Reaction mass of 4-methyl-1-oxaspiro[5.5]undec-3-ene and 4-methyl-1-oxaspiro[5.5]undec-4-ene and 4-methylidene-1-oxaspiro[5.5]undecane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2017 - 16 October 2017

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-methyl-1-oxaspiro[5.5]undec-4-ene

Cas Number:

62062-89-9

Molecular formula:

C11-H18O

IUPAC Name:

4-methyl-1-oxaspiro[5.5]undec-4-ene

Constituent 2

Reference substance name:

4-methyl-1-oxaspiro[5.5]undec-3-ene

Cas Number:

62062-94-6

Molecular formula:

C11H18O

IUPAC Name:

4-methyl-1-oxaspiro[5.5]undec-3-ene

Constituent 3

Reference substance name:

4-methylidene-1-oxaspiro[5.5]undecane

Cas Number:

62062-84-4

Molecular formula:

C11H18O

IUPAC Name:

4-methylidene-1-oxaspiro[5.5]undecane

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 17-EKIN-041); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2.5 hours at 37°C.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.6 - 37.1 °C
- Humidity (%): 65 - 85

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed once with phosphate buffered saline
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, 25 μL sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
- Cell viability: The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw): ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u +MTT).
The % viability for each sample and the positive control is calculated as follows: %Viability = (ODc/mean ODlt_u+MTT) * 100
- Skin irritation is expressed as the remaining cell viability after exposure to the test item.

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM:
25 μL directly on top of the tissue

NEGATIVE CONTOL:
- Amount applied: 25 μL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 μL
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3 for the test item, the negative and the positive control, each.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Cell viability: 10%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range (i.e., a mean of 1.115 ± 0.051) and the SD of the % viability was <18% (i.e., 4.5%)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 10%) and the SD of the % viability was <18% (i.e., 0.8%).
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <18% (i.e., 5%).

- Since the mean relative tissue viability for the substance was below 50%, it is considered to be irritant.

Any other information on results incl. tables

Table Individual OD measurements (570 nm)

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	1.2414 1.1881	1.2934 0.9755	1.2003 1.0412
Test item OD570measurement 1 OD570measurement 2	0.2161 0.2166	0.2142 0.1402	0.1344 0.1054
Positive control OD570measurement 1 OD570measurement 2	0.1637 0.1397	0.1728 0.1625	0.1501 0.1536

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Applicant's summary and conclusion

Interpretation of results:

other: Skin irritant Cat. 2

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

An in vitro skin irritation test with the substance was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is irritating in the in vitro skin irritation test.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. All validity criteria were met and the study was considered to be valid. The positive control had a mean cell viability of 10% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 12%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritant to the skin.