6-{4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenoxy}hexyl methacrylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep. 2016 - Feb. 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: crystalline

Details on test material:

- State of aggregation: Non-aggregated
- Particle size distribution: D50 = 12.2 µm
- Geometric standard deviation (GSD): 0.2
- Shape of particles: not assessed
- Surface area of particles: not determined
- Crystal structure: Crystalline, no further details known
- Coating: None
- Moisture content: 0.03%
- Residual solvent: Non detected
- Activation: Not applicable
- Stabilisation: With Phenothiazine (< 1000 ppm)
- Other:

Specific details on test material used for the study:

Designation in Test Facility: 16082904G
Date of Receipt: 29. Aug. 2016
Condition at Receipt: Room temperature, in proper conditions
Name: ROC-601
Batch no.: S23PSG0816
Appearance: white crystalline powder
Composition: 99.5% purity, see analytical certificate page 20
CAS No.: 439661-46-8
EINECS-No.: not yet assigned
Molecular formula: C20H26O5
Molecular weight: 346.42 g/mol
Purity: 99.5% by LC-UV
Homogeneity: uniform crystalline powder
Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date: 29. June 2016
Expiry date: July 2017
Storage: Room Temperature (20 ±5 °C), keep away from light. The test item was stored in the test facility in a closed vessel at 16.6 – 20.5 °C, protected from light.
SMILES Code: C(=O)(C(=C)C)OCCCCCCOc1ccc(C=CC(=O)OC)cc1

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava. Designation of the kit: EPI-200-SIT Day of delivery: 11. Oct. 2016 Batch no.: 23362

Details on animal used as source of test system:

The test system is a commercially available EpiDerm(TM)-Kit, procured by MatTek.
The EpiDerm(TM) 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 representing main lipid classes analogous to those found in vivo. The EpiDerm(TM) tissues are cultured on specially prepared cell culture inserts.

Justification for test system used:

Skin corrosion was not expected

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

Tissue #1: 24.8 mg
Tissue #2: 25.1 mg
Tissue #3: 25.2 mg
Negative control: 30 µL DPBS buffer
Positive control: 30 µL 5% SDS-solution

Duration of treatment / exposure:

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ±1 °C and 5.0 ±0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.

Duration of post-treatment incubation (if applicable):

After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ±1 °C and 5.0 ±0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue #1

Value:

92.1

Vehicle controls validity:

other: see negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: calculated in comparison to the negative control

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue #2

Value:

98

Vehicle controls validity:

other: see negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: calculated in comparison to the negative control

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue #3

Value:

97.3

Vehicle controls validity:

other: see negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: calculated in comparison to the negative control

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of tissues 1 - 3

Value:

95.8

Vehicle controls validity:

other: see negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Standard deviation ±3.2%

Other effects / acceptance of results:

Positive controls showed a mean viability of 2.9 ±0.1%

Any other information on results incl. tables

Measured Values: As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate. The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.041	0.041	0.040	0.040	0.038	0.039	0.040	0.038	0.040

 

The absorbance values of negative control, test item and positive control (OD 570 nm) are given in the following table:

 

Designation	Measurement	Negative Control	ROC-601	Positive Control
Tissue 1	1	2.021	1.835	0.097
	2	2.033	1.878	0.097
Tissue 2	1	2.050	1.974	0.098
	2	2.026	1.973	0.097
Tissue 3	1	1.994	1.972	0.096
	2	1.954	1.945	0.096

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given above. The mean of the three tissues was also calculated.

 

Designation	Negative Control	ROC-601	Positive Control
Mean – blank (tissue 1)	1.987	1.817	0.057
Mean – blank (tissue 2)	1.998	1.934	0.058
Mean – blank (tissue 3)	1.934	1.919	0.056
Mean of the three tissues	1.973	1.890	0.057

 

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:

 

Designation	ROC-601	Positive Control
% Formazan production (tissue 1)	92.1%	2.9%
% Formazan production (tissue 2)	98.0%	2.9%
% Formazan production (tissue 3)	97.3%	2.8%
% Formazan production (mean)	95.8%	2.9%
± SD of mean Formazan production (%)	3.2%	0.1%

 

Assessment and Validity

Skin Irritation Potential of the Test Item: The relative absorbance values were reduced to 95.8% after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-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	2.0
% Formazan production of positive control SDS	£20% of negative control	2.9%
SD of mean viability of the tissue replicates (%)	≤ 18%	1.7% (negative control) 0.1% (positive control) 3.2% (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:

GHS criteria not met

Conclusions:

The test item is considered as non-irritant to skin.

Executive summary:

The test substance ROC-601 was investigated for skin irritation potential in a study named “Determination of Skin Irritation Potential of ROC-601 in the Reconstructed Human Epidermis (RHE) Test Method following EU-Method B.46 resp. OECD 439”.

Three tissues of the human skin model EpiDerm^TM were treated with ROC-601 for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as negative control, 5% SDS solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed clear irritating effects. Relative absorbance was reduced to 2.9% (required: < 20%). Variation within the tissue replicates was acceptable (required: ≤ 18%).

After the treatment with the test item, the relative absorbance values were reduced to 95.8 %. This value is above the threshold for skin irritation potential (50%). Therefore, ROC-601 is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) Test Method.