1-ethylazepan-2-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar - 30 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-ethylazepan-2-one

EC Number:

606-384-1

Cas Number:

19797-08-1

Molecular formula:

C8H15NO

IUPAC Name:

1-ethylazepan-2-one

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempearture, protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, USA

Source strain:

other: EpiDerm Skin Model (EPI-200)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Ashland MA, USA)
- Tissue batch number: 28307, kit E and F

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure); 37.0 ± 1.0 °C (60 ± 5 min exposure)
- Temperature of post-treatment incubation: 37.0 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with phosphate buffered saline to remove residual test item; not further specified
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (1 mg/mL)
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.851 ± 0.085 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 1% Triton X-100. The ET-50 value was determined to be 7.24 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi. None of these were detected.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable as the test item did not show reducing capacity 1 h after MTT incubation.

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

PREDICTION MODEL / DECISION CRITERIA
Step 1
- The test item is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and the viability after 1 hour exposure is less than 15%.
- The test item 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%.
Step 2 (for test items identified as corrosive in Step 1)
- Sub-category 1A: if the viability after 3 minutes exposure is less than 25%
- A combination of sub-categories 1B and 1C : if the viability after 3 minutes exposure is greater than or equal to 25%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
- Concentration : 8 N

NEGATIVE CONTROL
- Amount applied: 50 μL

Duration of treatment / exposure:

3 min and 60 ± 5 min

Number of replicates:

duplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean values of 2 tissues

Run / experiment:

3 min exposure

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean values of 2 tissues

Run / experiment:

60 min exposure

Value:

25

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 item did not show reducing capacity after 1 h incubation with MTT
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water.

ACCEPTANCE OF RESULTS:
- The mean OD of the tissue replicates treated with the negative control are within the laboratory historical control data range, i.e. ≥ 1.258.and ≤ 2.615 for every exposure time (range: 1.871 to 2.120).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (6.2%).
- Acceptance criteria met for variability between replicate measurements: The Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 2.7% to 15%).

Any other information on results incl. tables

Table 2. Mean absorption values

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean  ± (OD570)		SD	A (OD570)	B (OD570)	Mean ± (OD570)		SD
Negative control	1.871	1.923	1.897	±	0.037	2.120	2.112	2.116	±	0.006
N-ethylcaprolactam	1.918	1.751	1.834	±	0.118	0.532	0.518	0.525	±	0.010
Positive control	0.212	0.244	0.228	±	0.023	0.143	0.122	0.132	±	0.015

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0414). Isopropanol was used to measure the background absorption.

Table 3. Mean tissue viability

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
N-ethylcaprolactam	97	25
Positive control	12	6.2

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

CLP: not classified