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

Endpoint summary

Description of key information

The test substance ROC-601 was investigated for skin irritation potential in a Reconstructed Human Epidermis (RHE) Test 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. 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.

Furthermore, the test substance ROC-601 was investigated for eye irritation potential in an EpiOcular^TM Human Cornea Model following OECD 492.

One valid experiment was performed. The solid test item ROC-601 was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control.

After treatment with the test item, the relative absorbance values (relative to control) were reduced to 99.8 %. This value is well above the threshold for eye irritation potential (≤ 60%). The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 25.8 % (< 50%).

Thus, under the conditions of the test, ROC-601 is considered as not eye irritant in the EpiOcular^TM Eye Irritation Test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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)

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

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

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

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

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

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%

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept. 2016 - Apr. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: See Certificate of Analysis
Purity: 99.5% by LC-UV
Homogeneity: Uniform crystalline powder
Expiry date: Jul. 2017
Storage: Room temperature (20 ±5 °C), keep away from light
CAS No.: 439661-46-8
EINECS-No.: Not yet assigned
Chemical Class: Organic substance: Para substituted cinnamic acid ester
Volatility: Unknown
pH-value: Unknown
LogP: Approx. 5.3 (KOWWIN v. 1.67 estimate)
Molecular weight: 346.42 g/mol
Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
SMILES Code: C(=O)(C(=C)C)OCCCCCCOc1ccc(C=CC(=O)OC)cc1
Storage: The test item was stored in the test facility in a closed vessel at room temperature (20 ±5 °C).

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The EpiOcular(TM) tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular(TM) tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm². EpiOcular(TM) tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia (Designation of the kit: OCL-212-EIT, Day of delivery: 11. Oct. 2016, Batch no.: 23738).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Tissue 1: 50.4 mg
Tissue 2: 50.7 mg

Duration of treatment / exposure:

6 hours at 37 ±1 °C, 5 ±1% CO2 and 80 – 100% relative humidity

Duration of post- treatment incubation (in vitro):

For post-treatment incubation, the tissues were incubated for 18 hours and 3 minutes at 37 ±1 °C, 5 ±1% CO2 and 80 – 100% relative humidity

Number of animals or in vitro replicates:

2 replicates

Irritation parameter:

other: % viability

Run / experiment:

Tissue #1

Value:

101.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: % viability

Run / experiment:

Tissue #2

Value:

98.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: % viability

Run / experiment:

Mean of two tissues

Value:

99.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Criterion (Demanded): Found
OD of negative control (≥ 0.8 and ≤ 2.5): 2.1
% Formazan production of positive control (< 50% of negative control): 25.8%
Variation within replicates (< 20%): 2.2% (negative control), 1.5% (positive control), 2.9% (test item)

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, ROC-601 is considered as not eye irritant in the EpiOcular(TM) Eye Irritation Test.

Executive summary:

In a study titled “Determination of Eye Irritation Potential ofROC-601using the EpiOcular^TM Human Cornea Model following OECD 492” the test substance ROC-601 was investigated for eye irritation potential.

One valid experiment was performed. The test item ROC-601 was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

The solid test item was applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control, methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.5, OD was 2.1. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 25.8 % (< 50%).

Variation within tissue replicates was acceptable (< 20%).

After treatment with the test item, the relative absorbance values were reduced to 99.8 %. This value is well above the threshold for eye irritation potential (≤ 60%).

Thus, under the conditions of the test, ROC-601 is considered as not eye irritant in the EpiOcular^TM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance ROC-601 was investigated for skin irritation potential in a Reconstructed Human Epidermis (RHE) Test following EU-Method B.46 resp. OECD 439 and found being non-irritant to skin. Furthermore, the test substance was assessed for eye damage/irritation within an EpiOcularTMHuman Cornea Model following OECD 492 and found not being irritating to eyes. Accordingly, the substance is not to be classified for skin corrosion/irritation or eye damage/irritation according to GHS or EU CLP (Regulation (EC) No 1272/2008).

Test data or information for respiratory irritation is not available.