Myrac Aldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 August 2017 - 14 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

253-617-4

EC Name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

37677-14-8

Molecular formula:

C13H20O

IUPAC Name:

4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde

Constituent 2

Reference substance name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

257-943-8

EC Name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

52475-89-5

Molecular formula:

C13H20O

IUPAC Name:

3-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde

Test material form:

liquid

Details on test material:

Batch No. 0007470718
Purity: 99.3% (sum of the two main constituents)
Name of the test item (as cited in the study report): MYRAC ALD BHT
Physical state: colourless-slightly yellow liquid
Storage Conditions: room temperature
Expiry Date: 23 December 2017

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 minimize 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number(s): 17-EKIN-032
- Pre-incubation: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

- Before the start of the test, the substance was checked for possible colour interference and possible direct MTT reduction.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.6-37.5 °C
- Humidity: 74-90%, containing 5.0 ± 0.5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 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

CELL VIABILITY MEASUREMENT:
After incubation in MTT, epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69.5 hours. The amount of extracted formazan was determined spectrophotometrically. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment with two OD measurements for each replicate.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA:
- 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.
- The mean relative tissue viability of the positive control should be ≤ 50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
- 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 material: 25 μL
Positive control: 25 μL, re-spread after 7 minutes contact time
Negative control: 25 μL

Duration of treatment / exposure:

15 minutes ± 0.5

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3 tissues for the treatment group, the positive control group and the negative control group each.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 4.8%

Remarks on result:

other: SD: 1.0%

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control (mean tissue viability of 4.8% with a standard deviation of 2.3%) showed that the test system functioned properly.

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 and the Standard Deviation value (SD) of the % viability was 1.3.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was 4.8% relative to the
negative control and the Standard Deviation value (SD) of the % viability was 2.3.
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <3%

Any other information on results incl. tables

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)
Negative Control Item	1.222	1.234	0.016	100
	1.227
	1.252
Positive Control Item	0.032	0.059	0.028	4.8
	0.087
	0.059
Test Item	0.162	0.156	0.013	13
	0.165
	0.142

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

other: Skin irritant.

Remarks:

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

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 13%. Since the mean relative tissue viability for the test substance was below 50% it is considered to be irritant.

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 a 42-hour 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. The positive control had a mean cell viability of 4.8% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 3%, 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 13%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritant.

Therefore, the substance has to be classified as a skin irritant according to CLP regulation (EC) n° 1272/2008.