTT extractant solution (isopropanol) was used to completely immerse each insert and the plate was covered with Nescofilm ™ to prevent isopropanol evaporation. The plates were placed in a refrigerator overnight to allow extraction to proceed.

After the extraction period was complete, the inserts were removed using forceps, and their contents were decanted back into the well that the insert came from. The contents of each well were mixed thoroughly using a pipette and for each tissue 3 x 200 μL aliquots of the blue formazan solution were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of isopropanol alone was added to the two wells designated as negative controls. The absorbency at 540 nm (OD540) of each well was measured using the Anthos 2001 microplate reader (Anthos Labtec Instruments, Salzburg, Austria).

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3-minute or 60-minute exposure

Number of replicates:

2 tissues

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

83.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minute exposure

Value:

66.1

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

QUALITY CRITERIA
The mean OD540 of the two negative control tissues were 1.895 for the 3-minute exposure and 1.824 for the 60-minute exposure. These both met test acceptance criteria (i.e. mean OD540 ≥ 0.8).
Potassium Hydroxide as an 8.0 N solution was used as a positive reference. A 3-minute application of 8.0 N Potassium Hydroxide revealed a mean relative tissue viability of 13.6%. The assay met the acceptance criteria (i.e. mean relative tissue viability of the 3 minute positive control was ≤ 30% ).
In addition to the positive and negative control quality criteria, a difference of > 30% between two tissues treated identically is regarded as a rejection criterion and re-testing of the chemical is recommended if the resulting viability is near to a classification eut-off. For all duplicate tissues in this study, the difference in viabilities between tissues was ≤ 30% in all cases.

Any other information on results incl. tables

Table 1: OD540 Values and % viabilities for the negative control, positive control and test material

Material	Exposure Time	OD540^1	Mean OD540^2	% Viability^3	Mean % Viability^4
Negative Control	3 Minutes	1.824	1.895 ± 0.100	N/A	100*
		1.965
	60 Minutes	1.773	1.824 ± 0.071		100*
		1.874
Positive Control	3 Minutes	0.297	0.258 ± 0.056	15.7	13.6 ± 3.0
		0.218		11.5
	60 Minutes	0.116	0.118 ± 0.002	6.4	6.5 ± 0.1
		0.119		6.5
Test Material	3 Minutes	1.601	1.583 ± 0.025	84.5	83.6 ± 1.3
		1.565		82.6
	60 Minutes	1.143	1.205 ± 0.087	62.7	66.1 ± 4.7
		1.266		69.4

1 = OD540 representing each EpiDerm tissue

2 = Mean OD540 of duplicate EpiDerm tissues ± standard deviation

3 = Viability representing each EpiDerm tissue expressed as a percentage of the mean OD540 of either the 3 minute or 60 minute negative control tissue

4 = Mean viability of duplicate treated EpiDerm tissues ± standard deviation

* = Mean viability of negative control tissues set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

The relative mean viability of the test material-treated tissues was ≥ 50% after the 3-minute exposure and ≥ 15% after the 60-minute exposure.
Monofluoroethylene carbonate was considered to be non-corrosive.

Executive summary:

The study was performed to assess the corrosivity potential of the test material using the EpiDerm Skin Model (MatTek, Ashland, MA, USA). The test is based on the hypothesis that corrosivity potential is related to toxicity to the EpiDerm tissue. The study was validated by the inclusion of a positive control material, 8.0 N Potassium Hydroxide.

The experimental design of the study consists of a test for Direct Reduction of MTT by the test material, followed by the main test.

For the main test, duplicate EpiDerm tissues were treated with 50 μL of Monofluoroethylene carbonate and exposed for 3 minutes and 60 minutes. The tissues were incubated at 37°C in a humidified atmosphere of 5% C02 in air for the appropriate exposure times.

Duplicate negative and positive control tissues, treated with 50 μL sterile distilled water or 50 μL 8.0 N Potassium Hydroxide respectively, were also exposed for 3 minutes and 60 minutes.

At the end of the exposure period each EpiDerm tissue was rinsed using Dulbecco' s phosphate buffered saline (DPBS) and placed into a 'holding plate', until all of the tissues had been treated and rinsed. They were then transferred to an MTT 'loading plate', and incubated at 37°C for 3 hours in a humidified atmosphere of 5% C02 in air. At the end of this time, each EpiDerm tissue was blotted dry and placed into an MTT 'extraction plate' in order to extract all of the reduced MTT from the tissues.

At the end of the extraction period, the extracted MTT solution was mixed for each EpiDerm tissue and 3 x 200 μL samples for each tissue were transferred to the appropriate wells of a 96 well plate. The absorbency at 540nm (OD540) of each well was measured with the Anthos 2001 microplate reader. Data are presented in the form of % viability (MTT conversion relative to negative controls) for each of the two exposure times.

The relative mean viability of Monofluoroethylene carbonate treated tissues was 83 .6% after 3 minutes exposure and 66.1 % after 60 minutes exposure.

The quality criteria required for acceptance of results in the test were satisfied.

Monofluoroethylene carbonate was considered to be non-corrosive in vivo.