3-methyl-1,1-diphenylurea

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.01.-23.01.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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:

3-methyl-1,1-diphenylurea

EC Number:

236-039-7

EC Name:

3-methyl-1,1-diphenylurea

Cas Number:

13114-72-2

Molecular formula:

C14H14N2O

IUPAC Name:

3-methyl-1,1-diphenylurea

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: 3-Methyl-1,1-diphenylurea- Physical state: white solid powder- Composition of test material, percentage of components: main component: 3-Methyl-1,1-diphenylurea CAS:13114-72-2 >98% (w/w) impurities: unlisted additives: unlisted-Molecular formula: C14H14N2O-Molecular weight: 226,27-Batch No.: 14015- Expiration date of the lot/batch: 11/2019- Stability under test conditions: stable- Storage condition of test material: in closed container, in dry room at room temperature (at laboratory conditions)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: normal human epidermal keratinocytes- Tissue batch number(s): The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Ashland, USA); Lot No. 21611, kit CTEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°CMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1mg/ml- Incubation time: 3h- Spectrophotometer: Libra S22- Wavelength: 570nm- Modifications to validated SOP: M/48/3 (VUOS-CETA, 2011)NUMBER OF REPLICATE TISSUES: 3 for every concetrationCONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- Fresh tissues - N. of replicates : 3- Method of calculation used: NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)-The cut-off values for the prediction of irritation are given below; these values are stated in EU Method B.46., part 2.2. INTERPRETATION OF RESULTS: The test substance is considered to be irritant to skin in accordance with UN GHS 1 category 2:(i) if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %.The test substance is considered to have no category:(ii) if the tissue viability after exposure and post-treatment incubation is more 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): 25 mgNEGATIVE CONTROL- PBS (phosphate buffered saline) prepared 06/01/2015, exp. 06/07/2015- Amount(s) applied (volume or weight): 25µLPOSITIVE CONTROL5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 110414ZSB, exp. 04/11/2015MEDIUMEPI-100-NMM-SIT/Maintenance Medium, MatTek, Lot No. 011515MHD, exp. 29/01/2015

Duration of treatment / exposure:

3h

Number of replicates:

3

Test system

Duration of treatment / exposure:

1h

Details on study design:

TEST SYSTEMThe reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. TEST PROCEDURE1. DIRECT MTT REDUCTION-FUNCTIONAL CHECK IN TUBESSome test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional check for this possibility is performed as follows: the test substance is added to mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.2. MTT TESTAfter pre-incubations (1 and 18±3hours), tissues were topically exposed to the test chemicals for 1 hour. Three tissues were used per every concentration of the test substance (C2), for the positive (PC) and negative (NC) controls. Tissues were then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium. After 24±2 hours incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18±2 hours. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank. EVALUATION OF RESULTSFor further classification the relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %. The cut-off values for the prediction of irritation are given below; these values are stated in EU Method B.46., part 2.2.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: OD570

Value:

2.22

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Value:

109.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

25 mg of the test substance was added to 1.0 ml of MTT medium. Solution was incubated for 1 hour (37±, 5±1 % CO₂, humidified). The test substance did not cause change of colour.

MTT TEST

25 mg ofthe test substancewasplaced directly atop the previously moistened tissue, so the test substance will cover its entiresurface. Length of exposition was 60±5 min. After that the test substance was removed, tissues were rinsed and post-incubated.

After post-incubations the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, theblue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.

OD₅₇₀measuring was performed after 2-hour extraction with shaking.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test substance was 109.6% of negative control average value, i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model. The test substance is considered to have no category in regard to skin irritation.

Executive summary:

Test substance, Akardit,was assayed for the in vitro skin irritation in human epidermal model EpiDerm^TM.The test was performed according to theMethod B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4, (1), (3).

After pre-incubation of tissues, 25 mg ofthe testwasplaced directly atop to the previously moistened tissue so it covered all tissue surfaces. Length of exposition was 60 minutes. Three tissues were used for every concentration and controls.

After removal of the test substance from tissues, tissues were post incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, average viability of tissues treated by the test substance was 109.6%, i.e.viability was >50 %.

The effect of the test substancewasnegative inEpiDerm^TMmodel (the tissue was not damaged).

According to the classification criteria given in chapter 3.9., the test substance is considered to have no category in regard to skin irritation.