Urea, N,N''-1,4-cyclohexanediylbis-, trans-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD Guideline 439 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[(1r,4r)-4-(carbamoylamino)cyclohexyl]urea

EC Number:

813-604-1

Cas Number:

68533-01-7

Molecular formula:

C8 H16 N4 O2

IUPAC Name:

[(1r,4r)-4-(carbamoylamino)cyclohexyl]urea

Test material form:

other: solid

Details on test material:

Name of test substance: NM01
Test-substance No.: 13/0143-2
Batch identification: 492-14-01
Purity: >95% (information given by the sponsor).
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
pH-value: Ca. 8 (undiluted test substance, moistened with water)
Physical state: Solid

Test animals

Species:

other: 3D human epidermis model (EpiDermTM)

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

25 μL bulk volume (about 6 mg)

Duration of treatment / exposure:

1 h

Observation period:

42 h

Details on study design:

TEST SYSTEM:
The present test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is designed to predict a skin corrosion or irritation potential of a chemical by using the three dimensional human epidermis model EpiDermTM. After application of the test material to the stratum corneum surface of the EpiDermTM tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol-extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to negative control values from tissues and expressed as relative tissue viability.
The EpiDermTM model consists of normal, 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 arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs(R), 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

DIRECT MTT REDUCTION:
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested
concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. In case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control.

BASIC PROCEDURE:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively. 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently treated with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: tissue viability, presented as the quotient of the mean OD570 divided by the respective OD570 NC value in %.

Value:

95

Remarks on result:

other:

Remarks:

Basis: mean. Max. score: 100.0. (migrated information)

Any other information on results incl. tables

Table 1: Individual and mean OD570 values, individual and mean viability values and standard deviations.

Test substance		tissue 1	tissue 2	tissue 3	mean           SD
NC	mean OD570	2.062	2.194	2.048	2.102
	viability [% of NC]	98.1	104.4	97.5	100	3.82
NM01	mean OD570	2.208	1.757	2.012	1.993
	viability [% of NC]	105.1	83.6	95.8	95	10.76
PC	mean OD570	0.048	0.050	0.057	0.052
	viability [% of NC]	2.3	2.4	2.7	2	0.23

Historical control data of NC and PC of irritation test (Period: Jan 2012 - May 2014).

Historic Range of NC: OD570 = 2.312, SD = 0.248

Historic Range of PC: OD570 = 0.0083, SD = 0.052

Viability (%): Mean = 3.5%, SD = 2.53%

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that NM01 does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Executive summary:

The potential of NM01 to cause dermal irritation was assessed by a single topical application of 25 μL bulk volume (about 6 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a

tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance was not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 95%. It was therefore concluded that NM01 does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.