Ethyl diphenylcarbamate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

Reconstructed Human Epidermis Test Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 3, 2016 - November 24, 2016

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)

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)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl diphenylcarbamate

EC Number:

210-047-0

EC Name:

Ethyl diphenylcarbamate

Cas Number:

603-52-1

Molecular formula:

C15H15NO2

IUPAC Name:

ethyl N,N-diphenylcarbamate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

The SkinEthic™ RHE model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 16-RHE-122
- Delivery date: November 22, 2016
- Date of initiation of testing: November 22, 2016
- Expiration date: November 28, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3h
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.2 (CV = 1.3%) specification OD > 0.7
- Barrier function: 5.2 h SPECIFICATION 4.0 h < ET50 < 10 h
- Morphology: 6 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg of the test item on 0.50 cm2 human skin model

NEGATIVE CONTROL
- Amount applied: 16 μL

POSITIVE CONTROL
- Amount applied: 16 μL
- Concentration: 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 15 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 105.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1% viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no.
- Direct-MTT reduction: A yellow solution was observed after 3 hours of incubation between 36.4ºC and 37.8ºC, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
In water: A colorless solution was obtained after 3 hours of incubation between 36.4ºC and 37.8ºC, 5% CO2.
In isopropanol: A colorless solution was obtained after 2 hours of incubation at room temperature.
Therefore, the test item will not interfere with the MTT assay and there is no need to add nonspecific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY:yes, a full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.Summary of proficiency chemicals testing according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the standard deviation of the viability of negative control epidermises is 21.3%, instead of ≤ 18% as initially scheduled. This is due to a mean OD value of the replicate No.3 treated with the negative control, which is slightly above the maximal OD value of 1.5. This higher value obtained in the negative control group could lead to an over-classification of the test item. Considering the results obtained (the cell viability of the three treated epidermises is superior to 100% with a standard deviation of 3.7%), this deviation is considered as without impact on the conclusion of the study (the test item is not irritant for the skin).
- Acceptance criteria met for positive control: yes.
- Acceptance criteria met for variability between replicate measurements: yes.

Any other information on results incl. tables

The results were expressed as a viability percentage compared with the negative control.  

viability % = (OD test item / OD negative control) x100

For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

Table 1. Individual and average values of OD after 42 minutes exposure

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD	Conclusion
Negative control	1	0.943	1.019	1.309	77.8	100.0	21.3
		1.039
		1.075
	2	1.308	1.334		101.9
		1.352
		1.343
	3	1.512	1.575		120.3
		1.557
		1.657
Positive control	4	0.012	0.013	0.013	1.0	1.0	0.1	Irritant
		0.013
		0.013
	5	0.011	0.011		0.8
		0.012
		0.011
	6	0.013	0.014		1.1
		0.014
		0.014
Test item PH-16/0551	18	1.369	1.426	1.381	108.7	105.5	3.7	Non irritant
		1.438
		1.471
	19	1.392	1.389		106.1
		1.392
		1.383
	20	1.349	1.329		101.5
		1.353
		1.285

# mean of 3 values (triplicate of the same extract)

OD: optical density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Acceptability criteria: SD≤18%.

The acceptance criteria were met.

 

Notes:

-         If the viability obtained for the test item is greater than 50%, the test item has to be considered as non irritant.

-         If the viability obtained for the test item is less than or equal to 50%, the test item has to be considered as irritant.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the treated tissues was 105.5%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). No other effects were observed. The test item is not irritant to the skin.

Executive summary:

An in vitro skin irritation test method was performed according OECD Guideline 439 and Test method B.46, with GLP, to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). The test item was applied at the dose of 16 mg, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41 hours and 15 minutes post-incubation period at 37ºC, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Negative and positive controls were run in parallel. The mean corrected percent viability of the treated tissues was 105.5%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). No other effects were observed. The test item has to be considered as Non-irritant to skin, corresponding to the UN GHS No category.