1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethylene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-Jan-2016 to 11-Jan-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:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

EC Number:

615-064-0

Cas Number:

700874-87-9

Molecular formula:

C4F8O2

IUPAC Name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

Test material form:

liquid: volatile

Specific details on test material used for the study:

MOVE 3 / Batch 150525V10 (2015)
- Name of test material (as cited in study report): MOVE3
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: keratinocytes. Prior to test the keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Cell source:

other: adult human donor

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. The RhE method according to OECD 439 is one of the OECD validated skin irritation test, which is recommended in international guidelines (e.g. OECD and EC). The RhE-based test methods are able to identify Cat. 2 and No Cat. chemicals and can thus serve as stand-alone skin irritation methods for non-corrosives in countries where optional Cat. 3 is not implemented. In case RhE-based test methods result in Cat. 2, an in vitro skin corrosion test, if not performed upfront, is required to determine the final classification (Cat. 2 (irritant) or Cat. 1(A, B or C) (corrosive)).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small model
- Tissue batch number(s): 16-EKIN-001
- Expiring date: 11 January 2016
- Experimental starting date: 04 January 2016
Experimental completion date: 11 January 2016

The test consists of topical application of MOVE3 on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C (Figure 1). Maintenance medium and Assay medium were supplied by the Tissue supplier.

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 71 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 36.7°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

TEST FOR INTERFERENCE OF THE TEST ITEM WITH MTT ENDPOINT:
1) TEST FOR COLOUR INTERFERENCE
MOVE3 was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, approximately 10 μl of MOVE3 was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
2) TEST FOR REDUCTION OF MTT
MOVE3 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 25 μl of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at room temperature. A negative control, sterile Milli-Q water was tested concurrently.

TREATMENT
The test was performed on a total of 3 tissues per test item together with negative and positive controls. An excess amount of test item was added into 12-well plates on top of the skin tissues. The test item was refilled continuously during the treatment for replacing the losses of test item due to volatility (the boiling point of test item is ca. 20°C). The refilling guaranteed the exposure of the tissues to the test item during the whole incubation period.
Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.
After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 71 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours


NUMBER OF REPLICATE TISSUES: 3 tissues per test item / negative and positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable

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

ACCEPTABILITY OF THE ASSAY
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A test item is considered irritant in the skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):TEST MATERIAL
- Amount(s) applied (volume or weight with unit): excess amount of test item was added. The test item was refilled continuously during the treatment for replacing the losses of test item due to volatility (the boiling point of test item is ca. 20°C). The refilling guaranteed the exposure of the tissues to the test item during the whole incubation period.
- Concentration: Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl 5% (aq) Sodium dodecyl sulphate. The positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): An excessive amount of the liquid test item was applied undiluted directly on top of the tissue. The test item was applied three times to completely cover the skin tissue during the whole incubation period. In order to reduce the volatilisation during the treatment, the test item was kept in the refrigerator (2-8°C) until the treatment.

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

Exposure:15 minutes
Post incubation period: 42 hours

Details on study design:

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 runs (see details below)

Value:

96

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The positive control had a mean cell viability of 7% after 15 ± 0.5 minutes exposure.
The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.
The standard deviation value of the percentage viability of three tissues treated identically was less than 8%.
The test system functioned properly and the test is valid.

Any other information on results incl. tables

Absorption Result
	Replicate 1 OD570	Replicate 2 OD570	Replicate 3 OD570	Mean OD570	SD
Negative control	0,832	0,758	0,721	0,770	0,057
MOVE3	0,744	0,772	0,710	0,742	0,031
Positive control	0,064	0,063	0,039	0,055	0,014
Tissue viability
	Replicate 1	Replicate 2	Replicate 3	Mean viability	SD
MOVE3	96,582	100,216	92,168	96,322	4,031
Positive control	8,308	8,178	5,063	7,183	1,837
Negative control	108,005	98,399	93,596	100,000	7,337

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that MOVE3 is non-irritant in the in vitro skin irritation test.

Executive summary:

This study assessed the ability of MOVE3 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of MOVE3 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

MOVE3 was applied undiluted directly on top of the skin tissue for 15 ± 0.5 minutes.

MOVE3 is a volatile liquid, therefore the test item was refilled continuously during the treatment for replacing the losses of test item due to volatility. The refilling guaranteed the exposure of the tissues to the test item during the whole incubation period.

After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with MOVE3 compared to the negative control tissues was 96%. Since the mean relative tissue viability for MOVE3 was above 50% after 15 ± 0.5 minutes treatment MOVE3 is considered to be non-irritant.

The positive control had a mean cell viability of 7% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

It is concluded that this test is valid and that MOVE3 is non-irritant in thein vitro skin irritation test.