1-methylheptyl acrylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2017 - 06 September 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:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-methylheptyl acrylate

EC Number:

256-005-5

EC Name:

1-methylheptyl acrylate

Cas Number:

42928-85-8

Molecular formula:

C11H20O2

IUPAC Name:

octan-2-yl prop-2-enoate

Test material form:

liquid

In vitro test system

Test system:

other: human skin model reconstructed human epidermis

Source species:

human

Cell type:

other: EpiskinTM Model Kit (0.38 cm2 tissues)

Cell source:

other: EpiSkin, Lyon, France

Details on animal used as source of test system:

not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: several
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: no data
- Wavelength: 570 nm
- Filter: no data
- Filter bandwidth: no data

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item or positive control) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank
- mean cOD Positive Control = mean ODPC - mean ODblank
DECISION CRITERIA
Mean relative viability is = or < 50%: category 2 (H315)
Mean relative viability is > 50% : no category but the concentration of the inflammatory mediator interleukine 1 alpha (IL-1a) is evaluated from the culture medium retained following the 42 hours recovery period. This quantification, based on an ELISA assay is performed in order to confirm a non-irritant result or to override the non-irritant result (see table 7.3.1/1 in Any Other Information on Materials and Methods).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 100%

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5% w/v in water

Duration of treatment / exposure:

Exposure period of 15 minutes, following rinsing.

Duration of post-treatment incubation (if applicable):

MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells

Number of replicates:

Triplicate tissues for each tested substance (test item, negative control and positive control).

Test animals

Species:

other: in vitro test

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

15 min

Run / experiment:

Mean

Value:

97

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6%

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: IL1a release

Run / experiment:

Mean

Value:

20.2

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no abnormality noted
- Direct-MTT reduction:The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties.
- Colour interference with MTT:as the water solution containing the test item did not change colour, the test item was found not to have a colouring potential.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean cOD of the three negative controls should be equal or above 0.6 and the Standard Deviation (SD) value of the % viability should be equal or below 18%,
- Acceptance criteria met for positive control: the relative mean viability of the positive control should be equal or below 40% of the relative mean viability of the negative control and the SD value of the % viability should be equal or below 18%.

Any other information on results incl. tables

7.3.1/2 Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

Group	Tissue n°	OD measurements		Mean OD blank	cOD			Mean cOD	Viability (%)
		1st	2nd		1st		2nd
Negative control	1	1.012	0.997		0.972	0.957		0.964	99
	2	1.001	1.012	0.041	0.961	0.972		0.966	99
	3	1.048	1.018		1.008	0.978		0.993	102
Positive control	1	0.087	0.087		0.047	0.047		0.047	5
	2	0.132	0.135	0.041	0.092	0.095		0.093	10
	3	0.090	0.089		0.050	0.049		0.049	5
Test item	1	1.019	1.019		0.979	0.979		0.979	100
	2	0.901	0.900	0.040	0.861	0.860		0.861	88
	3	1.019	1.045		0.979	1.005		0.992	102

OD= Optical Density

cOD= blank Corrected Optical Density

7.3.1/3 Mean tissue viability and standard deviation for the test item, the negative and positive controls

Group	cOD		Viability (%)
	Mean	SD	Mean	SD
Negative control	0.974	0.016	100	2
Positive control	0.063	0.026	6	3
Test item	0.944	0.072	97	7

 

SD= Standard Deviation

cOD= blank Corrected Optical Density

7.3.1/4 Determination of IL-1a concentration (pg/mL) in Episkin samples

Sample identification	IL-1a measured concentration (pg/mL)	Mean IL-1a measured concentration (pg/mL) n=3 tissues	Inter tissue %CV, n =3 tissues
Negative control 1	BLQ	NC	NC
Negative control 2	5.81
Negative control 3	10.8
Test sample 1	14.4	20.2	26.1
Test sample 2	24.7
Test sample 3	21.4

% CV : Coefficient of Variation

BLQ : Below Limit of Quantification (<5.00 pg/mL)

NC: Not Calculated

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is considered to be non-irritant to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test substance using the EPISKIN^TM reconstructed human epidermis model. The study was performed according to OECD guideline 439 in compliance with Good Laboratory Practice.

Preliminary tests were performed to detect the ability of the test substance to directly reduce MTT as well as its colouring potential. following these preliminary tests, triplicate tissues (Episkin epidermis surface area of 0.38 cm²) were treated with 10 µL of the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed to remove residual test substance before incubating for 42 hours at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the post-exposure incubation period each tissue was taken for MTT-loading (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm with acidified isopropanol solution as a blank.

In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

Negative ( Dulbecco's Phosphate Buffered Saline (DPBS)) and positive controls (5% sodium dodecyl sulphate (SDS) in distilled water) were used in the study. The mean absorbance of the triplicate negative control values was 0.974 which was above the minimum acceptance value of 0.6. The standard deviation (SD) of the % viability was 2 which was below the maximum acceptance value of 18. The mean % viability of the positive control was 6 +/- 3 of the negative control. This was below the maximum acceptance values of 40% viability and SD of 18. Therefore the study was considered as valid.

The relative mean viability of the test item treated tissues was 97 +/- 7 % after the 15-minute exposure period. As the mean viability was above 50% after MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The mean IL-1a concentration for treated tissues was 20.2 pg/mL. Due to this value being below 60 pg/mL, the results met the criteria for a non-irritant response.