1,3-Dioxolane, 4,5-dichloro-2,2,4-trifluoro-5-(trifluoromethoxy)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2016 - June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,5-dichloro-2,2,4-trifluoro-5-(trifluoromethoxy)-1,3-dioxolane

EC Number:

605-262-5

Cas Number:

161611-73-0

Molecular formula:

C4F6Cl2O3 C4F6Cl2O3

IUPAC Name:

4,5-dichloro-2,2,4-trifluoro-5-(trifluoromethoxy)-1,3-dioxolane

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 15-TV09366 - BIN 5/2 BIS C607
- Expiration date of the batch: 31 October 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: 31 October 2020

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Details on animal used as source of test system:

- Test system:
EpiDerm Skin Model: the model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

- Source:
MatTek Corporation, Ashland MA, U.S.A.

- Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

- DMEM (Dulbecco’s Modified Eagle’s Medium):
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

- MTT medium:
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

- Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 69 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200, Lot no.: 23697 kit U and T)
- Tissue batch number(s): 23697
- Production date: 04 April 2016
- Shipping date:04 April 2016
- Delivery date:04 April 2016
- Date of initiation of testing: 05 April 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C
See details in "Any other information on materials and methods incl. tables"

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item
See details in "Any other information on materials and methods incl. tables"

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours at 37°C in air containing 5% CO2.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength:570 nm
See details in "Any other information on materials and methods incl. tables"

NUMBER OF REPLICATE TISSUES: The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to DICHLORO TRIFLUORO METHOXY DIOXOLANE and two for a 1-hour exposure.

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% (sub-category 1A) , 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%. (sub-category 1B and 1C).
- The test substance is considered to be non-corrosive to skin 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: Fifty μl of the undiluted test item was added into the 6-well plates directly on top of the skin tissues on top of the tissue, after 45 minutes additional test item (100 μl) was applied to ensure that the tissue was covered during the whole exposure (since the test item was volatile).
No correction was made for the purity/composition of the test item.

NEGATIVE CONTROL
- Amount applied: 50 μl Milli-Q water

POSITIVE CONTROL
- Amount applied: 50 μl 8N KOH

Duration of treatment / exposure:

The possible corrosive potential of dichloro trifluoro methoxy dioxolane was tested by topical application for 3 minutes and for 1 hour.

Number of replicates:

Duplicate for each time application (3 min and 1 hour)
- Test item: one duplicate for the 3 min and 1 hour application
- Negative control: one duplicate for the 3 min and 1 hour application
- Positive control: one duplicate for the 3 min and 1 hour application

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application viability

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application viability

Value:

76

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction / Colour interference with MTT: DICHLORO TRIFLUORO METHOXY DIOXOLANE was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that DICHLORO TRIFLUORO METHOXY DIOXOLANE did not interfere with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: The mean relative tissue viability following 3-minute exposure to the positive control was 7%
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 28%, indicating that the test system functioned properly

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin corrosion test with dichloro trifluoro methoxy dioxolane

	3-minute application				1-hour application
	A (OD570)	B (OD570)	Mean (OD570)	SD	A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	2.144	2.239	2.192	± 0.067	2.033	2.081	2.057	± 0.034
DICHLORO TRIFLUORO METHOXY DIOXOLANE	1.852	1.473	1.662	± 0.268	1.811	1.314	1.562	± 0.352
Positive control	0.146	0.152	0.149	± 0.004	0.141	0.218	0.179	± 0.055

 

Table 2: Mean tissue viability in thein vitro skin corrosion test with dichloro trifluoro methoxy dioxolane

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
DICHLORO TRIFLUORO METHOXY DIOXOLANE	76	76
Positive control	7	9

 

 

Table 3: Coefficient of Variation between tissue replicates

	3-minute application	1-hour application
Negative control	4.3	2.3
DICHLORO TRIFLUORO METHOXY DIOXOLANE	20.5	17.4
Positive control	3.9	35.5

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

 

 

  

Table 4: individual OD measurements at 570 nm

	3-minute application (OD570)		1-minute application     (OD570)
	A	B	A	B
Negative control
OD570 measurement 1	2.1973	2.3017	2.1016	2.1050
OD570 measurement 2	2.1816	2.2773	2.0743	2.1339
OD570 measurement 3	2.1752	2.2608	2.0458	2.1257
DICHLORO TRIFLUORO METHOXY DIOXOLANE
OD570 measurement 1	1.8924	1.5280	1.8726	1.3696
OD570 measurement 2	1.8867	1.5056	1.8092	1.3236
OD570 measurement 3	1.8982	1.5067	1.8728	1.3701
Positive control
OD570 measurement 1	0.1862	0.1939	0.1827	0.2583
OD570 measurement 2	0.1874	0.1910	0.1816	0.2639
OD570 measurement 3	0.1870	0.1934	0.1792	0.2537

OD = Optical density

Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item dichloro trifluoro methoxy dioxolane is not corrosive in the in vitro skin corrosion test under the experimental conditions of a test performed according the OECD guideline 431.

Executive summary:

The ability of the test item dichloro trifluoro methoxy dioxolane (CAS 161611-73-0) to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) was investigated using the OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method (adopted 28 July 2015). This study was performed in compliance with GLP guidelines.

The test item was applied undiluted (50 μl) directly on top of the skin tissue. The skin was exposed during 3 minutes or 1 hour. The test item, negative control (water) and positive control (8N KOH) were tested in duplicate for each exposure time.

The positive control had a mean relative tissue viability of 7% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was ≤ 28%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with dichloro trifluoro methoxy dioxolane

compared to the negative control tissues was 76% and 76% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, dichlroro trifluoro metoxy dioxolane is considered not to be corrosive to the skin.

Finally, it is concluded that this test is valid and that dichloro trifluoro methoxy dioxolan is not corrosive in the in vitro skin corrosion test under the experimental conditions.