α-methylenebenzyl acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-19 to 2015-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 2014-10-13/14, Date of signature: 2015-04-08)

Test material

Test material information

Constituent 1

Reference substance name:

α-methylenebenzyl acetate

EC Number:

218-620-7

EC Name:

α-methylenebenzyl acetate

Cas Number:

2206-94-2

Molecular formula:

C10H10O2

IUPAC Name:

1-phenylethenyl acetate

Test material form:

other: liquid

Test animals

Species:

other: EpiDerm

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: positive and negative control tissues

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 60 μl (0.63 μl/cm2)
- Concentration (if solution): undiluted

CONTROLS: Controls were set up in parallel to the test item cultures in order to confirm the validity of the test.
Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and without MgCl2).
Positive control: Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in deionised H2O containing 5% SDS. Procured from MatTek In Vitro Life Science Laboratories, batch no.: 020615MHG

Duration of treatment / exposure:

60 minutes

Observation period:

Treated tissue had a total incubation time of 42 h

Number of animals:

Three tissues for each of test, negative control and positive control groups

Details on study design:

TEST SYSTEM:
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared.

PRE-EXPERIMENT
The test item was tested for the ability of direct MTT reduction. To test for this ability, 30 µL of the liquid test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5 ± 0.5% CO2 for 60 min. Untreated MTT solution was used as control.
The MTT solution didn’t change its colour within one hour, therefore, direct MTT reduction had not taken place, and no data correction was necessary


EXPERIMENTAL PROCEDURE

Pre-Incubation of Tissues
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 h.
After the pre-incubation (one hour), the other three wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 18 h.

Treatment
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only.
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 µL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used for treatment with the test item:
30 µL test item were applied and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in one minute intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min. 37 ± 1°C and 5 ± 0.5% CO2. 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute-intervals.
After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 24 h.

Medium Renewal
For three incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for 10 min. (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18 h for post-incubation.

MTT Assay
After a total incubation time of 42 h, a 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 h.
After this time, the MTT reagent was aspirated and replaced by DPBS buffer. This was also then aspirated and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 h at room temperature.
After 2 h, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, 2 replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: tissue viability

Value:

122.9

Remarks on result:

other:

Remarks:

Basis: other: mean optical density relative to control. Remarks: not irritating . (migrated information)

Any other information on results incl. tables

Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.038	0.037	0.037	0.038	0.036	0.037	0.037	0.037	0.037

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values negative control, test item and positive control (OD at 570 nm)

Designation	Measurement	Negative Control	Alpha Methylenebenzyl Acetate	Positive Control
Tissue 1	1	1.828	2.240	0.088
	2	1.794	2.250	0.085
Tissue 2	1	1.786	2.191	0.083
	2	1.780	2.212	0.083
Tissue 3	1	1.877	2.233	0.089
	2	1.840	2.228	0.089

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in the table above. Mean and relative standard deviation (comparison of the 3 tissues) were also calculated.

Mean Absorbance Values

Designation	Negative Control	Alpha Methylenebenzyl Acetate	Positive Control
Mean – blank (tissue 1)	1.774	2.208	0.050
Mean – blank (tissue 2)	1.746	2.165	0.046
Mean – blank (tissue 3)	1.822	2.194	0.052
Mean of the 3 tissues	1.781	2.189	0.049
Relative standard deviation of the 3 tissues	2.2%	1.0%	6.2%

 

Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table9.2‑a           % Formazan Production

Designation	Alpha Methylenebenzyl Acetate	Positive Control
% Formazan production (tissue 1)	124.0%	2.8%
% Formazan production (tissue 2)	121.6%	2.6%
% Formazan production (tissue 3)	123.2%	2.9%
% Formazan production (mean)	122.9%	2.8%

 

Assessment and Validity

Skin Irritation Potential of the Test Item

The relative absorbance values were increased to 122.9 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as not irritant to skin.

Validity and Acceptability

Validity criteria and results are stated in the following table:

Criterion	Demanded	Found
OD of negative control	≥0.8 and≤ 2.8	1.8
% Formazan production of positive control SDS	<20% of negative control	2.8 %
Variation within replicates (RSD)	< 18%	2.2 % (negative control) 6.2 % (positive control) 1.0 % (test item)

 

All validity criteria were met.

Values for negative control and for positive control were within the range of historical data of the test facility.

Therefore, the experiment is considered valid.

 

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

α-Methylenebenzyl acetate has been tested in a reliable in vitro study for skin irritation conducted according to OECD 439 and in compliance with GLP using EpiDerm tissue. The relative tissue viability of the test-item treated tissues, was not reduced relative to the negative controls. The positive control produced the expected reduction in viability. It is concluded that the test item is not irritant under the conditions of the test.

Executive summary:

α-Methylenebenzyl acetate has been tested for skin irritation in a study carried out according to OECD Guideline 439, compliant with GLP.

Following a 60 minute application of the test substance to the human skin model, it was concluded that the test item is not irritating to skin.