Monuron

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

1,1-dimethyl-3-phenylurea

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

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):

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: -
- Modifications to validated SOP: -

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 25 mg IsoQure UR 300 were added to 1 mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 87.4-110.8 % (negative control); 1.3-10.2% (positive control)
- Barrier function: The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration.
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- No. of replicates : each of the three tissues in 2 replicates
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

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

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

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

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 min

Number of replicates:

3

Test system

Details on study design:

TEST SITE
- Area of exposure: 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: 42 h

OBSERVATION TIME POINTS
(indicate if minutes, hours or days)

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 group

Value:

99.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min exposure, followed 42h

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, followed 42h

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: min exposure, followed 42h

Other effects / acceptance of results:

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 determined for all triplicates was below the limit of acceptance of 18%. Hence, all acceptance criteria required were fulfilled.

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 300 was 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.