1,3-Dioxole, 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:

25-Oct-2016 to 26-Jan-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 431 and EU Method B.40 BIS. Furthermore, functional model conditions and references to historical control data are included in the report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Adopted July 29, 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: UE Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

605-263-0

EC Number:

605-263-0

Cas Number:

161611-74-1

Molecular formula:

C4F6O3

IUPAC Name:

605-263-0

Test material form:

liquid

Details on test material:

- Physical state: Colourless liquid; odorless
- Storage condition of test material: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-Kit, MatTek Corporation (Ashland, MA, USA).
- Lot number: 24940 Kits J and K
- Production Date: no data
- Shipping date: no data
- Delivery date: no data
- Date received: no data
- Date of initiation of testing: December 06, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 and 60 minutes at 37 °C in a humidified atmosphere of 5% CO2

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. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570 nm): 1.744 +/- 0.093 [1.0-3.0]
- Barrier function: ET-50: 5.21 hrs [4.77-8.72 hrs]
- Morphology: 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.
- Contamination: No contamination

NUMBER OF REPLICATE TISSUES: 4 tissues per test item together with a negative control and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not needed

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability >= 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL of the undiluted test item.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of Milli-Q water was used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied

Duration of treatment / exposure:

3 and 60 minutes.

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

4 tissues per test item together with a negative control and positive control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

7%

Remarks on result:

other: No indication of corrosion

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

46

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

14%

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not reported
- Direct-MTT reduction: None
- Colour interference with MTT: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 7.3.1/1: Mean absorption in the in vitro skin corrosion test with Perfluoro methoxy dioxole

	3 -minute application					1 -hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.698	1.618	1.658	+/-	0.056	1.440	1.371	1.406	+/-	0.049
Test item	1.719	1.531	1.625	+/-	0.133	0.678	0.608	0.643	+/-	0.049
Positive control	0.125	0.114	0.120	+/-	0.008	0.234	0.170	0.202	+/-	0.046

OD = Optical Density

SD = Standard Deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0424). Isopropanol was used to measure the background absorption.

Table 7.3.1/2: Mean tissue viability in the in vitro skin corrosion test with Perfluoro methoxy dioxole

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100 (4.7)	100 (4.8)
Test item	98 (11)	46 (10)
Positive control	7 (9.0)	14 (28)

( ): Coefficient of variation between tissue replicates

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, Perfluoro methoxy dioxole is not corrosive to skin.

Executive summary:

An in vitro skin corrosion study was performed according to the most recent OECD Guideline 431, EU Method B.40 BIS and in compliance with GLP, using the EpiDerm™ Human Skin Model.

 

The test item was applied undiluted (50 μl for 3 minutes exposure and an excess amount for the 1-hour exposure) directly on top of the skin tissue. Since the test item was volatile, an excess amount of the test item was applied every 15 minutes for the 1-hour exposure.

The positive control had a mean relative tissue viability of 14% after the 1-hour exposure. This is within the acceptability range for the positive control (<15%). The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit >= 0.8 and upper acceptance limit =< 2.8) and the laboratory historical control data range (1.324 – 2.615 for 3 minute exposure and 1.361 – 2.352 for 1 hour exposure). In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was =< 11%, indicating that the test system functioned properly (acceptability value:≤30%).

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 98% and 46%, respectively. Because the mean relative tissue viability for Perfluoro methoxy dioxole was not below 50% compared to control after the 3-minute treatment and not below 15% after the 1-hour treatment Perfluoro methoxy dioxole is considered to be not corrosive.

Finally, it is concluded that this test is valid and that Perfluoro methoxy dioxole is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.