Cyclooctane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-08-31 to 2021-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU method B.40bis IN VITRO SKIN CORROSION: RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHOD

Version / remarks:

2019-07-31

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclooctane

EC Number:

206-031-8

EC Name:

Cyclooctane

Cas Number:

292-64-8

Molecular formula:

C8H16

IUPAC Name:

cyclooctane

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 (Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 21-RHE-104
- Expiry date: 2021-09-06
- Date of initiation of testing: 2021-08-31

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 20 mL DPBS, gentle rinsing
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 15 minutes)
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Filter: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Please refer to “Any other information on materials”.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Pre-tests for MTT-reducing capacity and colorant properties of the test item were conducted. No such properties were detected and therefore, no additional controls were needed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, 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 %.
- 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:

40 ± 3 µL per tissue

Duration of treatment / exposure:

test item and negative control: 3 min and 1 h
positive control: 1 h

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2 per substance and time point

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 1 h exposure

Value:

90.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 3 min exposure

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified.
- Direct-MTT reduction: No.
- Colour interference with MTT: No.

ACCEPTANCE OF RESULTS:
Please refer to “Any other information on results”.

Any other information on results incl. tables

The results of the reconstructed human epidermis model:

Group		Tissue 1		Tissue 2		Mean		CV
		OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	3 min	1.697	100.8 %	1.668	99.1 %	1.683	100.0 %	1.2 %
	1 hour	1.561	101.5 %	1.515	98.5 %	1.538	100.0 %	2.1 %
Positive Control	1 hour	0.009	0.6 %	0.008	0.5 %	0.009	0.6 %	16.7 %
Test item	3 min	1.653	98.2 %	1.779	105.7 %	1.716	102.0 %	5.2 %
	1 hour	1.362	88.6 %	1.413	91.9 %	1.388	90.3 %	2.5 %

 

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

	Acceptance Criterion	Result
Negative control OD	≥0.8 and ≤3.0	1.515 to 1.697

 

Acceptance Criteria stated by Episkin/SkinEthic Laboratories:

	Acceptance Criterion	Result
Mean OD negative control	≥0.8 and ≤3.0	1.683 (3 min) 1.538 (1 hour)
Mean viability positive control	< 15 % after 1-hour exposure	0.6 %
Range between identically treated tissues with test item	< 30 %	7.6 % (3 min) 3.7 % (1 hour)

 

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	≥1.605 (3 min) ≥1.408 (1 hour)	1.683 (3 min) 1.538 (1 hour)
Mean viability positive control	≤1.01 %	0.6 %

 

Negative Control, Positive Control and Test Substance Data Acceptance Criteria stated by the Testing Laboratory:

	Group	Acceptance Criterion	Result
Range between identically treated tissues	Negative control	< 30 %	1.7 % (3 min) 3.0 % (1 hour)
	Positive control	< 30 %	20.0 % (1 hour)
	Test substance	< 30 %	7.6 % (3 min) 3.7 % (1 hour)

The study met all acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not considered as corrosive to skin.

Executive summary:

The objective of the study conducted according to OECD TG 431 was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of the liquid test item, the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. After treatment with the positive control the mean viability value was 0.6 % and, thus, lower than the historically established threshold of 1.01 %. After treatment with the negative control the mean ODs were 1.683 (3 minutes exposure) and 1.538 (1 hour exposure) and, thus , higher than the historically established thresholds of 1.605 and 1.408, respectively. Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was ≥50 % after 3 minutes exposure (mean viability: 102.0 %) and ≥15 % after 1 hour exposure (mean viability: 90.3 %). Therefore, under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.