Ethane, 1,2-dichloro-1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoro-

Endpoint summary

Description of key information

Skin Irritation / Corrosion

An in vitro testing strategy according to the bottom up approach was applied in order to investigate the potential of MOVE 3 Adduct to be irritating to the skin.

According to the data, MOVE 3 Adduct was found not-irritant and not-corrosive to the skin.

Skin Irritation / Corrosion

No data available

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-NOV-2015 to 11-MAR-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): MOVE3 ADDUCT
- Chemical name: Ethane, 1,2-dichloro-1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoro-
- Physical state: colourless liquid
- Molecular weight: 302.94
- Molecular formula: C4Cl2F8O2
- Analytical purity: 98.0%
- Purity test date: 08-SEP-2015
- Lot/batch No.: 15-TV10790
- Expiration date of the lot/batch: 30-NOV-2020
- Storage condition of test material: at 2-8°C

Test system:

human skin model

Remarks:

(source: SkinEthic Laboratories (FRANCE))

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed human epidermal tissue (0.38 cm²)
- Tissue batch number(s): 16-EKIN-003
- Production date, shipping date, delivery date: no data available
- Date of initiation of testing: DEC-2015
- Remarks: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 24 hrs at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories (Lyon, FRANCE).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure and temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline (PBS) 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.
- Observable damage in the tissue due to washing, modifications to validated SOP: not applicable

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 hrs
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 270 nm
- Filter, filter bandwidth, linear OD range of spectrophotometer: no data available
- Remarks: after 3-h incubation with MTT, the tissues were placed on blotting paper to dry the tissues. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 70 hrs. The amount of extracted formazan was determined spectrophotometrically. 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 expo sure of the test item.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No data available

NUMBER OF REPLICATE TISSUES: the test was performed on a total of 3 tissues per test item together with negative and positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was checked for possible colour interference and possible direct MTT reduction before the study was started. To assess the colour interference, approximately 10 μL of test item was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 min. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed. To assess the ability of the test item to reduce MTT, 25 μL of test item was added to 2 mL MTT solution. The mixture was incubated for 3 hrs at 37°C. A negative control, sterile Milli-Q water was tested concurrently. Because no colour changes were observed it was concluded that the test item did not interact with the MTT endpoint.

EXPERIMENTS TO DERIVE FINAL PREDICTION:
After 3-h incubation with MTT, the tissues were placed on blotting paper to dry the tissues. Total biop sy was made by using a biopsy punch.

ACCEPTABILITY CRITERIA
The in vitro skin irritation test was considered acceptable if it met 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.

DECISION CRITERIA
- The test substance is considered to be corrosive / irritant to skin if the viability after 15 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-corrosive / non-irritant to skin if the viability after 15 minutes exposure is greater than to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied, concentration: no correction was made for the purity/composition of the test item. The liquid test item was applied undiluted (25 μL) directly on top of the tissue. Due to volatility after 7 min, additional 25 μL was added to be sure that the skin tissue was covered during the whole procedure.

VEHICLE
Not applicable

NEGATIVE CONTROL
- Amount(s) applied, concentration: 25 μL PBS

POSITIVE CONTROL
- Amount(s) applied, concentration: 25 μL 5% (aq) sodium dodecyl sulphate (SDS) in PBS (CAS Number 151-21-3); the positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

15 ± 0.5-min exposure period

Duration of post-treatment incubation (if applicable):

42 hrs

Number of replicates:

3 tissues per test item together with negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Remarks:

(15-min exposure)

Run / experiment:

Basis: mean

Value:

116

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 116%. Since the mean relative tissue viability for the test material was above 50% after 15 ± 0.5 minutes treatment the test material was considered to be non-irritant.
The positive control had a mean cell viability of 17% 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 10%, indicating that the test system functioned properly.

Table 2: Mean absorption in the in vitro skin irritation test with the test item

	Triplicate exposures			Mean (OD570)	Standard deviation
	A (OD570)	B (OD570)	C (OD570)	-	-
Negative control	0.969	0.916	0.808	0.898	0.082
Test substance	1.085	0.999	1.038	1.041	0.043
Positive control	0.146	0.158	0.158	0.154	0.007

OD = optical density

Table 3: Mean tissue viability in the in vitro skin irritation test with the test item

	Mean tissue viability (% of control)
Negative control	100
Test substance	116
Positive control	17

Interpretation of results:

other: Not irritant

Conclusions:

In conclusion, this test was considered to be valid and the test material was found to be non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN(TM). The study was conducted according to OECD guideline 439 and in compliance with good laboratory practices (GLP).

The test item was applied undiluted (2 x 25 µL) directly on top of the skin tissue for 15 +/- 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was 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 the test substance compared to the negative control tissues was 116%. Since the mean relative tissue viability for the test substance was above 50% after 15 +/- 0.5 minutes treatment the test item was considered to be non-irritant. The positive control had a mean cell viability of 17% 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 10%, indicating that the test system functioned properly.

It was concluded that this test was valid and that the test item was non-irritant in the in vitro skin irritation test under the experimental conditions described in this report. Therefore, the test material is not identified as requiring classifcation and labelling for Skin Irritation / Corrosion according to Category 1 or Category 2 of UN GHS and EC Regulation No. 1270/2008 (CLP / EU GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation / Corrosion

An in vitro testing strategy according to the bottom up approach was applied in order to investigate the potential of MOVE 3 Adduct to be irritating to the skin.

 

Basing on the available information there is no indication MOVE 3 Adduct could be corrosive to the skin as:

-      It was tested in an oral toxicity study in rat reporting no local signs of irritation or corrosion;

-      For the analogue compound (MOVE3)in vitroskin irritation data andin vivoSkin sensitization data are available reporting no indication of skin irritation or corrosion.

 

The potential of the MOVE 3 Adduct to be irritating to the skin was investigated in a study conducted according to OECD guideline 439 and in compliance with good laboratory practices (GLP).

The in vitro study described in the OECDguideline439 allows the identification of irritating chemicals requiring classification and labelling according to UN GHS Category 2 or Category 1.

According to the results, MOVE 3 Adduct was found not-irritant to the skin, not requiring classification and labelling according to UN GHS Category 2 or Category 1.

Eye Irritation / Corrosion

No data available

Justification for classification or non-classification

Skin Irritation / Corrosion

Basing on the available information MOVE 3 Adduct does not fulfill the classification criteria for Skin Irritation / Corrosion according to EC Regulation No.1272/2008 (CLP / EU GHS) and UN GHS.