Fenuron

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

adopted 28 July 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 06, 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Fenuron

EC Number:

202-941-4

EC Name:

Fenuron

Cas Number:

101-42-8

Molecular formula:

C9H12N2O

IUPAC Name:

fenuron

Test material form:

other: white to off-white powder

Details on test material:

- Particle size distribution: 100 % particle size < 12 µm (method: Laser Diffraction; Certificate of Analysis)
Particle size parameter determined with a Malvern Mastersizer 2000 (Non-GLP determination)
D10% = 1.18 µm
D50% = 7.72 µm
D90% = 16.00 µm
D: Diameter; 10, 50, 90: percentage cumulative
- Mass median aerodynamic diameter (MMAD): 1.595 µm
- Geometric standard deviation (GSD): calculated as 2.09

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH, Batch no. 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 5 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material : at room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture

OTHER SPECIFICS: IsoQure UR 300

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM (EPI-200, Lot no. 25876) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

Justification for test system used:

Skin irritation by cytotoxic action of substances with direct human skin contact refers to the production of reversible damage to the skin following the application of a test item for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)]. This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach.

Vehicle:

other: Dulbecco’s phosphate buffered saline

Remarks:

For better contact of the test item to the skin, the skin surface was moistened .

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200)
- Tissue batch number(s): Lot no. 25876, Keratinocyte strain 00267
- Date of initiation of testing: January 23, 2018 (start experimental phase)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Temperature of post-treatment incubation (if applicable): A post-treatment incubation period of the rinsed tissues in 0.9 mL fresh assay medium of 42 hours was performed; no data on temperature.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
- Observable damage in the tissue due to washing: Not specified.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: Each skin sample was placed in MTT assay solution of 1 mg/mL for 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Specification: MTT QC assay, 4 hours. n=3; Acceptance criteria: OD (540-570 nm) 1.0-3.0 ; Result: 1.554 ± 0.167.
- Barrier function: Specification: ET-50 assay. 100 µL 1% Triton X-100, 4 time-points. n=3, MTT assay; Acceptance criteria: ET-50 (4.77-8.72 hrs); Result: 6.95 hrs.
- Morphology: Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. The Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS).
- Contamination: Specification sterility: Long term antibiotic and antimycotic free culture; Acceptance criteria: No contamination; Result: Sterile
The cells used to produce EpiDerm™ tissue are screened for potential biological contaminants. Tests for each potential biological contaminant listed below were performed according to the test method given. Results of "Not detected" indicate that testing for the potential biological contaminant was not observed as determined by the stated test method.
HIV-1 virus - Oligonucleotide-directed amplification: Not detected
Hepatitis B virus - Oligonucleotide- directed amplification: Not detected
Hepatitis C virus - Oligonucleotide- directed amplification: Not detected
Bacteria, yeast. and other fungi - long term antibiotic. antimycotic free culture: Not detected

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No direct MYY interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 Independent test sequence was performed. The measurements were made for each of the three tissues in 2 replicates.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL
For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): D-PBS. 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Test system

Details on study design:

TEST SITE
- Area of exposure: skin surface
- % coverage: 0.63 cm2

REMOVAL OF TEST SUBSTANCE
- Washing: washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- Time after start of exposure: 60 minutes

SCORING SYSTEM:
- Method of calculation: According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 2 )
mean tissue viability > 50% non-irritant (NI).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

99.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min exposure, 42 h post-treatment incubation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (D-PBS)

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min exposure, 42 h post-treatment incubation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control (5% SDS )

Value:

7.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min exposure, 42 h post-treatment incubation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: 25 mg IsoQure UR 300 were added to 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. No change of colour was noted.
In conclusion, no possible interference with the MTT measurement (OD 540 nm), due to colour changes or direct interacting with the MTT assay reagent, was noted and no additional test had to be performed.
- Colour interference with MTT: 25 mg test item were mixed with 300 µL sterile deionised water and incubated in the dark at 37 °C, 5% CO2 and 95% relative humidity for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolouration of the test item was noted. In addition, 25 mg test item were mixed with 2 mL isopropanol and incubated at room temperature for three hours. No discolouration of the test item was noted. Hence, the criteria of the test on colour interference were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.
The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5. The mean optical density (OD) of 3 negative control tissues was 1.320 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as% of the negative control tissues is ≤ 20%. The viability of cells treated with the positive reference item, 5% SDS, was 7.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual% tissue viabilities of the 3 identically treated replicates is ≤ 18%. The standard deviation determined for all triplicates was below the limit of acceptance of 18%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions, IsoQure UR 300 tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).

Executive summary:

A key in vitro study was performed to determine cytotoxic properties of IsoQure UR 300 to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensional model of human skin. The EpiDerm^TM model was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. IsoQure UR 300 was applied as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS).D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium. The mean viability of cells exposed to IsoQure UR 300 was 99.7% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. IsoQure UR 300was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.320 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 7.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%. The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria were fulfilled.