3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene

Endpoint summary

Description of key information

Skin irritation (OECD 439, GLP): not irritating

Eye irritation (OECD 492, GLP): not irritating

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:

03 Feb - 10 Mar 2017

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:

adopted Jul 2015

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: LabCyte EPI-MODEL24 SIT

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: LabCyte EPI-MODEL24 SIT (Japan Tissue Engineering Co., Ltd.)
- Tissue batch number(s): LCE24-170206-A
- Delivery date: 07 Feb 2017
- Expiry date: 10 Feb 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed fifteen times or more with PBS (without Mg2+ and Ca2+). Remaining PBS was removed from outside of tissue insert with a sterile cotton swab.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.5 mg/mL
- Incubation time: 180 ± 5 min
- Spectrophotometer: Multimode Microplate Reader (FLUOstar OPTIMA, BMG LABTECH)
- Wavelength: 570 and 650 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the LabCyte EPI-MODEL 24 tissue was assessed by undertaking a MTT cell viability test. The OD was determined to be 1.1 (acceptance criteria 0.8 ≤ OD ≤ 2.5).
- Barrier function: According to the Supplier`s Data Sheet, the IC50 value was determined to be 0.26 (acceptance criteria: 0.14 ≤ IC50 ≤ 0.40).
- Morphology: Observing multilayered epidermis with a stratum corneum according to the Supplier`s Data Sheet.

NUMBER OF REPLICATE TISSUES: Triplicate tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-corrosive to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL

NEGATIVE CONTROL
- Amount(s) applied: 25 µL

POSITIVE CONTROL
- Amount(s) applied: 25 µL
- Concentration: 5% (w/v)

Duration of treatment / exposure:

15 min ± 30 s (administered in 1 min intervals)

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min ± 30 s

Value:

118

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not show a MTT-reducing capacity.
- Tissue-binding test: As a result of tissue-binding test, the staining ratio was -0.2%, thus below 5% and a correction of measurement value was not conducted.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD measured after treatment with the negative control was 0.810, thus in the acceptability range (0.8 ≤ OD ≤ 2.5).
- Acceptance criteria met for positive control: The cell viability was strongly reduced in tissues treated with the positive control. The mean cell viability was 2.7%.
- Acceptance criteria met for variability between replicate measurements: The standard deviations of the cell viabilities in the negative control, positive control and test substance group were 10.8, 0.1 and 1.8%, respectively, and within the acceptability range for the variability (SD ≤ 18.0) according to OECD TG 439.

Table 1: Summary of Results

Group	Tissue Number	OD	Mean OD	Cell Viability (%)	Mean Cell Viability (%)	SD
Negative control	1	0.902	0.810	111.4	100	10.8
	2	0.801		98.9
	3	0.727		89.8
Positive control	1	0.022	0.022	2.7	2.7	0.1
	2	0.022		2.7
	3	0.021		2.6
Test substance	1	0.960	0.956	118.5	118.0	1.8
	2	0.940		116.0
	3	0.967		119.4

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of the RHE test method the test substance did not show irritant properties.
CLP: not classified

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:

03 Feb - 10 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted Jul 2015

Deviations:

no

GLP compliance:

yes

Species:

human

Strain:

other: Epiocular™EIT (OCL-200)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: K6G73

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: 020917ALA

Duration of treatment / exposure:

30 ± 2 min (administere in 1 min intervals)

Duration of post- treatment incubation (in vitro):

12 ± 2 min

Number of animals or in vitro replicates:

duplicates for each treatment and control group; from each tissue, 2 absorbance measurements after MTT incubation were performed

Details on study design:

- RhCE tissue construct used, including batch number: Epiocular™EIT (OCL-200); Test Kit name: OCL-200EIT (manufactured on 16 Feb 2017 by MatTek Corporation); Lot number: 20978; Receipt Date: 20 Feb 2017

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to change the color of the MTT medium was assessed in a pre-experiment. Since the MTT solution colour did not change, the test substance is not presumed to have reduced the MTT. A tissue-binding test was performed with medium without MTT to determine the staining ratio using the following formula:
Staining ratio (%) = OD of the test substance (without MTT) / Mean OD of the negative control substance group (with MTT) x 100

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength: 570 nm

- Spectrophotometer: Multimode Microplate Reader (FLUOstar OPTIMA, BMG Labtech)

- Description of evaluation criteria used: The test substance is considered to be not irritating to eye if the tissue viability is > 60%. The test substance is considered to be irritating to eye if the tissue viability is ≤ 60%.

- Positive and negative control means and acceptance ranges: OD in the negative control substance group is > 0.8 and < 2.5. The cell viability in the positive control substance group is < 50%.
- Acceptable variability between tissue replicates for positive and negative controls and for the test substance: Differences of two tissue cell viabilities in each treatment group are < 20%

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Run / experiment:

30 min exposure

Value:

72.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in MTT medium did not led to a change in colour.
The mean staining ratio was below 60%, therefore the cell viability was corrected by the following formula:
Corrected cell viability (%) = Mean cell viability of the test substance group (%) - Mean staining ratio (%)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD of the negative control was 1.117, 1.052, 1.005 and 1.047, and thus within the acceptibility range (OD > 0.8 and < 2.5).
- Acceptance criteria met for positive control: Yes, the positive control reduced the cell viability at 36.0% and fulfilled the acceptance criteria.
- Acceptance criteria for variabilties: Differences of two tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 5.5%, 4.2% and 2.4%, respectively, and thus < 20%.

Table 1: Summary of Results

Group	Tissue	OD	Mean OD / Tissue	Mean OD of Tissue 1 and 2	Mean Cell Viability (%) / Tissue	Mean Cell Viability (%) of Tissue 1 and 2	Corrected Mean Cell Viability (%) *	Difference
Negative control	1	1.117	1.085	1.056	102.7	100	-	5.5
		1.052
	2	1.005	1.026		97.2
		1.047
Test substance	1	0.394	0.402	0.380	38.1	36.0	-	4.2
		0.410
	2	0.351	0.358		33.9
		0.364
Positive control	1	0.775	0.786	0.773	74.4	73.2	72.4	2.4
		0.797
	2	0.745	0.760		72.0
		0.774

* The cell viability was corrected by the staining ratio

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of the RhCE test method the test substance did not show irritant properties.
CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are in vitro data available regarding skin irritation and eye irritation / corrosion for 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene (CAS 25291-17-2) in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.1 and 8.2.

Skin

The skin irritation potential of the registered substance was determined by an in vitro skin irritation test using a human skin model according to OECD Guideline 439 and in compliance with GLP (Fujishima, 2017). In this study the test substance did not show irritating properties towards human-derived epidermal keratinocytes.

Therefore, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene is not considered to be a skin irritant.

 

Eye

The eye irritation/corrosion potential of the registered substance was determined by an in vitro eye irritation test using a reconstructed human cornea-like model according to OECD Guideline 492 and in compliance with GLP (Fujishima, 2017). In this study the test substance did not show irritating properties towards the EpiOcular tissue.

Therefore, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene is not considered to be an eye irritant.

Justification for classification or non-classification

The available data on skin irritation and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.