3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2018 - 11 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-280-8

EC Name:

-

Cas Number:

449177-94-0

Molecular formula:

C8H11F5O2

IUPAC Name:

3-methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane

Test material form:

liquid

Details on test material:

Lot No: 2231AM-68E
Expiry date: 01 January 2019
Storage conditions: room temperature in the dark

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 day culture period.

The EPISKIN model kit was supplied by MatTek, Lot Number 28607.

Pre-incubation: EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing:
For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.

For the 3 minute exposure time, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure.

The tissues were rinsed with Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. They were then blotted and transferred to the 24 well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours.

After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader and LT-com analysis software.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of the test item, 50 µL of 8.0 N Potassium Hydroxide (positive control) and 50 µL of sterile distilled water (negative control) were applied.

Duration of treatment / exposure:

3 minute and 60 minute exposure time

Duration of post-treatment incubation (if applicable):

The plate was incubated (37 °C, 5% CO2) for 3 hours.

Number of replicates:

2 replicates per exposure period

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

99.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

79.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean OD570 for the negative control treated tissues was 2.028 for the 3 Minute exposure period and 1.989 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 1.8% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1     Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.982	2.028	0.064	3.2	100*
		2.073
	60 Minutes	1.947	1.989	0.059	3.0
		2.030
Positive Control	3 Minutes	0.043	0.041	0.004	na	2.0
		0.038
	60 Minutes	0.040	0.036	0.006	na	1.8
		0.032
Test Item	3 Minutes	2.051	2.014	0.053	2.6	99.3
		1.976
	60 Minutes	1.546	1.576	0.042	2.7	79.2
		1.606

 

OD= Optical density

*=          The mean percentage viability of the negative control tissue is set at 100%

na=         Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes according to OECD method 431. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The relative mean viabilities for each treatment group were as follows:

 

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	2.0	99.3
60 minute	100*	1.8	79.2

 

In conclusion, the test item was considered to be non-corrosive to the skin.