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

Endpoint summary

Description of key information

Two key studies are available: one in vitro skin corrosion study (Eurilings, 2016) performed according to OECD 431 (Epiderm model) and one in vitro skin irritation study (Eurilings, 2016) performed according to OECD 439 (Episkin small model). Both studies were performed in compliance with GLP guidelines.

The in vitro skin corrosion study showed that dichloro trifluoro methoxy dioxolane is not corrosive to the skin under the conditions of the study.

The in vitro skin irritation study showed that dichloro trifluoro methoxy dioxolane is not irritant to the skin under the conditions of the study

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

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)

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

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

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

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

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.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2016 - June 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)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-019).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. 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.

- Source:
SkinEthic Laboratories, Lyon, France.

- Tissues:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 24 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

- MTT medium:
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

- 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 67 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 – 37.0°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:EPISKIN Small Model TM
- Tissue batch number: Batch no.: 16-EKIN-019,
- Production date: 16 may 2016
- Shipping date: 16 may 2016
- Delivery date:16 may 2016
- Date of initiation of testing:

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 of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline 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: 0.3 mg/ml in PBS
- Incubation time: 3 h at 37°C.
- 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 undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively.


PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin 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.
- The test substance is considered to be non-irritating to skin 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 applied: The liquid test item was applied undiluted (25 μl) directly on top of the tissue (added into 12-well plates).

NEGATIVE CONTROL
- Amount applied: 25 μl PBS

POSITIVE CONTROL
- Amount applied : 25 μl 5% SDS

Duration of treatment / exposure:

15 minutes

Number of replicates:

Triplicates
- Test item: one triplicate
- Negative control: one triplicate
- Positive control: one triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-min application

Value:

84

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: The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range, which fulfills the acceptance criterion
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure and 42 hours post incubation of 12%, which is below the acceptance criterion of 50%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 13% for the negative control, positive control and the test item which is below the acceptance criterion of 18%.

Table 1: Mean absorption in the in vitro skin irritation test with DICHLORO TRIFLUORO METHOXY DIOXOLANE

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD
Negative control	0.755	0.924	0.968	0.882	± 0.112
DICHLORO TRIFLUORO METHOXY DIOXOLANE	0.761	0.668	0.780	0.743	± 0.049
Positive control	0.115	0.136	0.069	0.107	± 0.055

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

 

Table 2: Mean tissue viability in the in vitro skin irritation test with DICHLORO TRIFLUORO METHOXY DIOXOLANE

	Mean tissue viability (percentage of negative control)	Standard deviation (percentage)
Negative control	100	13
DICHLORO TRIFLUORO METHOXY DIOXOLANE	84	5.5
Positive control	12	3.9

 

 

Table 4: individual OD measurements at 570 nm

	A	B	C
Negative control
OD570 measurement 1	0.8261	0.9820	1.0665
OD570 measurement 2	0.7665	0.9482	0.9509
DICHLORO TRIFLUORO METHOXY DIOXOLANE
OD570 measurement 1	0.8101	0.7477	0.8387
OD570 measurement 2	0.7946	0.7114	0.8046
Positive control
OD570 measurement 1	0.1570	0.1895	0.1112
OD570 measurement 2	0.1562	0.1653	0.1086

OD = Optical density

Duplicate exposures are indicated by A, B and C.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item dichloro trifluoro methoxy dioxolane was found non-irritant in the in vitro skin irritation test under the experimental conditions of a test performed according the OECD guideline 439.

Executive summary:

The ability of the test item dichloro trifluoro methoxy dioxolane to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)) was investigated using the OECD Guidelines for Testing of Chemicals, Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 28 July 2015). The test was performed in compliance with GHP guidelines.

The test item was applied undiluted (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. The percentage of tissue viability is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive control had a mean cell viability of 12% after 15 ± 0.5 minutes exposure and 42 hours post incubation which is below the acceptance criterion of 50%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range, which fulfills the acceptance criterion. The standard deviation value of the percentage viability of three tissues treated identically was less than 13% which is below the acceptance criterion of 18%, indicating that the test system functioned properly.

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 and 42 hours post incubation with the test item compared to the negative control tissues was 84%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

 

Finally, it is concluded that this test is valid and that the test item is non-irritant in the in vitro skin irritation test under the experimental conditions

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Dichloro trifluoro methoxy dioxolane has been tested in two validated in vitro tests: OECD 431 (sin vitro skin corrosion) and OECD 439 (in vitro skin irriation). The substance showed to be neither corrosive nor irritant to skin under the test conditions.

Based on the results of the in vitro studies and according to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, ECHA Guidance on the Application of the CLP Criteria (June 2015) and ECHA guidance Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015, no classification is needed for dichloro trifluoro metoxy dioxolane.