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

Endpoint summary

Description of key information

Skin irritation

In an OECD 439, the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model was tested. The relative mean viability of the test item treated tissues was determined to be 98.1% after the 15 minute exposure period and 42 hours post-exposure incubation period. The test item was therefore classified as non-irritant.

Skin corrosion

In an OECD 431, the corrosivity potential of the test item using the EpiDerm™ Human Skin Model was tested. 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 results indicated that the test item was non-corrosive. 

Eye irritation

In an OECD 437, the potential of the test item to induce serious eye damage upon application to the eye was investigated using the Bovine Corneal Opacity and Permeability (BCOP) test method. The in vitro irritancy score for the test item was determined to be 0.1 and therefore it can be concluded that the test item does not require classification for eye irritation.

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:

09 May 2018 - 11 May 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

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

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.

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

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2018 - 04 June 2018

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

GLP compliance:

yes (incl. QA statement)

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 EpiSkin Laboratories, Lyon, France. Lot Number 18-EKIN-022.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount / concentration applied:

10 µL (26.3 µL/cm2) of the test item was applied topically to triplicate tissues.
Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls.

Duration of treatment / exposure:

15 minutes

Number of animals:

Not applicable.

Details on study design:

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a water bottle containing DPBS with Ca and Mg. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium. The rinsed tissues were then incubated for 42 hours at 37 °C, 5% CO2 in air and placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.

The tissues were then transferred to the third column of 3 wells of the 12-well plate which contained 2 mL of a 0.3 mg/mL MTT solution and incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the incubation period, the tissues were dried and a total biopsy was made using the EPISKIN biopsy punch. The epidermis and collagen matrix were separated, placed in micro tubes containing acidified isopropanol (500 µL) and plugged to prevent evaporation. following mixing on a vortex mixer, the tubes were refrigerated at 1 to 10 °C until Day 6, allowing the extraction of formazan crystals out of the MTT loaded tissues.

On Day 6, the tubes were mixed again on a vortex mixere and duplicate samples (200 µL) taken to measure the optical density at 570 nm.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Overall (mean of 3 results)

Value:

98.1

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

The relative mean tissue viability for the positive control treated tissues was 13.2% relative to the negative control treated tissues and the standard deviation value of the viability was 14.2%. The positive control acceptance criteria were therefore satisfied.

The mean optical density for the negative control treated tissues was 0.749 and the standard deviation value of the viability was 6.4%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilites of the three identical test item treated tissues was 15.0%. The test item acceptance criterion was therefore satisfied.

Table 1: Mean OD₅₇₀ Values and Viabilites for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of triplicate tissues	±SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	±SD of Relative mean viability (%)
Negative Control Item	0.789	0.749	0.048	105.3	100*	6.4
	0.696			92.9
	0.761			101.6
Positive Control Item	0.024	0.099	0.106	3.2	13.2	14.2
	0.220			29.4
	0.052			6.9
Test Item	0.605	0.735	0.112	80.8	98.1	15.0
	0.794			106.0
	0.805			107.5

OD = Optical Density

SD = Standard Deviation

* = the mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability was determined to be 98.1% as this is >50% then the test item does not need to be classified for irritation.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes according to the OECD method 439. The relative mean viability of the test item treated tissues was determined to be 98.1% after the 15 minute exposure period and 42 hours post-exposure incubation period. The test item was therefore classified as non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2018 - 16 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.

Immediately after slaughter, the eyes were placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antiobiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs and refrigerated upon arrival. They were used within 24 hours of receipt.

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of test item or control item was directly applied. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.

Duration of treatment / exposure:

The corneas were Incubated at 32 ± 1 °C for 10 minutes.

Duration of post- treatment incubation (in vitro):

The holders were incubated, anterior chamber facing forward at 32 ± 1 °C for 120 minutes following the treatment.

Details on study design:

A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

The test item or control were applied directly to the corneas and incubated for 10 minutes at 32 ± 1 °C.

At the end of the exposure period, the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

The holders were incubated, anterior chamber facing forward at 32 ± 1 °C for 120 minutes.

After incubation. the holders were removed from the incubator and the medium replaced with fresh EMEM. A final opacity reading was taken and each cornea visually inspected.

Following the final opacity measurement the permability of the corneas to sodium fluorescein was evaluated by incubation with 1mL of sodium fluorescein solution (4 mg/mL) at 32 ± 1 °C for 90 minutes.

After incubation, the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of pre-labeled 96-well plate. The optical density was measured at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Irritation parameter:

cornea opacity score

Run / experiment:

Overall score (mean of 3 values)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Overall Score

Value:

0.1

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:

- Sodium chloride 0.9% w/v was used for the negative control. The test was considered acceptable if the negative control produced an opacity value of <3.0 and a permeability of <0.077. The values obtained for the negative control did meet the acceptance criterion at 0.78 for opacity and 0.060 for permeability.
- Neat ethanol was used for positive control purposes. The test was considered acceptable if the positive control produced an In Vitro Irritancy score between the range of 31.6 to 58.7. The In Vitro Irritancy Score obtained for the positive control was 45.3 and is therefore considered acceptable.

The condition of each cornea were also noted. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation minus Pre-Treatment	Corrected value		Corrected value
Negative Control	2	3	3	4	1		0.014
	3	6	5	6	0		0.015
	6	2	2	3	1		0.152
					0.7*		0.060#		1.6
Positive Control	7	5	35	35	30	29.3	1.735	1.675
	10	5	27	26	21	20.3	1.505	1.445
	11	4	33	30	26	25.3	1.010	0.950
						25.0•		1.356•	45.3
Test Item	12	4	4	3	-1	0.0	0.015	0.000
	14	2	3	2	0	0.0	0.052	0.000
	15	2	2	2	0	0.0	0.072	0.012
						0.0•		0.004•	0.1

OD = Optical density      * = Mean of post incubation – pre-treatment     •= Mean corrected value             # = mean permeability

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro irritancy score for the test item was determined to be 0.1 and therefore the test item does not require classification for eye irritation.

Executive summary:

The purpose of this test was to identify whether the test item would induce serious eye damage upon application to the eye using the Bovine Corneal Opacity and Permeability (BCOP) test method.

The test item along with a positive (ethanol) and negative (Sodium chloride 0.9% w/v) control were applied directly to the eye and the opacity and permeability recorded. The test results obtained for the negative and positive control were within the limits stated and therefore considered acceptable.

The in vitro irritancy score for the test item was determined to be 0.1 and therefore it can be concluded that the test item does not require classification for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The relative mean viability of the test item treated tissues was determined to be 98.1% after the 15 minute exposure period and 42 hours post-exposure incubation period. As there was no visible destruction of the skin tissues then the test item was therefore classified as non-irritant.

The relative mean viabilities determined after 3 and 60 minutes exposure time were 99.3% and 79.2% respectively.  No corrosive responses were noted in any of the samples and therefore the test item can be classified as non-corrosive. 

The in vitro irritancy score for the test item was determined to be 0.1 and therefore it can be concluded that the test item does not require classification for eye irritation.